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Integration and topology of membrane proteins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Responsible organisation
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins comprise around 20-30% of most proteomes. They play important roles in most biochemical pathways. All receptors and ion channels are membrane proteins, which make them attractive targets for drug design. Membrane proteins insert and fold co-translationally into the endoplasmic reticular membrane of eukaryotic cells. The protein-conducting channel that inserts the protein into the membrane is called Sec61 translocon, which is a hetero-oligomeric channel that allows transmembrane segments to insert laterally into the lipid bilayer. The focus of this thesis is how the translocon recognizes the transmembrane helices and integrates them into the membrane.

We have investigated the sequence requirements for the translocon-mediated integration of a transmembrane α-helix into the ER by challenging the Sec61 translocon with designed polypeptide segments in an in vitro expression system that allows a quantitative assessment of membrane insertion efficiency. Our studies suggest that helices might interact with each other already during the membrane-insertion step, possibly forming helical hairpins that partition into the membrane as a single unit. Further, the insertion efficiency for Nin-Cout vs. Nout-Cin transmembrane helices and the integration efficiency of Alzheimer’s Aβ-peptide fragments has been investigated.

Finally, detailed topology mapping was performed on two biologically interesting proteins with unknown topology, the human seipin protein and Drosophila melanogaster odorant receptor OR83b.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik , 2009. , 53 p.
Keyword [en]
insertion, Sec61, translocation
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-8575ISBN: 978-91-7155-827-5 (print)OAI: oai:DiVA.org:su-8575DiVA: diva2:200527
Public defence
2009-04-09, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2009-03-12 Created: 2009-02-20 Last updated: 2016-02-23Bibliographically approved
List of papers
1. Asn- and Asp-mediated interactions between transmembrane helices during translocon-mediated membrane protein assembly
Open this publication in new window or tab >>Asn- and Asp-mediated interactions between transmembrane helices during translocon-mediated membrane protein assembly
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2006 (English)In: EMBO reports, ISSN 1469-221X, Vol. 7, no 11, 1111-1116 p.Article in journal (Refereed) Published
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.

Keyword
endoplasmic reticulum, helix-helix interaction, membrane protein assembly, Sec61, transmembrane helix
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-25790 (URN)10.1038/sj.embor.7400818 (DOI)000242279500012 ()
Available from: 2009-03-19 Created: 2009-02-20 Last updated: 2016-02-23Bibliographically approved
2. Molecular code for protein insertion in the endoplasmic reticulum membrane is similar for N-in-C-out and N-out-C-in transmembrane helices
Open this publication in new window or tab >>Molecular code for protein insertion in the endoplasmic reticulum membrane is similar for N-in-C-out and N-out-C-in transmembrane helices
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2008 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 41, 15702-15707 p.Article in journal (Refereed) Published
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.

Keyword
membrane protein, positive-inside rule, Saccharomyces cerevisiae, topology, translocon
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-14917 (URN)10.1073/pnas.0804842105 (DOI)000260240900014 ()18840693 (PubMedID)
Available from: 2008-11-10 Created: 2008-11-10 Last updated: 2017-12-13Bibliographically approved
3. Stable insertion of Alzheimer Aβ peptide into the ER membrane strongly correlates with its length
Open this publication in new window or tab >>Stable insertion of Alzheimer Aβ peptide into the ER membrane strongly correlates with its length
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2007 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, no 20, 3809-3813 p.Article in journal (Refereed) Published
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.

Keyword
amyloid beta-protein precursor, A beta-peptide, translocon mediated, membrane insertion
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-25792 (URN)10.1016/j.febslet.2007.07.003 (DOI)000248903600008 ()
Available from: 2009-03-19 Created: 2009-02-20 Last updated: 2017-12-13Bibliographically approved
4. Membrane topology of the human seipin protein
Open this publication in new window or tab >>Membrane topology of the human seipin protein
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2006 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 580, no 9, 2281-2284 p.Article in journal (Refereed) Published
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.

Keyword
seipin, N-linked glycosylation, oligosaccharyl transferase, topology mapping, BCSL gene
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-25793 (URN)10.1016/j.febslet.2006.03.040 (DOI)000237012800021 ()
Available from: 2009-03-19 Created: 2009-02-20 Last updated: 2017-12-13Bibliographically approved
5. Membrane topology of the Drosophila OR83b odorant receptor
Open this publication in new window or tab >>Membrane topology of the Drosophila OR83b odorant receptor
Show others...
2007 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, no 29, 5601-5604 p.Article in journal (Refereed) Published
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.

Keyword
odorant receptor, membrane protein, topology, Drosophila melanogaster
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-25794 (URN)10.1016/j.febslet.2007.11.007 (DOI)000253488000010 ()
Available from: 2009-03-19 Created: 2009-02-20 Last updated: 2017-12-13Bibliographically approved

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