Change search
Refine search result
1 - 44 of 44
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1. Bañó-Polo, Manuel
    et al.
    Baeza-Delgado, Carlos
    Tamborero, Silvia
    Hazel, Anthony
    Grau, Brayan
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Whitley, Paul
    Gumbart, James C.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mingarro, Ismael
    Transmembrane but not soluble helices fold inside the ribosome tunnel2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 5246Article in journal (Refereed)
    Abstract [en]

    Integral membrane proteins are assembled into the ER membrane via a continuous ribosome-translocon channel. The hydrophobicity and thickness of the core of the membrane bilayer leads to the expectation that transmembrane (TM) segments minimize the cost of harbouring polar polypeptide backbones by adopting a regular pattern of hydrogen bonds to form a-helices before integration. Co-translational folding of nascent chains into an a-helical conformation in the ribosomal tunnel has been demonstrated previously, but the features governing this folding are not well understood. In particular, little is known about what features influence the propensity to acquire a-helical structure in the ribosome. Using in vitro translation of truncated nascent chains trapped within the ribosome tunnel and molecular dynamics simulations, we show that folding in the ribosome is attained for TM helices but not for soluble helices, presumably facilitating SRP (signal recognition particle) recognition and/or a favourable conformation for membrane integration upon translocon entry.

  • 2. Ch'ng, Jun-Hong
    et al.
    Sirel, Madle
    Zandian, Arash
    del Pilar Quintana, Maria
    Chan, Sherwin Chun Leung
    Moll, Kirsten
    Tellgren-Roth, Åsa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, Peter
    Qundos, Ulrika
    Wahlgren, Mats
    Epitopes of anti-RIFIN antibodies and characterization of rif-expressing Plasmodium falciparum parasites by RNA sequencing2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 43190Article in journal (Refereed)
    Abstract [en]

    Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing.

  • 3. Cuviello, Flavia
    et al.
    Tellgren-Roth, Åsa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ruud Selin, Frida
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Monné, Magnus
    Bisaccia, Faustino
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ostuni, Angela
    Membrane insertion and topology of the amino-terminal domain TMD0 of multidrug-resistance associated protein 6 (MRP6)2015In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 589, no 24, p. 3921-3928Article in journal (Refereed)
    Abstract [en]

    The function of the ATP-binding cassette transporter MRP6 is unknown but mutations in its gene cause pseudoxanthoma elasticum. We have investigated the membrane topology of the N-terminal transmembrane domain TMD0 of MRP6 and the membrane integration and orientation propensities of its transmembrane segments (TMs) by glycosylation mapping. Results demonstrate that TMD0 has five TMs, an Nout-Cin topology and that the less hydrophobic TMs have strong preference for their orientation in the membrane that affects the neighboring TMs. Two disease-causing mutations changing the number of positive charges in the loops of TMD0 did not affect the membrane insertion efficiencies of the adjacent TMs.

  • 4.
    Enquist, Karl
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fransson, Mawritz
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Boekel, Carolina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bengtsson, Inger
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Geiger, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pettersson, Aron
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Sofia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane-integration characteristics of two ABC transporters, CFTR and P-glycoprotein2009In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 387, no 5, p. 1153-1164Article in journal (Refereed)
    Abstract [en]

    To what extent do corresponding transmembrane helices in related integral membrane proteins have different membrane-insertion characteristics? Here, we compare, side-by-side, the membrane insertion characteristics of the 12 transmembrane helices in the adenosine triphosphate-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and the cystic fibrosis transmembrane conductance regulator (CFTR). Our results show that 10 of the 12 CFTR transmembrane segments can insert independently into the ER membrane. In contrast, only three of the P-gp transmembrane segments are independently stable in the membrane, while the majority depend on the presence of neighboring loops and/or transmembrane segments for efficient insertion. Membrane-insertion characteristics can thus vary widely between related proteins.

  • 5. Gadalla, Salah-Eldin
    et al.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lara Vasquez, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ericsson, Christer
    Zhao, Jian
    Nister, Monica
    EpCAM associates with endoplasmic reticulum aminopeptidase 2 (ERAP2) in breast cancer cells2013In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 439, no 2, p. 203-208Article in journal (Refereed)
    Abstract [en]

    Epithelial cell adhesion molecule (EpCAM) is an epithelial and cancer cell marker and there is a cumulative and growing evidence of its signaling role. Its importance has been recognized as part of the breast cancer stem cell phenotype, the tumorigenic breast cancer stem cell is EpCAM(+). In spite of its complex functions in normal cell development and cancer, relatively little is known about EpCAM-interacting proteins. We used breast cancer cell lines and performed EpCAM co-immunoprecipitation followed by mass spectrometry in search for novel potentially interacting proteins. The endoplasmic reticulum aminopeptidase 2 (ERAP2) was found to co-precipitate with EpCAM and to co-localize in the cytoplasm/ER and the plasma membrane. ERAP2 is a proteolytic enzyme set in the endoplasmic reticulum (ER) where it plays a central role in the trimming of peptides for presentation by MHC class I molecules. Expression of EpCAM and ERAP2 in vitro in the presence of dog pancreas rough microsomes (ER vesicles) confirmed N-linked glycosylation, processing in ER and the size of EpCAM. The association between ERAP2 and EpCAM is a unique and novel finding that provides new ideas on EpCAM processing and on how antigen presentation may be regulated in cancer.

  • 6. Goel, Suchi
    et al.
    Palmkvist, Mia
    Moll, Kirsten
    Joannin, Nicolas
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Akhouri, Reetesh R.
    Moradi, Nasim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Westman, Mattias
    Angeletti, Davide
    Kjellin, Hanna
    Lehtio, Janne
    Blixt, Ola
    Ideström, Lars
    Gahmberg, Carl G.
    Storry, Jill R.
    Hult, Annika K.
    Olsson, Martin L.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wahlgren, Mats
    RIFINs are adhesins implicated in severe Plasmodium falciparum malaria2015In: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 21, no 4, p. 314-317Article in journal (Refereed)
    Abstract [en]

    Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum-encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), bind to RBCs-preferentially of blood group A-to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population.

