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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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Gradient fractionation of vps-ts sec6-4 mutants. Strains were grown at 24°C in SD with required amino acids for maintaining plasmids (vps4-ts sec6, EHY348; pep12-ts sec6, EHY413) or in YPD (vps27-ts sec6, EHY374; vps4Δ sec6, EHY327). Strains grown in SD were shifted to YPD at 24°C for 2 h before 30- or 60-min shifts to 37°C in prewarmed YPD, pH 4.5. (Bottom right) A single culture of vps4Δ sec6 cells was split in half; one half was maintained at 24°C and the other half shifted to 32°C (semi-permissive for sec6-4) for 1h. Cells were fractionated as for Fig. 1, and gradient fractions were assayed for invertase and exoglucanase activities.
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fig2: Gradient fractionation of vps-ts sec6-4 mutants. Strains were grown at 24°C in SD with required amino acids for maintaining plasmids (vps4-ts sec6, EHY348; pep12-ts sec6, EHY413) or in YPD (vps27-ts sec6, EHY374; vps4Δ sec6, EHY327). Strains grown in SD were shifted to YPD at 24°C for 2 h before 30- or 60-min shifts to 37°C in prewarmed YPD, pH 4.5. (Bottom right) A single culture of vps4Δ sec6 cells was split in half; one half was maintained at 24°C and the other half shifted to 32°C (semi-permissive for sec6-4) for 1h. Cells were fractionated as for Fig. 1, and gradient fractions were assayed for invertase and exoglucanase activities.

Mentions: A vps mutant that lacks the sorting receptor for CPY, vps10Δ (Marcusson et al., 1994), was capable of sorting invertase properly (Fig. 1 E). However, unlike for all other mutants fractionated invertase sorting in the vps10Δ sec6-4 mutant varied between experiments. Lowering the pH of the growth medium to pH 4.5 (from pH 6.5 in standard rich medium reduced missorting, suggesting that a lower pH may be more optimal for proper invertase sorting. However, for all mutants except vps10Δ sec6, lowering the pH of the medium made no difference other than resulting in slightly lower levels of invertase activity. All results shown in Figs. 1 and 2 are from experiments in which cells were shifted into pH 4.5 medium.


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)

Gradient fractionation of vps-ts sec6-4 mutants. Strains were grown at 24°C in SD with required amino acids for maintaining plasmids (vps4-ts sec6, EHY348; pep12-ts sec6, EHY413) or in YPD (vps27-ts sec6, EHY374; vps4Δ sec6, EHY327). Strains grown in SD were shifted to YPD at 24°C for 2 h before 30- or 60-min shifts to 37°C in prewarmed YPD, pH 4.5. (Bottom right) A single culture of vps4Δ sec6 cells was split in half; one half was maintained at 24°C and the other half shifted to 32°C (semi-permissive for sec6-4) for 1h. Cells were fractionated as for Fig. 1, and gradient fractions were assayed for invertase and exoglucanase activities.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199237&req=5

fig2: Gradient fractionation of vps-ts sec6-4 mutants. Strains were grown at 24°C in SD with required amino acids for maintaining plasmids (vps4-ts sec6, EHY348; pep12-ts sec6, EHY413) or in YPD (vps27-ts sec6, EHY374; vps4Δ sec6, EHY327). Strains grown in SD were shifted to YPD at 24°C for 2 h before 30- or 60-min shifts to 37°C in prewarmed YPD, pH 4.5. (Bottom right) A single culture of vps4Δ sec6 cells was split in half; one half was maintained at 24°C and the other half shifted to 32°C (semi-permissive for sec6-4) for 1h. Cells were fractionated as for Fig. 1, and gradient fractions were assayed for invertase and exoglucanase activities.
Mentions: A vps mutant that lacks the sorting receptor for CPY, vps10Δ (Marcusson et al., 1994), was capable of sorting invertase properly (Fig. 1 E). However, unlike for all other mutants fractionated invertase sorting in the vps10Δ sec6-4 mutant varied between experiments. Lowering the pH of the growth medium to pH 4.5 (from pH 6.5 in standard rich medium reduced missorting, suggesting that a lower pH may be more optimal for proper invertase sorting. However, for all mutants except vps10Δ sec6, lowering the pH of the medium made no difference other than resulting in slightly lower levels of invertase activity. All results shown in Figs. 1 and 2 are from experiments in which cells were shifted into pH 4.5 medium.

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