Distinct processes mediate glycoprotein and glycopeptide export from the endoplasmic reticulum in Saccharomyces cerevisiae.

Römisch, Karin; Schekman, Randy

doi:10.1073/pnas.89.15.7227

Cited by 50 publications

(53 citation statements)

References 23 publications

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“…Our findings also suggest that the first experiment to demonstrate secretory bulk flow, based on a short peptide as a bulk flow marker (Wieland et al, 1987), was far more representative of the in vivo situation than is perceived at present (Romisch and Schekman, 1992;Kuehn and Schekman, 1997). Given this notion, it is not surprising that ERD2 mutants with defective ER retention affinity cause drastic transcriptional induction of the KAR2 gene to compensate for the loss of BiP through increased ER export (Semenza et al, 1990).…”

Section: Bulk Flow Is Efficientmentioning

confidence: 56%

Secretory Bulk Flow of Soluble Proteins Is Efficient and COPII Dependent

Phillipson¹,

Pimpl²,

daSilva³

et al. 2001

The Plant Cell

159

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COPII-coated vesicles, first identified in yeast and later characterized in mammalian cells, mediate protein export from the endoplasmic reticulum (ER) to the Golgi apparatus within the secretory pathway. In these organisms, the mechanism of vesicle formation is well understood, but the process of soluble cargo sorting has yet to be resolved. In plants, functional complements of the COPII-dependent protein traffic machinery were identified almost a decade ago, but the selectivity of the ER export process has been subject to considerable debate. To study the selectivity of COPII-dependent protein traffic in plants, we have developed an in vivo assay in which COPII vesicle transport is disrupted at two distinct steps in the pathway. First, overexpression of the Sar1p-specific guanosine nucleotide exchange factor Sec12p was shown to result in the titration of the GTPase Sar1p, which is essential for COPII-coated vesicle formation. A second method to disrupt COPII transport at a later step in the pathway was based on coexpression of a dominant negative mutant of Sar1p (H74L), which is thought to interfere with the uncoating and subsequent membrane fusion of the vesicles because of the lack of GTPase activity. A quantitative assay to measure ER export under these conditions was achieved using the natural secretory protein barley ␣ -amylase and a modified version carrying an ER retention motif. Most importantly, the manipulation of COPII transport in vivo using either of the two approaches allowed us to demonstrate that export of the ER resident protein calreticulin or the bulk flow marker phosphinothricin acetyl transferase is COPII dependent and occurs at a much higher rate than estimated previously. We also show that the instability of these proteins in post-ER compartments prevents the detection of the true rate of bulk flow using a standard secretion assay. The differences between the data on COPII transport obtained from these in vivo experiments and in vitro experiments conducted previously using yeast components are discussed. INTRODUCTIONIn yeast and mammalian cells, the export of proteins from the endoplasmic reticulum (ER) occurs in COPII-coated vesicles. The process of COPII vesicle formation is well understood and can be reconstituted using purified yeast components (Barlowe et al., 1994). Vesicle formation in vitro depends solely on the ER donor membrane, the GTPase Sar1p, the Sar1p-specific guanosine nucleotide exchange factor Sec12p, the cytosolic COPII coat components, and GTP (Barlowe et al., 1994). Considerably less is known about the sorting of soluble cargo molecules during ER export in eukaryotes (Klumperman, 2000), and conflicting reports from the plant field have contributed to the ongoing discussions (Crofts et al., 1999;Gomord and Faye, 2000;Pagny et al., 2000;.To support protein synthesis and folding, the ER lumen maintains high levels of soluble residents such as the lumenal binding protein (BiP), protein disulfide isomerase, or calreticulin. The concentration of nonresidents in the ER lume...