  • 7.
    Hedin, Linnea E.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bernsel, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hennerdal, Aron
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Illergård, Kristoffer
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Enquist, Karl
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kauko, Anni
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cristobal, Susana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lerch-Bader, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 396, no 1, p. 221-229Article in journal (Refereed)
    Abstract [en]

    In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.

  • 8.
    Hessa, Tara
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bernsel, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sato, Yoko
    Lerch Bader, Mirjam
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A quantitative analysis of translocon-mediated insertion of transmembrane alpha-helicesManuscript (Other academic)
  • 9.
    Hessa, Tara
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bihlmaier, Karl
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundin, Carolina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Boekel, Jorrit
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andersson, Helena
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Recognition of transmembrane helices by the endoplasmic reticulum translocon2005In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 433, no 7024, p. 377-381Article in journal (Refereed)
    Abstract [en]

    Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein - lipid interactions are critical during translocon-mediated membrane insertion.

  • 10.
    Hessa, Tara
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Meindl-Beinker, Nadja M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bernsel, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sato, Yoko
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lerch-Bader, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen H.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Molecular code for transmembrane-helix recognition by the Sec61 translocon2007In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 450, no 7172, p. 1026-1030Article in journal (Refereed)
    Abstract [en]

    Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position- dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon- mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.

  • 11. Jaud, Simon
    et al.
    Fernández-Vidal, Mónica
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Meindl-Beinker, Nadja M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hübner, Nadja C.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tobias, Douglas J.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen H.
    Insertion of short transmembrane helices by the Sec61 translocon2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 28, p. 11588-11593Article in journal (Refereed)
    Abstract [en]

    The insertion efficiency of transmembrane (TM) helices by the Sec61 translocon depends on helix amino acid composition, the positions of the amino acids within the helix, and helix length. We have used an in vitro expression system to examine systematically the insertion efficiency of short polyleucine segments (L(n), n = 4 ... 12) flanked at either end by 4-residue sequences of the form XXPX-L(n)-XPXX with X = G, N, D, or K. Except for X = K, insertion efficiency (p) is <10% for n < 8, but rises steeply to 100% for n = 12. For X = K, p is already close to 100% for n = 10. A similar pattern is observed for synthetic peptides incorporated into oriented phospholipid bilayer arrays, consistent with the idea that recognition of TM segments by the translocon critically involves physical partitioning of nascent peptide chains into the lipid bilayer. Molecular dynamics simulations suggest that insertion efficiency is determined primarily by the energetic cost of distorting the bilayer in the vicinity of the TM helix. Very short lysine-flanked leucine segments can reduce the energetic cost by extensive hydrogen bonding with water and lipid phosphate groups (snorkeling) and by partial unfolding.

  • 12. Karamyshev, Andrey L.
    et al.
    Kelleher, Daniel J.
    Gilmore, Reid
    Johnson, Arthur E.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mapping the interaction of the STT3 subunit of the oligosaccharyl transferase complex with nascent polypeptide chains2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 49, p. 40489-40493Article in journal (Refereed)
    Abstract [en]

    Many secretory and membrane proteins are N-glycosylated by the oligosaccharyl transferase complex during their translocation across the endoplasmic reticulum membrane. Several experimental observations suggest that the highly conserved STT3 subunit contains the active site of the oligosaccharyl transferase. Here, we report a detailed study of the interaction between the active site of the STT3 protein and nascent polypeptide chains using an in vitro photocrosslinking technique. Our results show that the addition of a glycan moiety in a stretch of similar to 15 residues surrounding a QK*T cross-linking site impairs the interaction between the nascent chain and STT3.

  • 13. Karamyshev, Andrey L.
    et al.
    Patrick, Anna E.
    Karamysheva, Zemfira N.
    Griesemer, Dustin S.
    Hudson, Henry
    Tjon-Kon-Sang, Sandra
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Otto, Hendrik
    Liu, Qinghua
    Rospert, Sabine
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Johnson, Arthur E.
    Thomas, Philip J.
    Inefficient SRP Interaction with a Nascent Chain Triggers a mRNA Quality Control Pathway2014In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 156, no 1-2, p. 146-157Article in journal (Refereed)
    Abstract [en]

    Misfolded proteins are often cytotoxic, unless cellular systems prevent their accumulation. Data presented here uncover a mechanism by which defects in secretory proteins lead to a dramatic reduction in their mRNAs and protein expression. When mutant signal sequences fail to bind to the signal recognition particle (SRP) at the ribosome exit site, the nascent chain instead contacts Argonaute2 (Ago2), and the mutant mRNAs are specifically degraded. Severity of signal sequence mutations correlated with increased proximity of Ago2 to nascent chain and mRNA degradation. Ago2 knockdown inhibited degradation of the mutant mRNA, while overexpression of Ago2 or knockdown of SRP54 promoted degradation of secretory protein mRNA. The results reveal a previously unappreciated general mechanismof translational quality control, in which specific mRNA degradation preemptively regulates aberrant protein production (RAPP).

  • 14.
    Kim, Hyun
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane topology of the STT3 subunit of the oligosaccharyl transferase complex2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 21, p. 20261-20267Article in journal (Refereed)
    Abstract [en]

    The highly conserved membrane protein STT3 is part of the oligosaccharyl transferase complex in the endoplasmic reticulum of eukaryotic cells. Various experimental observations strongly suggest that STT3 contains the active site of the complex. Here, we report a detailed topology study of STT3 from two different organisms, Saccharomyces cerevisiae and mouse, using in vivo and in vitro topology mapping assays. Our results suggest that STT3 has 11 transmembrane helices and an overall N-cyt-C-lum orientation.

