| 1. |
- Karamyshev, Andrey L., et al.
(författare)
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Mapping the interaction of the STT3 subunit of the oligosaccharyl transferase complex with nascent polypeptide chains
- 2005
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:49, s. 40489-40493
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Kim, Hyun, et al.
(författare)
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Membrane topology of the STT3 subunit of the oligosaccharyl transferase complex
- 2005
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:21, s. 20261-20267
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Lara, Patricia, et al.
(författare)
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Murine astrotactins 1 and 2 have a similar membrane topology and mature via endoproteolytic cleavage catalyzed by a signal peptidase
- 2019
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 294:12, s. 4538-4545
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Lara, Patricia, et al.
(författare)
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Refined topology model of the STT3/Stt3 protein subunit of the oligosaccharyltransferase complex
- 2017
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 292:27, s. 11349-11360
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Nilsson, IngMarie, et al.
(författare)
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Distant downstream sequence determinants can control N-tail translocation during protein insertion into the endoplasmic reticulum membrane
- 2000
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 275:9, s. 6207-6213
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Nilsson, IngMarie, et al.
(författare)
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Glycosylation efficiency of Asn-Xaa-Thr sequons depends both on the distance from the C terminus and on the presence of a downstream transmembrane segment
- 2000
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 275:23, s. 17338-17343
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Nilsson, IngMarie, et al.
(författare)
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How hydrophobic is alanine?
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:32, s. 29389-29393
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Nilsson, IngMarie, et al.
(författare)
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Inhibition of protein translocation across the endoplasmic reticulum membrane by sterols
- 2001
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:45, s. 41748-41754
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Saenz, Alejandra, et al.
(författare)
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Folding and Intramembraneous BRICHOS Binding of the Prosurfactant Protein C Transmembrane Segment
- 2015
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:28, s. 17628-17641
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
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