Abstract
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.
Highlights
Proteins destined for the secretory pathway are translated and translocated into the endoplasmic reticulum (ER), which provides a specialized environment for their folding, disulfide bond formation, and N-linked glycosylation
Cells were lysed in Triton X-100 and the detergent lysates were immunoprecipitated with the polyclonal antibody 40336, which recognizes all forms of gp160
We found that the dramatically delayed cleavage of the signal peptide of HIV-1 gp160 functions as folding regulator to ensure correct formation of disulfide bonds and functional structure
Summary
Proteins destined for the secretory pathway are translated and translocated into the endoplasmic reticulum (ER), which provides a specialized environment for their folding, disulfide bond formation, and N-linked glycosylation. Targeting of soluble and type-I transmembrane proteins to the ER is mediated via cleavable signal peptides, near-N-terminal hydrophobic stretches of 14–50 amino acids that are recognized by SRP (von Heijne, 1985; Kurzchalia et al, 1986; Lutcke et al, 1992; Walter and Blobel, 1981; Blobel and Dobberstein, 1975; Hegde and Bernstein, 2006). Cleavable signal peptides are variable in sequence but share characteristics of an N-terminal region with typically 0–2 basic residues, a membrane-spanning hydrophobic a-helix (H) region, and a C-terminal region that often contains a signal-peptide cleavage site (von Heijne, 1983; von Heijne, 1984). Signal-peptide cleavage is mediated by the signal-peptidase complex, which, like oligosaccharyl transferase, associates with the translocon (Gorlich et al, 1992; Gilmore, 1993).
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