  • 15.
    Lara, Patricia
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tellgren-Roth, Åsa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Behesti, Hourinaz
    Horn, Zachi
    Schiller, Nina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Enquist, Karl
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cammenberg, Malin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Liljenström, Amanda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hatten, Mary E.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Murine astrotactins 1 and 2 have a similar membrane topology and mature via endoproteolytic cleavage catalyzed by a signal peptidase2019In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 294, no 12, p. 4538-4545Article in journal (Refereed)
    Abstract [en]

    Astrotactin 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking, and synaptic plasticity in the brain as well as in planar polarity pathways in the skin. Here we used glycosylation mapping and protease protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes and found that Astn2 has a cleaved N-terminal signal peptide, an N-terminal domain located in the lumen of the rough microsomal membranes (topologically equivalent to the extracellular surface in cells), two transmembrane helices, and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of signal peptide cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second transmembrane helix; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.

  • 16.
    Lara, Patricia
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reithinger, Johannes
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Holgado, Aurora
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Maojun, You
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hammed, Abdessalem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mattle, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Refined topology model of the STT3/Stt3 protein subunit of the oligosaccharyltransferase complex2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 27, p. 11349-11360Article in journal (Refereed)
    Abstract [en]

    The oligosaccharyltransferase complex, localized in the endoplasmic reticulum (ER) of eukaryotic cells, is responsible for the N-linked glycosylation of numerous protein substrates. The membrane protein STT3 is a highly conserved part of the oligosaccharyltransferase and likely contains the active site of the complex. However, understanding the catalytic determinants of this system has been challenging, in part because of a discrepancy in the structural topology of the bacterial versus eukaryotic proteins and incomplete information about the mechanism of membrane integration. Here, we use a glycosylation mapping approach to investigate these questions. We measured the membrane integration efficiency of the mouse STT3-A and yeast Stt3p transmembrane domains (TMDs) and report a refined topology of the N-terminal half of the mouse STT3-A. Our results show that most of the STT3 TMDs are well inserted into the ER membrane on their own or in the presence of the natural flanking residues. However, for the mouse STT3-A hydrophobic domains 4 and 6 and yeast Stt3p domains 2, 3a, 3c, and 6 we measured reduced insertion efficiency into the ER membrane. Furthermore, we mapped the first half of the STT3-A protein, finding two extra hydrophobic domains between the third and the fourthTMD. This result indicates that the eukaryotic STT3 has 13 transmembrane domains, consistent with the structure of the bacterial homolog of STT3 and setting the stage for future combined efforts to interrogate this fascinating system.

  • 17. Lee, Hunsang
    et al.
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ostuni, Angela
    Presto, Jenny
    Johansson, Janne
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Live-cell topology assessment of URG7, MRP6(102) and SP-C using glycosylatable green fluorescent protein in mammalian cells2014In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 450, no 4, p. 1587-1592Article in journal (Refereed)
    Abstract [en]

    Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6(102), SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in C-in, form. MRP6(102) and SP-C(Leu/Val) are inserted into the membrane in C-out form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.

  • 18.
    Lerch-Bader, Mirjam
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundin, Carolina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Contribution of positively charged flanking residues to the insertion of transmembrane helices into the endoplasmic reticulum2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 11, p. 4127-4132Article in journal (Refereed)
    Abstract [en]

    Positively charged residues located near the cytoplasmic end of hydrophobic segments in membrane proteins promote membrane insertion and formation of transmembrane alpha-helices. A quantitative understanding of this effect has been lacking, however. Here, using an in vitro transcription-translation system to study the insertion of model hydrophobic segments into dog pancreatic rough microsomes, we show that a single Lys or Arg residue typically contributes approximately -0.5 kcal/mol to the apparent free energy of membrane insertion (DeltaG(app)) when placed near the cytoplasmic end of a hydrophobic segment and that stretches of 3-6 Lys residues can contribute significantly to DeltaG(app) from a distance of up to approximately 13 residues away.

  • 19.
    Lundin, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Sofia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johnson, Arthur E.
    Näslund, Jan
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stable insertion of Alzheimer Aβ peptide into the ER membrane strongly correlates with its length2007In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, no 20, p. 3809-3813Article in journal (Refereed)
    Abstract [en]

    Alzheimer's disease is characterized by the deposition of amyloid P-peptide (All) plaques in the brain. Full-length amyloid-beta precursor protein (APP) is processed by alpha- and beta-secretases to yield soluble APP derivatives and membrane-bound C-terminal fragments, which are further processed by gamma-secretase to a non-amyloidogenic 3 kDa product or to All fragments. As different A beta fragments contain different parts of the APP transmembrane helix, one may speculate that they are retained more or less efficiently in the membrane. Here, we use the translocon-mediated insertion of different APP-derived polypeptide segments into the endoplasmic reticulum membrane to assess the propensities for membrane retention of All fragments. Our results show a strong correlation between the length of an A beta-derived segment and its ability to integrate into the microsomal membrane.

  • 20.
    Lundin, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen H
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Molecular code for protein insertion in the endoplasmic reticulum membrane is similar for N-in-C-out and N-out-C-in transmembrane helices2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 41, p. 15702-15707Article in journal (Refereed)
    Abstract [en]

    Transmembrane alpha-helices in integral membrane proteins can have two orientations in the membrane: N(in)-C(out) or N(out)-C(in). Previous studies of model N(out)-C(in) transmembrane segment have led to a detailed, quantitative picture of the "molecular code" that relates amino acid sequence to membrane insertion efficiency in vivo [Hessa T, et al. (2007) Molecular code for transmembrane helix recognition by the Sec61 translocon. Nature 450:1026-1030], but whether the same code applies also to N(in)-C(out) transmembrane helices is unknown. Here, we show that the contributions of individual amino acids to the overall efficiency of membrane insertion are similar for the two kinds of helices and that the threshold hydrophobicity for membrane insertion can be up to approximately 1 kcal/mol lower for N(in)-C(out) compared with N(out)-C(in) transmembrane helices, depending on the neighboring helices.

