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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 sec6 and vpsΔ sec6 mutants. sec6 (EHY227), vps1Δ sec6 (EHY225), pep12Δ sec6 (EHY232), vps10Δ sec6 (EHY282), vps27Δ sec6 (EHY309), and vps4Δ sec6 (EHY327) cells were grown at 24°C in YPD for 12–14 h and then shifted to 37°C in prewarmed YPD, pH 4.5, for 1 h or for the times indicated. Cells were fractionated as described in Materials and methods. Membrane pellets (100,000 g) were loaded into the bottoms of 15–30% Nycodenz/0.8 M sorbitol linear gradients and floated to equilibrium. Fractions were collected from the top and assayed for enzyme activities. Where two graphs are shown for a strain (vps1Δ sec6 and vps27Δ sec6), activities were obtained from a single gradient. The density profiles were similar for all gradients.
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fig1: Gradient fractionation of sec6 and vpsΔ sec6 mutants. sec6 (EHY227), vps1Δ sec6 (EHY225), pep12Δ sec6 (EHY232), vps10Δ sec6 (EHY282), vps27Δ sec6 (EHY309), and vps4Δ sec6 (EHY327) cells were grown at 24°C in YPD for 12–14 h and then shifted to 37°C in prewarmed YPD, pH 4.5, for 1 h or for the times indicated. Cells were fractionated as described in Materials and methods. Membrane pellets (100,000 g) were loaded into the bottoms of 15–30% Nycodenz/0.8 M sorbitol linear gradients and floated to equilibrium. Fractions were collected from the top and assayed for enzyme activities. Where two graphs are shown for a strain (vps1Δ sec6 and vps27Δ sec6), activities were obtained from a single gradient. The density profiles were similar for all gradients.

Mentions: The sec6-4 mutant was among the first group of conditional yeast secretory mutants isolated in a screen for mutants that are blocked for growth and secretion and accumulate secretory organelles at a restrictive temperature (Novick et al., 1980). Sec6p is part of a protein complex (the “Exocyst”) involved in the polarized fusion of exocytic vesicles with the plasma membrane (TerBush et al., 1996). The mutant grows as well as wild-type cells at 24°C, but growth ceases at 37°C and the cells accumulate abundant 100-nm vesicles. These vesicles can be separated into two populations by isodensity gradient centrifugation (Fig. 1 A; Harsay and Bretscher, 1995). The more abundant, lighter density, vesicles contain the plasma membrane and cell wall proteins Pma1p and Bgl2p, respectively, whereas the denser vesicles contain the periplasmic enzymes invertase and acid phosphatase. Both vesicle populations also contain an exoglucanase activity, which most likely comprises two different enzymes (Harsay and Bretscher, 1995). Wild-type cells contain very few secretory vesicles at steady state, so it is difficult to distinguish vesicles from other organelles in cell fractionation experiments. Therefore, to analyze the effects of various mutations on the two exocytic pathways, we performed all fractionations with cells having a sec6-4 mutant background.


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 sec6 and vpsΔ sec6 mutants. sec6 (EHY227), vps1Δ sec6 (EHY225), pep12Δ sec6 (EHY232), vps10Δ sec6 (EHY282), vps27Δ sec6 (EHY309), and vps4Δ sec6 (EHY327) cells were grown at 24°C in YPD for 12–14 h and then shifted to 37°C in prewarmed YPD, pH 4.5, for 1 h or for the times indicated. Cells were fractionated as described in Materials and methods. Membrane pellets (100,000 g) were loaded into the bottoms of 15–30% Nycodenz/0.8 M sorbitol linear gradients and floated to equilibrium. Fractions were collected from the top and assayed for enzyme activities. Where two graphs are shown for a strain (vps1Δ sec6 and vps27Δ sec6), activities were obtained from a single gradient. The density profiles were similar for all gradients.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199237&req=5

fig1: Gradient fractionation of sec6 and vpsΔ sec6 mutants. sec6 (EHY227), vps1Δ sec6 (EHY225), pep12Δ sec6 (EHY232), vps10Δ sec6 (EHY282), vps27Δ sec6 (EHY309), and vps4Δ sec6 (EHY327) cells were grown at 24°C in YPD for 12–14 h and then shifted to 37°C in prewarmed YPD, pH 4.5, for 1 h or for the times indicated. Cells were fractionated as described in Materials and methods. Membrane pellets (100,000 g) were loaded into the bottoms of 15–30% Nycodenz/0.8 M sorbitol linear gradients and floated to equilibrium. Fractions were collected from the top and assayed for enzyme activities. Where two graphs are shown for a strain (vps1Δ sec6 and vps27Δ sec6), activities were obtained from a single gradient. The density profiles were similar for all gradients.
Mentions: The sec6-4 mutant was among the first group of conditional yeast secretory mutants isolated in a screen for mutants that are blocked for growth and secretion and accumulate secretory organelles at a restrictive temperature (Novick et al., 1980). Sec6p is part of a protein complex (the “Exocyst”) involved in the polarized fusion of exocytic vesicles with the plasma membrane (TerBush et al., 1996). The mutant grows as well as wild-type cells at 24°C, but growth ceases at 37°C and the cells accumulate abundant 100-nm vesicles. These vesicles can be separated into two populations by isodensity gradient centrifugation (Fig. 1 A; Harsay and Bretscher, 1995). The more abundant, lighter density, vesicles contain the plasma membrane and cell wall proteins Pma1p and Bgl2p, respectively, whereas the denser vesicles contain the periplasmic enzymes invertase and acid phosphatase. Both vesicle populations also contain an exoglucanase activity, which most likely comprises two different enzymes (Harsay and Bretscher, 1995). Wild-type cells contain very few secretory vesicles at steady state, so it is difficult to distinguish vesicles from other organelles in cell fractionation experiments. Therefore, to analyze the effects of various mutations on the two exocytic pathways, we performed all fractionations with cells having a sec6-4 mutant background.

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