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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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Clathrin-coated vesicles transport invertase. (A) Percoll gradient fractionation of a wt strain (EHY191) for identifying fractions enriched for clathrin-containing membranes. A 25–55% Percoll step gradient was formed in a buffer optimized for clathrin coat stabilization. Fractions were collected from the top and assayed for invertase activity and by Western blotting to detect clathrin light chain (Clc1p) and GDPase (Gda1p). (B) Immunoisolated clathrin-coated vesicles (from fraction #14 in the gradient in A) contain invertase and Vps10p but not Pma1p (immunoblots and enzyme assays are from the same immunoisolation experiment). (C) Thin section EM of immunoisolated clathrin-coated vesicles. Bar, 100 nm.
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fig8: Clathrin-coated vesicles transport invertase. (A) Percoll gradient fractionation of a wt strain (EHY191) for identifying fractions enriched for clathrin-containing membranes. A 25–55% Percoll step gradient was formed in a buffer optimized for clathrin coat stabilization. Fractions were collected from the top and assayed for invertase activity and by Western blotting to detect clathrin light chain (Clc1p) and GDPase (Gda1p). (B) Immunoisolated clathrin-coated vesicles (from fraction #14 in the gradient in A) contain invertase and Vps10p but not Pma1p (immunoblots and enzyme assays are from the same immunoisolation experiment). (C) Thin section EM of immunoisolated clathrin-coated vesicles. Bar, 100 nm.

Mentions: Invertase may be transported by clathrin-coated vesicles, or clathrin may play a less direct role in invertase transport by, for example, recycling Golgi proteins required for invertase transport. Previous immunoisolations of clathrin-coated vesicles suggested that clathrin is not involved directly in invertase transport (Deloche et al., 2001). However, invertase in transport vesicles in wild-type cells may represent a very small fraction of the total invertase in the high speed spin membrane fractions used. Furthermore, it is possible that insufficient osmotic support was provided to prevent leakage of invertase, which we found to be released easily from membranes. Therefore, we immunoisolated clathrin-coated vesicles from gradient fractions of membranes isolated from wild-type cells (Fig. 8) . A high buoyant density fraction of invertase cofractionated with clathrin light chain but not with the cis/medial Golgi marker, Gda1p (Fig. 8 A). Immunoisolation of clathrin-coated vesicles from fraction #14 isolated up to 30% of the invertase (Fig. 8 B); nearly identical results were obtained in immunoisolations from neighboring gradient fractions (fractions #13–17; unpublished data). Anti-GST antibodies isolated from the same serum from which anti-Clc1p antibodies were purified was used as a negative control. The clathrin-coated vesicle cargo protein Vps10p was somewhat more efficiently isolated in the same experiment (Fig. 8 B), as expected, because at steady state invertase should be distributed more evenly among secretory organelles than a recycling receptor. Pma1p contained in the same gradient fraction was not likewise immunoisolated; thus, only a subset of exocytic cargo molecules are trafficked via clathrin-coated vesicles. Thin section EM of the isolated membranes showed that essentially all membranes bound to the beads were ∼40-nm vesicles (Fig. 8 C). We had difficulty detecting coats on the surfaces of the vesicles, most likely because they did not preserve or stain well during processing for microscopy.


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)

Clathrin-coated vesicles transport invertase. (A) Percoll gradient fractionation of a wt strain (EHY191) for identifying fractions enriched for clathrin-containing membranes. A 25–55% Percoll step gradient was formed in a buffer optimized for clathrin coat stabilization. Fractions were collected from the top and assayed for invertase activity and by Western blotting to detect clathrin light chain (Clc1p) and GDPase (Gda1p). (B) Immunoisolated clathrin-coated vesicles (from fraction #14 in the gradient in A) contain invertase and Vps10p but not Pma1p (immunoblots and enzyme assays are from the same immunoisolation experiment). (C) Thin section EM of immunoisolated clathrin-coated vesicles. Bar, 100 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199237&req=5

fig8: Clathrin-coated vesicles transport invertase. (A) Percoll gradient fractionation of a wt strain (EHY191) for identifying fractions enriched for clathrin-containing membranes. A 25–55% Percoll step gradient was formed in a buffer optimized for clathrin coat stabilization. Fractions were collected from the top and assayed for invertase activity and by Western blotting to detect clathrin light chain (Clc1p) and GDPase (Gda1p). (B) Immunoisolated clathrin-coated vesicles (from fraction #14 in the gradient in A) contain invertase and Vps10p but not Pma1p (immunoblots and enzyme assays are from the same immunoisolation experiment). (C) Thin section EM of immunoisolated clathrin-coated vesicles. Bar, 100 nm.
Mentions: Invertase may be transported by clathrin-coated vesicles, or clathrin may play a less direct role in invertase transport by, for example, recycling Golgi proteins required for invertase transport. Previous immunoisolations of clathrin-coated vesicles suggested that clathrin is not involved directly in invertase transport (Deloche et al., 2001). However, invertase in transport vesicles in wild-type cells may represent a very small fraction of the total invertase in the high speed spin membrane fractions used. Furthermore, it is possible that insufficient osmotic support was provided to prevent leakage of invertase, which we found to be released easily from membranes. Therefore, we immunoisolated clathrin-coated vesicles from gradient fractions of membranes isolated from wild-type cells (Fig. 8) . A high buoyant density fraction of invertase cofractionated with clathrin light chain but not with the cis/medial Golgi marker, Gda1p (Fig. 8 A). Immunoisolation of clathrin-coated vesicles from fraction #14 isolated up to 30% of the invertase (Fig. 8 B); nearly identical results were obtained in immunoisolations from neighboring gradient fractions (fractions #13–17; unpublished data). Anti-GST antibodies isolated from the same serum from which anti-Clc1p antibodies were purified was used as a negative control. The clathrin-coated vesicle cargo protein Vps10p was somewhat more efficiently isolated in the same experiment (Fig. 8 B), as expected, because at steady state invertase should be distributed more evenly among secretory organelles than a recycling receptor. Pma1p contained in the same gradient fraction was not likewise immunoisolated; thus, only a subset of exocytic cargo molecules are trafficked via clathrin-coated vesicles. Thin section EM of the isolated membranes showed that essentially all membranes bound to the beads were ∼40-nm vesicles (Fig. 8 C). We had difficulty detecting coats on the surfaces of the vesicles, most likely because they did not preserve or stain well during processing for microscopy.

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