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A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway.

Harsay E, Schekman R - J. Cell Biol. (2002)

Bottom Line: Exocytic vesicles that accumulate in a temperature-sensitive sec6 mutant at a restrictive temperature can be separated into at least two populations with different buoyant densities and unique cargo molecules.These results suggest that at least one branch of the yeast exocytic pathway transits through endosomes before reaching the cell surface.Consistent with this possibility, we show that immunoisolated clathrin-coated vesicles contain invertase.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.

ABSTRACT
Exocytic vesicles that accumulate in a temperature-sensitive sec6 mutant at a restrictive temperature can be separated into at least two populations with different buoyant densities and unique cargo molecules. Using a sec6 mutant background to isolate vesicles, we have found that vacuolar protein sorting mutants that block an endosome-mediated route to the vacuole, including vps1, pep12, vps4, and a temperature-sensitive clathrin mutant, missort cargo normally transported by dense exocytic vesicles, such as invertase, into light exocytic vesicles, whereas transport of cargo specific to the light exocytic vesicles appears unaffected. Immunoisolation experiments confirm that missorting, rather than a changed property of the normally dense vesicles, is responsible for the altered density gradient fractionation profile. The vps41Delta and apl6Delta mutants, which block transport of only the subset of vacuolar proteins that bypasses endosomes, sort exocytic cargo normally. Furthermore, a vps10Delta sec6 mutant, which lacks the sorting receptor for carboxypeptidase Y (CPY), accumulates both invertase and CPY in dense vesicles. These results suggest that at least one branch of the yeast exocytic pathway transits through endosomes before reaching the cell surface. Consistent with this possibility, we show that immunoisolated clathrin-coated vesicles contain invertase.

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Nycodenz gradient fractionation (performed as in the legend to Figs. 1 and 2) of a chc1-ts sec6-4 strain (EHY242). The gradient profiles of invertase and exoglucanase show a missorting of these exocytic cargo molecules, indicating that clathrin plays a role in invertase transport, as was observed for vps sec6-4 strains (Figs. 1 and 2).
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fig7: Nycodenz gradient fractionation (performed as in the legend to Figs. 1 and 2) of a chc1-ts sec6-4 strain (EHY242). The gradient profiles of invertase and exoglucanase show a missorting of these exocytic cargo molecules, indicating that clathrin plays a role in invertase transport, as was observed for vps sec6-4 strains (Figs. 1 and 2).

Mentions: Clathrin is involved in TGN-to-endosome transport of lysosomal precursors in mammalian cells (Kornfeld and Mellman, 1989), and it plays a similar role in the transport of vacuolar hydrolases in yeast (Seeger and Payne, 1992). A mutant with a temperature-sensitive chc1 (clathrin heavy chain) allele rapidly and severely missorts vacuolar hydrolases after a shift to a restrictive temperature but over time appears to regain the ability to properly sort vacuolar proteins (Seeger and Payne, 1992). A sec6-4 mutant with this temperature-sensitive chc1 allele missorted invertase into light-density vesicles (Fig. 7) , consistent with other vps mutants that block transport to endosomes. Gradients obtained after both the 30- and 60-min shifts also displayed a small peak of invertase in the same fractions where invertase peaked in vps27Δ sec6-4 (Fig. 1) and vps27-ts sec6-4 (Fig. 2) gradients, indicating that invertase may accumulate in both light and intermediate-density vesicles in this mutant.


A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway.

Harsay E, Schekman R - J. Cell Biol. (2002)

Nycodenz gradient fractionation (performed as in the legend to Figs. 1 and 2) of a chc1-ts sec6-4 strain (EHY242). The gradient profiles of invertase and exoglucanase show a missorting of these exocytic cargo molecules, indicating that clathrin plays a role in invertase transport, as was observed for vps sec6-4 strains (Figs. 1 and 2).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199237&req=5

fig7: Nycodenz gradient fractionation (performed as in the legend to Figs. 1 and 2) of a chc1-ts sec6-4 strain (EHY242). The gradient profiles of invertase and exoglucanase show a missorting of these exocytic cargo molecules, indicating that clathrin plays a role in invertase transport, as was observed for vps sec6-4 strains (Figs. 1 and 2).
Mentions: Clathrin is involved in TGN-to-endosome transport of lysosomal precursors in mammalian cells (Kornfeld and Mellman, 1989), and it plays a similar role in the transport of vacuolar hydrolases in yeast (Seeger and Payne, 1992). A mutant with a temperature-sensitive chc1 (clathrin heavy chain) allele rapidly and severely missorts vacuolar hydrolases after a shift to a restrictive temperature but over time appears to regain the ability to properly sort vacuolar proteins (Seeger and Payne, 1992). A sec6-4 mutant with this temperature-sensitive chc1 allele missorted invertase into light-density vesicles (Fig. 7) , consistent with other vps mutants that block transport to endosomes. Gradients obtained after both the 30- and 60-min shifts also displayed a small peak of invertase in the same fractions where invertase peaked in vps27Δ sec6-4 (Fig. 1) and vps27-ts sec6-4 (Fig. 2) gradients, indicating that invertase may accumulate in both light and intermediate-density vesicles in this mutant.

Bottom Line: Exocytic vesicles that accumulate in a temperature-sensitive sec6 mutant at a restrictive temperature can be separated into at least two populations with different buoyant densities and unique cargo molecules.These results suggest that at least one branch of the yeast exocytic pathway transits through endosomes before reaching the cell surface.Consistent with this possibility, we show that immunoisolated clathrin-coated vesicles contain invertase.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.

ABSTRACT
Exocytic vesicles that accumulate in a temperature-sensitive sec6 mutant at a restrictive temperature can be separated into at least two populations with different buoyant densities and unique cargo molecules. Using a sec6 mutant background to isolate vesicles, we have found that vacuolar protein sorting mutants that block an endosome-mediated route to the vacuole, including vps1, pep12, vps4, and a temperature-sensitive clathrin mutant, missort cargo normally transported by dense exocytic vesicles, such as invertase, into light exocytic vesicles, whereas transport of cargo specific to the light exocytic vesicles appears unaffected. Immunoisolation experiments confirm that missorting, rather than a changed property of the normally dense vesicles, is responsible for the altered density gradient fractionation profile. The vps41Delta and apl6Delta mutants, which block transport of only the subset of vacuolar proteins that bypasses endosomes, sort exocytic cargo normally. Furthermore, a vps10Delta sec6 mutant, which lacks the sorting receptor for carboxypeptidase Y (CPY), accumulates both invertase and CPY in dense vesicles. These results suggest that at least one branch of the yeast exocytic pathway transits through endosomes before reaching the cell surface. Consistent with this possibility, we show that immunoisolated clathrin-coated vesicles contain invertase.

Show MeSH
Related in: MedlinePlus