The protein translocation channel mediates glycopeptide export across the endoplasmic reticulum membrane - PubMed (original) (raw)

The protein translocation channel mediates glycopeptide export across the endoplasmic reticulum membrane

P Gillece et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Peptides and misfolded secretory proteins are transported efficiently from the endoplasmic reticulum (ER) lumen to the cytosol, where the proteins are degraded by proteasomes. Protein export depends on Sec61p, the ribosome-binding core component of the protein translocation channel in the ER membrane. We found that prebinding of ribosomes abolished export of a glycopeptide from yeast microsomes. Deletion of SSH1, which encodes a ribosome-binding Sec61p homologue in the ER, had no effect on glycopeptide export. A collection of cold-sensitive sec61 mutants displayed a variety of phenotypes: two mutants strongly defective in misfolded protein export from the ER, sec61-32 and sec61-41, displayed only minor peptide export defects. Glycopeptide export was severely impaired, however, in several sec61 mutants that were only marginally defective in misfolded protein export. In addition, a mutation in SEC63 strongly reduced peptide export from the ER. ER-luminal ATP was required for both misfolded protein and glycopeptide export. We conclude that the protein translocation channel in the ER membrane mediates glycopeptide transport across the ER membrane.

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Figures

Figure 1

Figure 1

Ribosomes inhibit glycopeptide export from the ER. Yeast cytosol (18 mg/ml) was separated by centrifugation into ribosomes and a ribosome-free supernatant fraction and the ribosomal pellet resuspended in the original volume B88. Wild-type microsomes [RSY255, 2 μl microsomes (_A_280 = 30) per sample] were translocated with 125I-Ac-NYT-NH2, washed, and incubated for 10 min at 24°C in 12.5 μl B88, 12.5 μl B88 containing nontranslating ribosomes, or 12.5 μl ribosome-free cytosol, as indicated. Glycopeptide export was assayed subsequently for 30 min at 24°C in the absence (−cytosol) or presence (all others) of cytosol and ATP, as described in Methods. Samples contained 2–5 × 104 Con A precipitable cpm, were done in duplicate, and the experiment was repeated three times.

Figure 2

Figure 2

Ssh1p is not required for glycopeptide export. Wild-type (SSH1) and Δ_ssh1_ microsomes were assayed for glycopeptide export as described in Methods. At individual time points, duplicate samples were transferred to ice, the membranes sedimented by centrifugation, and glycopeptide in the supernatatant fraction analyzed by Con A precipitation and γ-counting.

Figure 3

Figure 3

Specific sec61 mutants are defective in glycopeptide export from the ER. Microsomes were prepared from SEC61 wild-type and mutant strains grown at their respective permissive temperatures (see Methods). (Upper) Glycopeptide export. Each strain was assayed in duplicate for glycopeptide export for 30 min at 24°C in the presence or individual absence of ATP and cytosol, as described in Methods. Release at 30 min is shown. Nonspecific release in the absence of ATP was less than 5% and subtracted. Mutants with significantly different effects on glycopeptide and misfolded protein export are marked with asterisks. (Lower) Misfolded protein export. Mutant α-factor precursor (pΔgpαf) was translocated into wild type or sec61 mutant microsomes and Δgpαf export and degradation initiated by the addition of ATP and cytosol, as described in Methods. After 30 min at 24°C, proteins were precipitated with TCA, resolved by gel electrophoresis, and Δgpαf quantified by using a Bio-Rad phosphorimager. Samples were done in duplicate and the experiment repeated twice.

Figure 4

Figure 4

Glycopeptide export from the ER depends on Sec63p. Microsomes were prepared from wild-type and sec62 or sec63 mutant cells grown at the permissive temperature (see Methods) and glycopeptide export assayed as described in Fig. 1.

Figure 5

Figure 5

Glycopeptide export across the ER membrane requires ATP in the ER lumen. (Upper) DIDS inhibits glycopeptide export from the ER. Wild-type (RSY255) microsomes were translocated with 125I-Ac-NYT-NH2, washed with B88 containing the indicated concentration of DIDS, and glycopeptide export in the presence of the indicated concentrations of DIDS assayed in duplicate as described in Fig. 1. (Lower) Δ_sac1_ microsomes are defective in glycopeptide export from the ER. Microsomes were prepared from wild-type (SAC1) and Δ_sac1_ cells and glycopeptide export assayed as described in Fig. 1.

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