  • 21.
    Lundin, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Käll, Lukas
    Kreher, Scott A.
    Kapp, Katja
    Sonnhammer, Erik L.
    Carlson, John R.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane topology of the Drosophila OR83b odorant receptor2007In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, no 29, p. 5601-5604Article in journal (Refereed)
    Abstract [en]

    By analogy to mammals, odorant receptors (ORs) in insects, such as Drosophila melanogaster, have long been thought to belong to the G-protein coupled receptor (GPCR) superfamily. However, recent work has cast doubt on this assumption and has tentatively suggested an inverted topology compared to the canonical N-out - C-in 7 transmembrane (TM) GPCR topology, at least for some Drosophila ORs. Here, we report a detailed topology mapping of the Drosophila OR83b receptor using engineered glycosylation sites as topology markers. Our results are inconsistent with a classical GPCR topology and show that OR83b has an intracellular N-terminus, an extracellular C-terminus, and 7TM helices. (c) 2007 Federation of European Biochemical Societies.

  • 22.
    Lundin, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordström, Richard
    Wagner, Klaus
    Windpassinger, Christian
    Andersson, Helena
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane topology of the human seipin protein2006In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 580, no 9, p. 2281-2284Article in journal (Refereed)
    Abstract [en]

    The Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene encodes an integral membrane protein, called seipin, of unknown function localized to the endoplasmic reticulum of eukaryotic cells. Seipin is associated with the heterogeneous genetic disease BSCL2, and mutations in an N-glycosylation motif links the protein to two other disorders, autosomal-dominant distal hereditary motor neuropathy type V and Silver syndrome. Here, we report a topological study of seipin using an in vitro topology mapping assay. Our results suggest that the predominant form of seipin is 462 residues long and has an N-cyt-C-cyt orientation with a long luminal loop between the two transmembrane helices.

  • 23.
    Marani, Paula
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Bologna, Italy.
    Wagner, Samuel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Baars, Louise
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Genevaux, Pierre
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Casadio, Rita
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    New Escherichia coli outer membrane proteins identified through prediction and experimental verification2006In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 15, no 4, p. 884-889Article in journal (Refereed)
    Abstract [en]

    Many new Escherichia coli outer membrane proteins have recently been identified by proteomics techniques. However, poorly expressed proteins and proteins expressed only under certain conditions may escape detection when wild-type cells are grown under standard conditions. Here, we have taken a complementary approach where candidate outer membrane proteins have been identified by bioinformatics prediction, cloned and overexpressed, and finally localized by cell fractionation experiments. Out of eight predicted outer membrane proteins, we have confirmed the outer membrane localization for five-YftM, YaiO, YfaZ, CsgF, and YliI - and also provide preliminary data indicating that a sixth -YfaL- may be an outer membrane autotransporter.

  • 24.
    Meindl-Beinker, Nadia M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundin, Carolina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen H.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Asn- and Asp-mediated interactions between transmembrane helices during translocon-mediated membrane protein assembly2006In: EMBO reports, ISSN 1469-221X, Vol. 7, no 11, p. 1111-1116Article in journal (Refereed)
    Abstract [en]

    Inter-helix hydrogen bonding involving asparagine (Asn, N), glutamine (Gin, Q), aspartic acid (Asp, D) or glutamic acid (Glu, E) can drive efficient di- or trimerization of transmembrane helices in detergent micelles and lipid bilayers. Likewise, Asn-Asn and Asp-Asp pairs can promote the formation of helical hairpins during translocon-mediated membrane protein assembly in the endoplasmic reticulum. By in vitro translation of model integral membrane protein constructs in the presence of rough microsomes, we show that Asn- or Asp-mediated interactions with a neighbouring transmembrane helix can enhance the membrane insertion efficiency of a marginally hydrophobic transmembrane segment. Our observations suggest that inter-helix hydrogen bonds can form during Sec61 translocon-assisted insertion and thus could be important for membrane protein assembly.

  • 25. Mingarro, Ismael
    et al.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Whitley, Paul
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Different conformations of nascent polypeptides during translocation across the ER membrane2000In: BMC Cell Biology, ISSN 1471-2121, E-ISSN 1471-2121, Vol. 1, no 3Article in journal (Refereed)
    Abstract [en]

    Background

    In eukaryotic cells, proteins are translocated across the ER membrane through a continuous ribosome-translocon channel. It is unclear to what extent proteins can fold already within the ribosome-translocon channel, and previous studies suggest that only a limited degree of folding (such as the formation of isolated α-helices) may be possible within the ribosome.

    Results

    We have previously shown that the conformation of nascent polypeptide chains in transit through the ribosome-translocon complex can be probed by measuring the number of residues required to span the distance between the ribosomal P-site and the lumenally disposed active site of the oligosaccharyl transferase enzyme (J. Biol. Chem 271: 6241-6244).Using this approach, we now show that model segments composed of residues with strong helix-forming properties in water (Ala, Leu) have a more compact conformation in the ribosome-translocon channel than model segments composed of residues with weak helix-forming potential (Val, Pro).

    Conclusions

    The main conclusions from the work reported here are (i) that the propensity to form an extended or more compact (possibly α-helical) conformation in the ribosome-translocon channel does not depend on whether or not the model segment has stop-transfer function, but rather seems to reflect the helical propensities of the amino acids as measured in an aqueous environment, and (ii) that stop-transfer sequences may adopt a helical structure and integrate into the ER membrane at different times relative to the time of glycan addition to nearby upstream glycosylation acceptor sites.

  • 26.
    Nilsson, IngMarie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Texas A&M University System Health Science Center, Texas.
    Johnson, Arthur E.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cleavage of a tail-anchored protein by signal peptidase2002In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 516, no 1-3, p. 106-108Article in journal (Refereed)
    Abstract [en]

    Tail-anchored proteins are post-translationally targeted and inserted into the endoplasmic reticulum membrane. They do not use the co-translational sign at-recognition particle (SRP)-dependent pathway, but rather utilize an ill-defined, ATP-dependent mechanism. Here, we show that a tail-anchored protein can be cleaved by signal peptidase and that the sequence requirements for efficient cleavage seem to be the same as for cleavage of co-translationally targeted SRP-dependent proteins.

