Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires ...core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.
Integral membrane proteins are assembled into the ER membrane via a continuous ribosome-translocon channel. The hydrophobicity and thickness of the core of the membrane bilayer leads to the ...expectation that transmembrane (TM) segments minimize the cost of harbouring polar polypeptide backbones by adopting a regular pattern of hydrogen bonds to form α-helices before integration. Co-translational folding of nascent chains into an α-helical conformation in the ribosomal tunnel has been demonstrated previously, but the features governing this folding are not well understood. In particular, little is known about what features influence the propensity to acquire α-helical structure in the ribosome. Using in vitro translation of truncated nascent chains trapped within the ribosome tunnel and molecular dynamics simulations, we show that folding in the ribosome is attained for TM helices but not for soluble helices, presumably facilitating SRP (signal recognition particle) recognition and/or a favourable conformation for membrane integration upon translocon entry.
Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is ...the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein-lipid interactions are critical during translocon-mediated membrane insertion.
The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes to the SRP receptor in the endoplasmic reticulum ...membrane, initiating translocation of the nascent chain through the Sec61 translocon. Although signal sequences do not have homology, they have similar structural regions: a positively charged N-terminus, a hydrophobic core and a more polar C-terminal region that contains the cleavage site for the signal peptidase. Here, we have used site-specific photocrosslinking to study SRP–signal sequence interactions. A photoreactive probe was incorporated into the middle of wild-type or mutated signal sequences of the secretory protein preprolactin by in vitro translation of mRNAs containing an amber-stop codon in the signal peptide in the presence of the Nε-(5-azido-2 nitrobenzoyl)-Lys-tRNAamb amber suppressor. A homogeneous population of SRP–ribosome-nascent chain complexes was obtained by the use of truncated mRNAs in translations performed in the presence of purified canine SRP. Quantitative analysis of the photoadducts revealed that charged residues at the N-terminus of the signal sequence or in the early part of the mature protein have only a mild effect on the SRP–signal sequence association. However, deletions of amino acid residues in the hydrophobic portion of the signal sequence severely affect SRP binding. The photocrosslinking data correlate with targeting efficiency and translocation across the membrane. Thus, the hydrophobic core of the signal sequence is primarily responsible for its recognition and binding by SRP, while positive charges fine-tune the SRP–signal sequence affinity and targeting to the translocon.
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•SRP cotranslationally recognizes signal sequences of secretory proteins.•Signal sequences have three regions: a positively charged N-terminus, a hydrophobic core and a polar C-terminal cleavage-site region.•Alterations at the N-terminus of the signal sequence or near the cleavage site have little effect on the association with mammalian SRP.•The hydrophobic core is primarily responsible for recognition by SRP.
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.
α-helical integral membrane proteins critically depend on the correct insertion of their transmembrane α helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the ...transmembrane α helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (Ncyt-Clum or Nlum-Ccyt) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the “missing hydrophobicity” in polytopic membrane proteins.
► ER-insertion propensity of a TMH depends on the orientational bias of nearby TMHs ► This helps explain the “missing hydrophobicity” in polytopic membrane proteins ► Membrane insertion of TMH8 in the human ASBT protein depends on TMH9
Transmembrane α-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 ≈1 kcal/mol lower for N in –C out compared with N out –C in transmembrane helices, depending on the neighboring helices. membrane protein positive-inside rule Saccharomyces cerevisiae topology translocon
In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many ...transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.
Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating ...polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel van den Berg B, et al. (2004) Nature 427:36–44; Zimmer J, et al. (2008) Nature 455:936–943; Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107:17182–17187. To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid’s contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal/(mol· Å 2 ), the solvation parameter for transfer from translocon to bilayer is 6–10 cal/(mol· Å 2 ), pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a thermodynamic partitioning model and insights into the physical properties of the translocon.
The insertion efficiency of transmembrane (TM) helices by the Sec61 translocon depends on helix amino acid composition, the positions of the amino acids within the helix, and helix length. We have ...used an in vitro expression system to examine systematically the insertion efficiency of short polyleucine segments (Ln, n = 4 ... 12) flanked at either end by 4-residue sequences of the form XXPX-Ln-XPXX with X = G, N, D, or K. Except for X = K, insertion efficiency (p) is <10% for n < 8, but rises steeply to 100% for n = 12. For X = K, p is already close to 100% for n = 10. A similar pattern is observed for synthetic peptides incorporated into oriented phospholipid bilayer arrays, consistent with the idea that recognition of TM segments by the translocon critically involves physical partitioning of nascent peptide chains into the lipid bilayer. Molecular dynamics simulations suggest that insertion efficiency is determined primarily by the energetic cost of distorting the bilayer in the vicinity of the TM helix. Very short lysine-flanked leucine segments can reduce the energetic cost by extensive hydrogen bonding with water and lipid phosphate groups (snorkeling) and by partial unfolding.
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