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Membrane Protein Biogenesis in Saccharomyces cerevisiae
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membranes are hydrophobic barriers that define the outer boundaries and internal compartments of living cells. Membrane proteins are the gates in these barriers, and they perform vital functions in the highly regulated transport of matter and information across membranes. Membrane proteins destined for the endoplasmic reticulum are targeted either co- or post-translationally to the Sec61 translocon, the major translocation machinery in eukaryotic cells, which allows for lateral partitioning of hydrophobic segments into the lipid bilayer. This thesis aims to acquire insights into the mechanism of membrane protein insertion and the role of different translocon components in targeting, insertion and topogenesis, using the yeast Saccharomyces cerevisiae as a model organism.

By measuring the insertion efficiency of a set of model proteins, we studied the sequence requirements for Sec61-mediated insertion of an α-helical transmembrane segment and established a ‘biological hydrophobicity scale’ in yeast, which describes the individual contributions of the 20 amino acids to insertion. Systematic mutagenesis and photo-crosslinking of the Sec61 translocon revealed key residues in the lateral gate that modulate the threshold hydrophobicity for membrane insertion and transmembrane segment orientation. Further, my studies demonstrate that the translocon-associated Sec62 is important not only for post-translational targeting, but also for the insertion and topogenesis of moderately hydrophobic signal anchor proteins and the C-terminal translocation of multi-spanning membrane proteins. Finally, nuclearly encoded mitochondrial membrane proteins were found to evade mis-targeting to the endoplasmic reticulum by containing short C-terminal tails.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2013. , 72 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-95376ISBN: 978-91-7447-798-6 (print)OAI: oai:DiVA.org:su-95376DiVA: diva2:661735
Public defence
2013-12-13, Nordenskiöld Lecture Hall, Geo-Science Building, Svante Arrhenius väg 12, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 4: Manuscript; Paper 5: Manuscript

Available from: 2013-11-21 Created: 2013-10-26 Last updated: 2014-07-25Bibliographically approved
List of papers
1. Analysis of transmembrane helix integration in the endoplasmic reticulum in S. cerevisiae
Open this publication in new window or tab >>Analysis of transmembrane helix integration in the endoplasmic reticulum in S. cerevisiae
2009 (English)In: Journal of molecular biology, ISSN 1089-8638, Vol. 386, no 5, 1222-8 p.Article in journal (Refereed) Published
Abstract [en]

What sequence features in integral membrane proteins determine which parts of the polypeptide chain will form transmembrane alpha-helices and which parts will be located outside the lipid bilayer? Previous studies on the integration of model transmembrane segments into the mammalian endoplasmic reticulum (ER) have provided a rather detailed quantitative picture of the relation between amino acid sequence and membrane-integration propensity for proteins targeted to the Sec61 translocon. We have now carried out a comparative study of the integration of N out-C in-orientated 19-residue-long polypeptide segments into the ER of the yeast Saccharomyces cerevisiae. We find that the 'threshold hydrophobicity' required for insertion into the ER membrane is very similar in S. cerevisiae and in mammalian cells. Further, when comparing the contributions to the apparent free energy of membrane insertion of the 20 natural amino acids between the S. cerevisiae and the mammalian ER, we find that the two scales are strongly correlated but that the absolute difference between the most hydrophobic and most hydrophilic residues is approximately 2-fold smaller in S. cerevisiae.

Keyword
membrane protein, membrane insertion, endoplasmic reticulum, hydrophobicity, Saccharomyces cerevisiae
National Category
Natural Sciences
Research subject
Biophysics; Biochemistry
Identifiers
urn:nbn:se:su:diva-33928 (URN)10.1016/j.jmb.2009.01.027 (DOI)000264383500004 ()19452628 (PubMedID)
Available from: 2009-12-30 Created: 2009-12-30 Last updated: 2013-11-05Bibliographically approved
2. Sec62 Protein Mediates Membrane Insertion and Orientation of Moderately Hydrophobic Signal Anchor Proteins in the Endoplasmic Reticulum (ER)
Open this publication in new window or tab >>Sec62 Protein Mediates Membrane Insertion and Orientation of Moderately Hydrophobic Signal Anchor Proteins in the Endoplasmic Reticulum (ER)
2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 25, 18058-18067 p.Article in journal (Refereed) Published
Abstract [en]

Nascent chains are known to be targeted to the endoplasmic reticulum membrane either by a signal recognition particle (SRP)-dependent co-translational or by an SRP-independent post-translational translocation route depending on signal sequences. Using a set of model and cellular proteins carrying an N-terminal signal anchor sequence of controlled hydrophobicity and yeast mutant strains defective in SRP or Sec62 function, the hydrophobicity-dependent targeting efficiency and targeting pathway preference were systematically evaluated. Our results suggest that an SRP-dependent co-translational and an SRP-independent post-translational translocation are not mutually exclusive for signal anchor proteins and that moderately hydrophobic ones require both SRP and Sec62 for proper targeting and translocation to the endoplasmic reticulum. Further, defect in Sec62 selectively reduced signal sequences inserted in an N-in-C-out (type II) membrane topology, implying an undiscovered role of Sec62 in regulating the orientation of the signal sequence in an early stage of translocation.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-92652 (URN)10.1074/jbc.M113.473009 (DOI)000320721900012 ()
Note