  • 27.
    Nilsson, IngMarie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Texas A&M University System Health Science Center, Texas.
    Johnson, Arthur E.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    How hydrophobic is alanine?2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 32, p. 29389-29393Article in journal (Refereed)
    Abstract [en]

    By a number of measures, alanine is poised at the threshold between those amino acids that promote the membrane integration of transmembrane alpha-helices and those that do not. We have measured the preference of alanine to partition into the lipid-water interface region over the central acyl chain region of the endoplasmic reticulum (ER) membrane both by its ability to promote the formation of so-called helical hairpins, i.e. a pair of transmembrane helices separated by a tight turn, and by mapping the position relative to the membrane of the lumenal end of a transmembrane alpha-helix that ends with a block of 10 alanines. Both measures show that Ala has a weak but distinct preference for the interface region, which is in agreement with recent biophysical measurements on pentaeptide partitioning in simple water-lipid or water-octanol systems (Jayasinghe, S., Hristova, K., and White, S. H. ( 2001) J. Mol. Biol. 312, 927 - 934). Considering the complexity of the translocon-mediated insertion of membrane proteins into the ER, the agreement between the biochemical and biophysical measurements is striking and suggests that protein-lipid interactions are already important during the very early steps of membrane protein assembly in the ER.

  • 28.
    Nilsson, IngMarie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Texas A&M University System Health Science Center, Texas.
    Kelleher, Daniel J.
    Miao, Yiwei
    Shao, Yuanlong
    Kreibich, Gert
    Gilmore, Reid
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johnson, Arthur E.
    Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex2003In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 161, no 4, p. 715-725Article in journal (Refereed)
    Abstract [en]

    In eukaryotic cells, polypeptides are N glycosylated after passing through the membrane of the ER into the ER lumen. This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme. Here, we report the first cross-linking of an OST subunit to a nascent chain that is undergoing translocation through, or integration into, the ER membrane. A photoreactive probe was incorporated into a nascent chain using a modified Lys-tRNA and was positioned in a cryptic glycosylation site (-Q-K-T- instead of -N-K-T-) in the nascent chain. When translocation intermediates with nascent chains of increasing length were irradiated, nascent chain photocross-linking to translocon components, Sec61alpha and TRAM, was replaced by efficient photocross-linking solely to a protein identified by immunoprecipitation as the STT3 subunit of the OST No cross-linking was observed in the absence of a cryptic sequence or in the presence of a competitive peptide substrate of the OST. As no significant nascent chain photocross-linking to other OST subunits was detected in these fully assembled translocation and integration intermediates, our results strongly indicate that the nascent chain portion of the OST active site is located in STT3.

  • 29.
    Nilsson, IngMarie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hessa, Tara
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johnson, Arthur E.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Karamyshev, Andrey L.
    The Code for Directing Proteins for Trans location across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence2015In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 6, p. 1191-1201Article in journal (Refereed)
    Abstract [en]

    The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes to the SRP receptor in the endoplasmic reticulum membrane, initiating translocation of the nascent chain through the Sec61 translocon. Although signal sequences do not have homology, they have similar structural regions: a positively charged N-terminus, a hydrophobic core and a more polar C-terminal region that contains the cleavage site for the signal peptidase. Here, we have used site-specific photocrosslinking to study SRP signal sequence interactions. A photoreactive probe was incorporated into the middle of wild-type or mutated signal sequences of the secretory protein preprolactin by in vitro translation of mRNAs containing an amber-stop codon in the signal peptide in the presence of the N-epsilon-(5-azido-2 nitrobenzoyl)-Lys-tRNA(amb) amber suppressor. A homogeneous population of SRP ribosome-nascent chain complexes was obtained by the use of truncated mRNAs in translations performed in the presence of purified canine SRP. Quantitative analysis of the photoadducts revealed that charged residues at the N-terminus of the signal sequence or in the early part of the mature protein have only a mild effect on the SRP signal sequence association. However, deletions of amino acid residues in the hydrophobic portion of the signal sequence severely affect SRP binding. The photocrosslinking data correlate with targeting efficiency and translocation across the membrane. Thus, the hydrophobic core of the signal sequence is primarily responsible for its recognition and binding by SRP, while positive charges fine-tune the SRP signal sequence affinity and targeting to the translocon.

  • 30.
    Nilsson, IngMarie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Texas A & M University System Health Science Center, Texas.
    Ohvo-Rekilä, Henna
    Slotte, J. Peter
    Johnson, Arthur E.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Inhibition of protein translocation across the endoplasmic reticulum membrane by sterols2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 45, p. 41748-41754Article in journal (Refereed)
    Abstract [en]

    Cholesterol and related sterols are known to modulate the physical properties of biological membranes and can affect the activities of membrane-bound protein complexes. Here, we report that an early step in protein translocation across the endoplasmic reticulum (ER) membrane is reversibly inhibited by cholesterol levels significantly lower than those found in the plasma membrane. By UV-induced chemical cross-linking we further show that high cholesterol levels prevent cross-linking between ribosome-nascent chain complexes and components of the Sec61 translocon, but have no effect on cross-linking to the signal recognition particle. The inhibiting effect on translocation is different between different sterols. Our data suggest that the protein translocation machinery may be sensitive to changes in cholesterol levels in the ER membrane.

  • 31.
    Nilsson, IngMarie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glycosylation efficiency of Asn-Xaa-Thr sequons depends both on the distance from the C terminus and on the presence of a downstream transmembrane segment2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 23, p. 17338-17343Article in journal (Refereed)
    Abstract [en]

    Statistical studies of N-glycosylated proteins have indicated that the frequency of nonglycosylated Asn-Xaa-(Thr/Ser) sequons increases toward the C terminus (Gavel, Y., and von Heijne, G. (1990) Protein Eng. 3, 433-442), Using in vitro transcription/translation of a truncated model protein in the presence of dog pancreas microsomes, we find that glycosylation efficiency of Asn-Xaa-Thr sequons indeed is reduced when the sequon is within similar to 60 residues of the C terminus. Surprisingly, the presence of a hydrophobic stop transfer sequence between the Asn-Xaa-Thr sequon and the C terminus results in a very different dependence of glycosylation efficiency on the distance to the C terminus, where the presence of the stop transfer segment inside the ribosome appears to cause a drastic drop in the level of glycosylation. We speculate that this may reflect a change in the structure of the ribosome/translocon complex induced by the stop transfer segment.