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Section: Bulk Flow Is Efficientmentioning

confidence: 56%

Secretory Bulk Flow of Soluble Proteins Is Efficient and COPII Dependent

Phillipson¹,

Pimpl²,

daSilva³

et al. 2001

The Plant Cell

159

View full text Add to dashboard Cite

show abstract

“…Cells were grown at 24°C (RSY155, RSY529, RSY524, RSY1132) or 30°C (all others) and microsomes prepared as described (14). Cytosol for glycopeptide export was prepared by bead beating from RSY255 (11). Cytosol for misfolded protein export and degradation was prepared from WCG4a by liquid nitrogen lysis.…”

Section: Methodsmentioning

confidence: 99%

“…Hydrophobic acceptor peptides enter microsomes by an undefined route, possibly by partitioning into the lipid bilayer, and release on the ER-luminal side where they are core glycosylated. In contrast to secretory proteins, however, glycopeptides are not packaged into ER-toGolgi transport vesicles; instead, they are transported directly across the ER membrane to the cytosol in an ATP-and cytosol-dependent fashion (11). Most TAP substrates and free polymannose oligosaccharides are also efficiently transported from the ER lumen to the cytosol in an ATP-dependent fashion (5,6,8).…”

mentioning

confidence: 99%

“…Most TAP substrates and free polymannose oligosaccharides are also efficiently transported from the ER lumen to the cytosol in an ATP-dependent fashion (5,6,8). Initially, retrograde transport across the ER membrane was assumed to be restricted to small molecules and was viewed as a disposal pathway for end products of ''ER degradation'' of misfolded secretory proteins (11). Recently it became clear, however, that cytosolic proteasomes are responsible for this degradation process, and that misfolded proteins themselves are exported across the ER membrane to the cytosol before degradation (for review, see ref.…”

mentioning

confidence: 99%

“…Peptide transport from the ER to the cytosol was first discovered by using a synthetic acceptor peptide for oligosaccharyl transferase in a yeast-based cell free assay system designed to study ER-to-Golgi transport (11). Hydrophobic acceptor peptides enter microsomes by an undefined route, possibly by partitioning into the lipid bilayer, and release on the ER-luminal side where they are core glycosylated.…”

mentioning

confidence: 99%

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

Gillece

Pilon

Römisch

2000

Proc. Natl. Acad. Sci. U.S.A.

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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 ribosomebinding 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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Endoplasmic reticulum-to-cytosol transport of free polymannose oligosaccharides in permeabilized HepG2 cells.

1995

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Free polymannose oligosaccharides have recently been localized to both the vesicular and cytosolic compartments of HepG2 cells. Here we investigated the possibility that free oligosaccharides originating in the lumen of the endoplasmic reticulum (ER) are transported directly into the cystosol. Incubation of permeabilized cells in the absence of ATP at 37 degrees C led to the intravesicular accumulation of free Man9GlcNAc2 which was generated from dolichol‐linked oligosaccharide in the ER. This oligosaccharide remained stable within the permeabilized cells unless ATP was added to the incubations at which time the Man9GlcNac2 was partially converted to Man8GlcNAc2, and both these components were released from an intravesicular compartment into the cytosolic compartment of permeabilized cells. In contrast, when permeabilized cells, primed with either free triglucosyl‐oligosaccharide or a glycotripeptide, were incubated with ATP both these structures remained associated with the intravesicular compartment. As the conditions in which free oligosaccharides were transported out of the intravesicular compartment into the cytosolic compartment did not permit vesicular transport of glycoproteins from the ER to the Golgi apparatus our data demonstrate the presence of a transport process for the delivery of free polymannose oligosaccharides from the ER to the cytosol.

show abstract

Distinct processes mediate glycoprotein and glycopeptide export from the endoplasmic reticulum in Saccharomyces cerevisiae.

Cited by 50 publications

References 23 publications

Secretory Bulk Flow of Soluble Proteins Is Efficient and COPII Dependent

Secretory Bulk Flow of Soluble Proteins Is Efficient and COPII Dependent

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

Endoplasmic reticulum-to-cytosol transport of free polymannose oligosaccharides in permeabilized HepG2 cells.

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