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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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Thin section EM of vesicles immunoisolated with anti-Pma1p monoclonal antibody #17 bound to Dynabeads protein G. A vps4Δ sec6-4 strain (EHY327) was fractionated as in the legend to Fig. 5, and membranes from the light invertase peak fraction were immunoisolated using undersaturating beads. Bar, 200 nm.
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fig6: Thin section EM of vesicles immunoisolated with anti-Pma1p monoclonal antibody #17 bound to Dynabeads protein G. A vps4Δ sec6-4 strain (EHY327) was fractionated as in the legend to Fig. 5, and membranes from the light invertase peak fraction were immunoisolated using undersaturating beads. Bar, 200 nm.

Mentions: Thin section electron microscopic examination of immunoisolated membranes from vps4Δ sec6-4 cells indicated 100-nm vesicles and some tubular membranes bound to the beads (Fig. 6) .


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)

Thin section EM of vesicles immunoisolated with anti-Pma1p monoclonal antibody #17 bound to Dynabeads protein G. A vps4Δ sec6-4 strain (EHY327) was fractionated as in the legend to Fig. 5, and membranes from the light invertase peak fraction were immunoisolated using undersaturating beads. Bar, 200 nm.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Thin section EM of vesicles immunoisolated with anti-Pma1p monoclonal antibody #17 bound to Dynabeads protein G. A vps4Δ sec6-4 strain (EHY327) was fractionated as in the legend to Fig. 5, and membranes from the light invertase peak fraction were immunoisolated using undersaturating beads. Bar, 200 nm.
Mentions: Thin section electron microscopic examination of immunoisolated membranes from vps4Δ sec6-4 cells indicated 100-nm vesicles and some tubular membranes bound to the beads (Fig. 6) .

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