  • 32.
    Nilsson, IngMarie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Witt, Susanne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kiefer, Hans
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mingarro, Ismael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Distant downstream sequence determinants can control N-tail translocation during protein insertion into the endoplasmic reticulum membrane2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 9, p. 6207-6213Article in journal (Refereed)
    Abstract [en]

    We have studied the membrane insertion of ProW, an Escherichia coli inner membrane protein with seven transmembrane segments and a large periplasmic N-terminal tail, into endoplasmic reticulum (ER)-derived dog pancreas microsomes. Strikingly, significant levels of N-tail translocation is seen only when a minimum of four of the transmembrane segments are present; for constructs with fewer transmembrane segments, the N-tail remains mostly nontranslocated and the majority of the molecules adopt an "inverted" topology where normally nontranslocated parts are translocated and vice versa, N-tail translocation can also be promoted by shortening of the N-tail and by the addition of positively charged residues immediately downstream of the first transmembrane segment. We conclude that as many as four consecutive transmembrane segments may be collectively involved in determining membrane protein topology in the ER and that the effects of downstream sequence determinants may vary depending on the size and charge of the N-tail, We also provide evidence to suggest that the ProW N-tail is translocated across the ER membrane in a C-to-N-terminal direction.

  • 33. Orrell, Kathleen E.
    et al.
    Tellgren-Roth, Åsa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Di Bernardo, Mercedes
    Zhang, Zhifen
    Cuviello, Flavia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundqvist, Jasmin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Melnyk, Roman A.
    Direct Detection of Membrane-Inserting Fragments Defines the Translocation Pores of a Family of Pathogenic Toxins2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 18, p. 3190-3199Article in journal (Refereed)
    Abstract [en]

    Large clostridial toxins (LCTs) are a family of homologous proteins toxins that are directly responsible for the symptoms associated with a number of clostridial infections that cause disease in humans and in other animals. LCTs damage tissues by delivering a glucosyltransferase domain, which inactivates small GTPases, across the endosomal membrane and into the cytosol of target cells. Elucidating the mechanism of translocation for LCTs has been hampered by difficulties associated with identifying marginally hydrophobic segments that insert into the bounding membrane to form the translocation pore. Here, we directly measured the membrane-insertion partitioning propensity for segments spanning the putative pore-forming region using a translocon-mediated insertion assay and synthetic peptides. We identified membrane-inserting segments, as well as a conserved and functionally important negatively charged residue that requires protonation for efficient membrane insertion. We provide a model of the LCT pore, which provides insights into translocation for this enigmatic family of a-helical translocases.

  • 34. Ostuni, A.
    et al.
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Armentano, M. F.
    Miglionico, R.
    Salvia, A. M.
    Monnich, M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Carmosino, M.
    Lasorsa, F. M.
    Monne, M.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bisaccia, F.
    The hepatitis B x antigen anti-apoptotic effector URG7 is localized to the endoplasmic reticulum membrane2013In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 587, no 18, p. 3058-3062Article in journal (Refereed)
    Abstract [en]

    Hepatitis B x antigen up-regulates the liver expression of URG7 that contributes to sustain chronic virus infection and to increase the risk for hepatocellular carcinoma by its anti-apoptotic activity. We have investigated the subcellular localization of URG7 expressed in HepG2 cells and determined its membrane topology by glycosylation mapping in vitro. The results demonstrate that URG7 is N-glycosylated and located to the endoplasmic reticulum membrane with an N-lumen-C-cytosol orientation. The results imply that the anti-apoptotic effect of URG7 could arise from the C-terminal cytosolic tail binding a pro-apoptotic signaling factor and retaining it to the endoplasmic reticulum membrane.

  • 35. Pasche, Boris
    et al.
    Knobloch, Thomas J.
    Bian, Yansong
    Liu, Junjian
    Phukan, Sharbani
    Rosman, Diana
    Kaklamani, Virginia
    Baddi, Lisa
    Siddiqui, Farida S
    Frankel, Wendy
    Prior, Thomas W.
    Schuller, David E.
    Agrawal, Amit
    Lang, Jas
    Dolan, M. Eileen
    Vokes, Everett E.
    Lane, William S.
    Huang, Chiang-Ching
    Caldes, Trinidad
    Di Cristofano, Antonio
    Hampel, Heather
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fodde, Riccardo
    Murty, V. V. V. S.
    de la Chapelle, Albert
    Weghorst, Christopher M.
    Somatic acquisition and signaling of TGFBR1*6A in cancer2005In: Journal of the American Medical Association (JAMA), ISSN 0098-7484, E-ISSN 1538-3598, Vol. 294, no 13, p. 1634-1646Article in journal (Refereed)
    Abstract [en]

