| 31. |
- Lundin, Carolina, et al.
(författare)
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Molecular code for protein insertion in the endoplasmic reticulum membrane is similar for N-in-C-out and N-out-C-in transmembrane helices
- 2008
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 105:41, s. 15702-15707
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 32. |
- Lundin, Carolina, et al.
(författare)
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Stable insertion of Alzheimer Aβ peptide into the ER membrane strongly correlates with its length
- 2007
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 581:20, s. 3809-3813
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 33. |
- Marani, Paula, et al.
(författare)
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New Escherichia coli outer membrane proteins identified through prediction and experimental verification
- 2006
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 15:4, s. 884-889
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 34. |
- Meindl-Beinker, Nadia M., et al.
(författare)
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Asn- and Asp-mediated interactions between transmembrane helices during translocon-mediated membrane protein assembly
- 2006
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Ingår i: EMBO reports. - : EMBO. - 1469-221X .- 1469-3178. ; 7:11, s. 1111-1116
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 35. |
- Mingarro, Ismael, et al.
(författare)
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Different conformations of nascent polypeptides during translocation across the ER membrane
- 2000
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Ingår i: BMC Cell Biology. - : Springer Science and Business Media LLC. - 1471-2121. ; 1:3
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundIn 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.ResultsWe 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).ConclusionsThe 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.
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| 36. |
- Nilsson, IngMarie, et al.
(författare)
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Cleavage of a tail-anchored protein by signal peptidase
- 2002
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Ingår i: FEBS Letters. - 0014-5793 .- 1873-3468. ; 516:1-3, s. 106-108
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 37. |
- Nilsson, IngMarie
(författare)
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Conformational properties of transmembrane polypeptide segments in the ER membrane
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- We have developed a new experimental approach where the active site of oligosaccharyl transferase is used as a point of reference against which the position of a transmembrane segment in the membrane can be measured. This so-called glycosylation mapping technique allows any transmembrane segment to be positioned relative to a known reference transmembrane helix with known position in the membrane. We have studied the position of transmembrane polypeptides in the ER membrane and determined the effects of single proline and charged residues on the position of a transmembrane helix in the endoplasmic reticulum membrane using the glycosylation mapping technique. We have found that proline residues can break a transmembrane helix when inserted near the end of the helix, but not when placed more centrally and only when the helix is sufficiently long. Compared to the helix-disrupting effects seen with proline residues, the charged amino acid residues cause less drastic changes. We suggest that charged residues do not break the helical conformation but just affect the position of the helix in the membrane. The difference between the effects of positively and negatively charged residues can be explained by the so-called snorkel effect, i.e. that the very long side-chains of Arg and Lys can reach up along the transmembrane helix to allow the terminal charged moiety to reside in the lipid head group region. Our results from a turn propensity investigation suggest that in a very long transmembrane helix, twice as long as a normal helix, a single proline residue introduced near the center of the helix can induce the formation of two transmembrane segments separated by a tight turn. The glycosylation mapping technique may be generally useful for determining the position of transmembrane helices in the membrane and the ends of different transmembrane segments.
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| 38. |
- 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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| 39. |
- 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
-
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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| 40. |
- 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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