AuthorCount:3;

Available from: 2013-08-14 Created: 2013-08-14 Last updated: 2017-12-06Bibliographically approved
3. A short C-terminal tail prevents mis-targeting of hydrophobic mitochondrial membrane proteins to the ER
Open this publication in new window or tab >>A short C-terminal tail prevents mis-targeting of hydrophobic mitochondrial membrane proteins to the ER
Show others...
2013 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 587, no 21, 3480-6 p.Article in journal (Refereed) Published
Abstract [en]

Sdh3/Shh3, a subunit of mitochondrial succinate dehydrogenase, contains transmembrane domains with a hydrophobicity comparable to that of endoplasmic reticulum (ER) proteins. Here, we show that a C-terminal reporter fusion to Sdh3/Shh3 results in partial mis-targeting of the protein to the ER. This mis-targeting is mediated by the signal recognition particle (SRP) and depends on the length of the C-terminal tail. These results imply that if nuclear-encoded mitochondrial proteins contain strongly hydrophobic transmembrane domains and a long C-terminal tail, they have the potential to be recognized by SRP and mis-targeted to the ER.

Keyword
Endoplasmic reticulum, mitochondria, signal sequence, co-translational translocation, succinate dehydrogenase
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Biochemistry; Molecular Biology
Identifiers
urn:nbn:se:su:diva-95375 (URN)10.1016/j.febslet.2013.08.041 (DOI)000325978700018 ()24055247 (PubMedID)
Note

AuthorCount: 6;

Funding agencies:

National Research Foundation of Korea 2012-0001935, C00048 

Available from: 2013-11-01 Created: 2013-10-26 Last updated: 2017-12-06Bibliographically approved
4. The Sec62/63 translocon facilitates membrane insertion and C-terminal translocation of multi-spanning membrane proteins
Open this publication in new window or tab >>The Sec62/63 translocon facilitates membrane insertion and C-terminal translocation of multi-spanning membrane proteins
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Majority of membrane proteins are co-translationally translocated. The Sec62/Sec63 complex which mediates post-translational translocation of a subset of primarily secretory proteins into the endoplasmic reticulum (ER),  therefore has been thought uninvolved in targeting and translocation of membrane proteins. By systematic analysis of single and multi-spanning membrane proteins with broad sequence context; varying hydrophobicity, flanking charged residues and orientation of transmembrane (TM) segments, in a set of Sec62 mutant yeast strains, we show that mutations in the N-terminal cytosolic domain of Sec62 impair interaction with Sec63 and lead to defects in membrane insertion and the C-terminal translocation of membrane proteins. These results reveal an unappreciated function of the Sec62/Sec63 translocon as a general membrane chaperone that regulates topogenesis of membrane proteins in the eukaryotic cell.

Keyword
endoplasmic reticulum, co-translational translocation, Sec61, topology, yeast
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-95674 (URN)
Available from: 2013-11-01 Created: 2013-11-01 Last updated: 2013-11-05
5. Structural and Functional Profiling of the Lateral Gate of the Sec61 Translocon
Open this publication in new window or tab >>Structural and Functional Profiling of the Lateral Gate of the Sec61 Translocon
Show others...
2014 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 22, 15845-15855 p.Article in journal (Refereed) Published
Abstract [en]

The evolutionarily conserved Sec61 translocon mediates the translocation and membrane insertion of proteins. For the integration of proteins into the membrane, the Sec61 translocon opens laterally to the lipid bilayer. Previous studies suggest that the lateral opening of the channel is mediated by the helices TM2b and TM7 of a pore-forming subunit of the Sec61 translocon. To map key residues in TM2b and TM7 in yeast Sec61 that modulate lateral gating activity, we performed alanine scanning and in vivo site-directed photocross-linking experiments. Alanine scanning identified two groups of critical residues in the lateral gate, one group that leads to defects in the translocation and membrane insertion of proteins and the other group that causes faster translocation and facilitates membrane insertion. Photocross-linking data show that the former group of residues is located at the interface of the lateral gate. Furthermore, different degrees of defects for the membrane insertion of single- and double-spanning membrane proteins were observed depending on whether the mutations were located in TM2b or TM7. These results demonstrate subtle differences in the molecular mechanism of the signal sequence binding/opening of the lateral gate and membrane insertion of a succeeding transmembrane segment in a polytopic membrane protein.

Keyword
Endoplasmic Reticulum (ER), Membrane, Membrane Protein, Protein Translocation, Yeast
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-106102 (URN)10.1074/jbc.M113.533794 (DOI)000337465400063 ()
Note

AuthorCount:5;

Available from: 2014-07-25 Created: 2014-07-21 Last updated: 2017-12-05Bibliographically approved

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