    Context: TGFBR1*6A is a common polymorphism of the type I transforming growth factor 0 receptor (TGFBR1). Epidemiological studies suggest that TGFBR1*6A may act as a tumor susceptibility allele. How TGFBR1*6A contributes to cancer development is largely unknown.. Objectives: To determine whether TGFBR1*6A is somatically acquired by primary tumors and metastases during cancer development and whether the 3-amino acid deletion that differentiates TGFBR1*6A from TGFBR1 is part of the mature receptor or part of the signal sequence and to investigate TGFBR1*6A signaling in cancer cells. Design, Setting, and Patients: Tumor And germline tissues from 531 patients with a diagnosis of head and neck, colorectal, or breast cancer recruited from 3 centers in the United States and from 1 center in Spain from June 1, 1994, through June 30, 2004, In vitro translation assays, MCF-7 breast cancer cells stably transfected with TGFBR1*6A, TGFBR1, or the vector alone, DLD-1 colorectal cancer cells that endogenously carry TGFBR1*6A, and SW48 colorectal cancer cells that do not carry TGFBR1*6A. Main Outcome Measures: TGFBR1*6A somatic acquisition in cancer. Determination of the amino terminus of the mature TGFBR1*6A and TGFBR1 receptors. Determination of TGF-beta-dependent cell proliferation. Results: TGFBR1*6A was somatically acquired in 13 of 44 (29.5%) colorectal cancer metastases, in 4 of 157 (2.5%) of colorectal tumors, in 4 of 226 (1.8%) head and neck primary tumors, and in none of the 104 patients with breast cancer. TGFBR1*6A somatic acquisition is not associated with loss of heterozygosity, microsatellite instability, or a mutator phenotype. The signal sequences of TGFBR1 and TGFBR1*6A are cleaved at the same site resulting in identical mature receptors. TGFBR1*6A may switch TGF-beta growth inhibitory signals into growth stimulatory signals in MCF-7 breast cancer cells and in DLD-1 colorectal cancer cells. Conclusions: TGFBR1*6A is somatically acquired in 29.5% of liver metastases from colorectal cancer and may bestow cancer cells with a growth advantage in the presence of TGF-beta. The functional consequences of this conversion appear to be mediated by the TGFBR1*6A signal sequence rather than by the mature receptor. The results highlight a new facet of TGF-beta signaling in cancer and suggest that TGFBR1*6A may represent a potential therapeutic target in cancer.

  • 36. Renault, Hugues
    et al.
    De Marothy, Minttu
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Jonasson, Gabriella
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nelson, David R.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andre, Francois
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Werck-Reichhart, Danièle
    Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants2017In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 34, no 8, p. 2041-2056Article in journal (Refereed)
    Abstract [en]

    Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct membrane topology. Insertion in the membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein topology rather than change in enzyme activity.

  • 37. Saenz, Alejandra
    et al.
    Presto, Jenny
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Akinyi-Oloo, Laura
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Garcia-Fojeda, Belen
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Jan
    Casals, Cristina
    Folding and Intramembraneous BRICHOS Binding of the Prosurfactant Protein C Transmembrane Segment2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 28, p. 17628-17641Article in journal (Refereed)
    Abstract [en]

    Surfactant protein C (SP-C) is a novel amyloid protein found in the lung tissue of patients suffering from interstitial lung disease (ILD) due to mutations in the gene of the precursor protein pro-SP-C. SP-C is a small alpha-helical hydrophobic protein with an unusually high content of valine residues. SP-C is prone to convert into beta-sheet aggregates, forming amyloid fibrils. Nature's way of solving this folding problem is to include a BRICHOS domain in pro-SP-C, which functions as a chaperone for SP-C during biosynthesis. Mutations in the pro-SP-C BRICHOS domain or linker region lead to amyloid formation of the SP-C protein and ILD. In this study, we used an in vitro transcription/translation system to study translocon-mediated folding of the WT pro-SP-C poly-Val and a designed poly-Leu transmembrane (TM) segment in the endoplasmic reticulum (ER) membrane. Furthermore, to understand how the pro-SP-C BRICHOS domain present in the ER lumen can interact with the TM segment of pro-SP-C, we studied the membrane insertion properties of the recombinant form of the pro-SP-C BRICHOS domain and two ILD-associated mutants. The results show that the co-translational folding of the WT pro-SP-C TM segment is inefficient, that the BRICHOS domain inserts into superficial parts of fluid membranes, and that BRICHOS membrane insertion is promoted by poly-Val peptides present in the membrane. In contrast, one BRICHOS and one non-BRICHOS ILD-associated mutant could not insert into membranes. These findings support a chaperone function of the BRICHOS domain, possibly together with the linker region, during pro-SP-C biosynthesis in the ER.

  • 38. Snapp, Erik Lee
    et al.
    McCaul, Nicholas
    Quandte, Matthias
    Cabartova, Zuzana
    Bontjer, Ilja
    Källgren, Carolina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Land, Aafke
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Sanders, Rogier W.
    Braakman, Ineke
    Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide2017In: eLIFE, E-ISSN 2050-084X, Vol. 6, article id e26067Article in journal (Refereed)
    Abstract [en]

    Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

  • 39. Stefansson, Anne
    et al.
    Armulik, Annika
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Staffan
    Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 20, p. 21200-21205Article in journal (Refereed)
    Abstract [en]

    Previous studies on the membrane-cytoplasm interphase of human integrin subunits have shown that a conserved lysine in subunits alpha(2), alpha(5), beta(1), and beta(2) is embedded in the plasma membrane in the absence of interacting proteins (Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. (1999) in J. Biol. Chem. 274, 37030-37034). Using a glycosylation mapping technique, we here show that alpha(10) and beta(8), two subunits that deviate significantly from the integrin consensus sequences in the membrane-proximal region, were found to have the conserved lysine at a similar position in the lipid bilayer. Thus, this organization at the C-terminal end of the transmembrane (TM) domain seems likely to be general for all 24 integrin subunits. Furthermore, we have determined the N-terminal border of the TM domains of the alpha(2), alpha(5), alpha(10), beta(1), and beta(8) subunits. The TM domain of subunit beta(8) is found to be 22 amino acids long, with a second basic residue (Arg(684)) positioned just inside the membrane at the exoplasmic side, whereas the lipid-embedded domains of the other subunits are longer, varying from 25 (alpha(2)) to 29 amino acids (alpha(10)). These numbers implicate that the TM region of the analyzed integrins (except beta(8)) would be tilted or bent in the membrane. Integrin signaling by transmembrane conformational change may involve alteration of the position of the segment adjacent to the conserved lysine. To test the proposed "piston" model for signaling, we forced this region at the C-terminal end of the alpha(5) and beta(1) TM domains out of the membrane into the cytosol by replacing Lys-Leu with Lys-Lys. The mutation was found to not alter the position of the N-terminal end of the TM domain in the membrane, indicating that the TM domain is not moving as a piston. Instead the shift results in a shorter and therefore less tilted or bent TM alpha-helix.

