| 1. |
- Hessa, Tara, et al.
(author)
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Analysis of transmembrane helix integration in the endoplasmic reticulum in S. cerevisiae
- 2009
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In: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 386:5, s. 1222-8
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Journal article (peer-reviewed)abstract
- 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.
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| 2. |
- Jung, Sung-jun, et al.
(author)
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The Sec62/63 translocon facilitates membrane insertion and C-terminal translocation of multi-spanning membrane proteins
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Other publication (other academic/artistic)abstract
- 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.
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| 3. |
- Jung, Sung-Jun, et al.
(author)
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The Sec62-Sec63 translocon facilitates translocation of the C-terminus of membrane proteins
- 2014
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In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 127:19, s. 4270-4278
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Journal article (peer-reviewed)abstract
- The Sec62-Sec63 complex mediates post-translational translocation of a subset of primarily secretory proteins into the endoplasmic reticulum (ER) in yeast. Therefore, it has been thought that membrane proteins, which are mainly co-translationally targeted into the ER, are not handled by the Sec62-Sec63 translocon. By systematic analysis of single and multi-spanning membrane proteins with broad sequence context [with differing hydrophobicity, flanking charged residues and orientation of transmembrane (TM) segments], we show that mutations in the N-terminal cytosolic domain of yeast Sec62 impair its interaction with Sec63 and lead to defects in membrane insertion and translocation of the C-terminus of membrane proteins. These results suggest that there is an unappreciated function of the Sec62-Sec63 translocon in regulating topogenesis of membrane proteins in the eukaryotic cell.
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| 4. |
- Reithinger, Johannes H, et al.
(author)
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A short C-terminal tail prevents mis-targeting of hydrophobic mitochondrial membrane proteins to the ER
- 2013
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In: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 587:21, s. 3480-6
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Journal article (peer-reviewed)abstract
- 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.
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| 5. |
- Reithinger, Johannes H., et al.
(author)
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Sec62 Protein Mediates Membrane Insertion and Orientation of Moderately Hydrophobic Signal Anchor Proteins in the Endoplasmic Reticulum (ER)
- 2013
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:25, s. 18058-18067
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Journal article (peer-reviewed)abstract
- 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.
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| 6. |
- Reithinger, Johannes H., et al.
(author)
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Structural and Functional Profiling of the Lateral Gate of the Sec61 Translocon
- 2014
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 289:22, s. 15845-15855
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Journal article (peer-reviewed)abstract
- 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.
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