  • 40. Vargas-Uribe, Mauricio
    et al.
    Rodnin, Mykola V.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Holgado, Aurora
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kyrychenko, Alexander
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Posokhov, Yevgen O.
    Makhatadze, George
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ladokhin, Alexey S.
    Thermodynamics of Membrane Insertion and Refolding of the Diphtheria Toxin T-Domain2015In: Journal of Membrane Biology, ISSN 0022-2631, E-ISSN 1432-1424, Vol. 248, no 3, p. 383-394Article in journal (Refereed)
    Abstract [en]

    The diphtheria toxin translocation (T) domain inserts into the endosomal membrane in response to the endosomal acidification and enables the delivery of the catalytic domain into the cell. The insertion pathway consists of a series of conformational changes that occur in solution and in the membrane and leads to the conversion of a water-soluble state into a transmembrane state. In this work, we utilize various biophysical techniques to characterize the insertion pathway from the thermodynamic perspective. Thermal and chemical unfolding measured by differential scanning calorimetry, circular dichroism, and tryptophan fluorescence reveal that the free energy of unfolding of the T-domain at neutral and mildly acidic pH differ by 3-5 kcal/mol, depending on the experimental conditions. Fluorescence correlation spectroscopy measurements show that the free energy change from the membrane-competent state to the interfacial state is approximately -8 kcal/mol and is pH-independent, while that from the membrane-competent state to the transmembrane state ranges between -9.5 and -12 kcal/mol, depending on the membrane lipid composition and pH. Finally, the thermodynamics of transmembrane insertion of individual helices was tested using an in vitro assay that measures the translocon-assisted integration of test sequences into the microsomal membrane. These experiments suggest that even the most hydrophobic helix TH8 has only a small favorable free energy of insertion. The free energy for the insertion of the consensus insertion unit TH8-TH9 is slightly more favorable, yet less favorable than that measured for the entire protein, suggesting a cooperative effect for the membrane insertion of the helices of the T-domain.

  • 41.
    Virkki, Minttu
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Boekel, Carolina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Illergård, Kristoffer
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Peters, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Shu, Nanjiang
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Tsirigos, Konstantinos D.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation2014In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, no 13, p. 2529-2538Article in journal (Refereed)
    Abstract [en]

    While early structural models of helix-bundle integral membrane proteins posited that the transmembrane a-helices [transmembrane helices (TMHs)] were orientated more or less perpendicular to the membrane plane, there is now ample evidence from high-resolution structures that many TMHs have significant tilt angles relative to the membrane. Here, we address the question whether the tilt is an intrinsic property of the TMH in question or if it is imparted on the TMH during folding of the protein. Using a glycosylation mapping technique, we show that four highly tilted helices found in multi-spanning membrane proteins all have much shorter membrane-embedded segments when inserted by themselves into the membrane than seen in the high-resolution structures. This suggests that tilting can be induced by tertiary packing interactions within the protein, subsequent to the initial membrane-insertion step.

  • 42. Wanngren, Johanna
    et al.
    Lara Vasques, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Maioli, Silvia
    Moradi, Nasim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chen, Lu
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tjernberg, Lars O.
    Lundkvist, Johan
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karlström, Helena
    Changed membrane integration and catalytic site conformation are two mechanisms behind the increased Aβ42/Aβ40 ratio by presenilin 1 familial Alzheimer-linked mutations.2014In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 4, p. 393-406Article in journal (Refereed)
    Abstract [en]

    The enzyme complex γ-secretase generates amyloid β-peptide (Aβ), a 37-43-residue peptide associated with Alzheimer disease (AD). Mutations in presenilin 1 (PS1), the catalytical subunit of γ-secretase, result in familial AD (FAD). A unifying theme among FAD mutations is an alteration in the ratio Aβ species produced (the Aβ42/Aβ40 ratio), but the molecular mechanisms responsible remain elusive. In this report we have studied the impact of several different PS1 FAD mutations on the integration of selected PS1 transmembrane domains and on PS1 active site conformation, and whether any effects translate to a particular amyloid precursor protein (APP) processing phenotype. Most mutations studied caused an increase in the Aβ42/Aβ40 ratio, but via different mechanisms. The mutations that caused a particular large increase in the Aβ42/Aβ40 ratio did also display an impaired APP intracellular domain (AICD) formation and a lower total Aβ production. Interestingly, seven mutations close to the catalytic site caused a severely impaired integration of proximal transmembrane/hydrophobic sequences into the membrane. This structural defect did not correlate to a particular APP processing phenotype. Six selected FAD mutations, all of which exhibited different APP processing profiles and impact on PS1 transmembrane domain integration, were found to display an altered active site conformation. Combined, our data suggest that FAD mutations affect the PS1 structure and active site differently, resulting in several complex APP processing phenotypes, where the most aggressive mutations in terms of increased Aβ42/Aβ40 ratio are associated with a decrease in total γ-secretase activity.

  • 43.
    Öjemalm, Karin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Halling, Katrin K.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Orientational Preferences of Neighboring Helices Can Drive ER Insertion of a Marginally Hydrophobic Transmembrane Helix2012In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 45, no 4, p. 529-540Article in journal (Refereed)
    Abstract [en]

    alpha-helical integral membrane proteins critically depend on the correct insertion of their transmembrane alpha helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the transmembrane alpha helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (N-cyt-C-lum or N-lum-C-cyt) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the missing hydrophobicity in polytopic membrane proteins.

  • 44.
    Öjemalm, Karin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Higuchi, Takashi
    Jiang, Yang
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen H.
    Suga, Hiroaki
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum2011In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 31, p. E359-E364Article in journal (Refereed)
    Abstract [en]

    Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel [van den Berg B, et al. (2004) Nature427:36–44; Zimmer J, et al. (2008) Nature455:936–943; Egea PF, Stroud RM (2010)Proc Natl Acad Sci USA 107:17182–17187]. To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid’s contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal∕ðmol · Å2Þ, the solvation parameter for transfer from translocon to bilayer is 6 –10 cal∕ðmol · Å2Þ, pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a termodynamic partitioning model and insights into the physical properties of the translocon.

1 - 44 of 44
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf