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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 mutants blocked in the ALP-transporting pathway to the vacuole. apl6Δ sec6 (EHY351) and vps41-ts sec6 (EHY350) cells (A and B) were shifted to 37°C for 1 h; vam3-ts sec6 (EHY436) cells were shifted to 38°C for 30 min (C) or 60 min (D). Cells were fractionated as described in the legend to Fig. 1, and gradient fractions were assayed for enzyme activities.
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fig9: Gradient fractionation of mutants blocked in the ALP-transporting pathway to the vacuole. apl6Δ sec6 (EHY351) and vps41-ts sec6 (EHY350) cells (A and B) were shifted to 37°C for 1 h; vam3-ts sec6 (EHY436) cells were shifted to 38°C for 30 min (C) or 60 min (D). Cells were fractionated as described in the legend to Fig. 1, and gradient fractions were assayed for enzyme activities.

Mentions: All of the VPS proteins discussed so far are required for the transport of CPY to the vacuole via an endosomal intermediate. However, the vacuolar membrane protein ALP is transported by an alternate route that bypasses endosome(s) (Cowles et al., 1997b; Piper et al., 1997). Vps1p is required for the normal transport of both CPY and ALP to the vacuole, but Pep12p, Vps4p, and Vps27p are not required for ALP transport. Proteins required for the transport of ALP but not of CPY include Vps41p (Cowles et al., 1997b) and components of the AP-3 adaptin complex (Cowles et al., 1997a; Stepp et al., 1997). We fractionated an apl6Δ sec6-4 mutant, which lacks the β-subunit of the AP-3 complex, to determine whether blocking the ALP pathway to the vacuole affects invertase transport. As shown in Fig. 9 A, this mutant sorted invertase properly into dense vesicles. The slightly lowered level of exoglucanase activity in the light vesicles was reproducible in two experiments; however, these vesicles contained abundant ATPase activity, so the light vesicles were formed properly (unpublished data). Similar results for invertase sorting were obtained for vps41-ts sec6-4 (Fig. 9 B) and vps41Δ sec6-4 (unpublished data), indicating that blocking the ALP pathway does not have a significant effect on the exocytic pathway. Although the sorting of CPY is not affected by a vps41-ts mutation after short shifts to a restrictive temperature, CPY missorting is severe in vps41Δ (Cowles et al., 1997b; Radisky et al., 1997), whereas invertase sorting is only slightly affected. Such differences in missorting are also noted among vacuolar hydrolases that traffic by the CPY (endosome-mediated) pathway. In vps41Δ, the secretion of the soluble vacuolar hydrolase, PrA, is much less severe than that of CPY (Radisky et al., 1997).


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 mutants blocked in the ALP-transporting pathway to the vacuole. apl6Δ sec6 (EHY351) and vps41-ts sec6 (EHY350) cells (A and B) were shifted to 37°C for 1 h; vam3-ts sec6 (EHY436) cells were shifted to 38°C for 30 min (C) or 60 min (D). Cells were fractionated as described in the legend to Fig. 1, and gradient fractions were assayed for enzyme activities.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199237&req=5

fig9: Gradient fractionation of mutants blocked in the ALP-transporting pathway to the vacuole. apl6Δ sec6 (EHY351) and vps41-ts sec6 (EHY350) cells (A and B) were shifted to 37°C for 1 h; vam3-ts sec6 (EHY436) cells were shifted to 38°C for 30 min (C) or 60 min (D). Cells were fractionated as described in the legend to Fig. 1, and gradient fractions were assayed for enzyme activities.
Mentions: All of the VPS proteins discussed so far are required for the transport of CPY to the vacuole via an endosomal intermediate. However, the vacuolar membrane protein ALP is transported by an alternate route that bypasses endosome(s) (Cowles et al., 1997b; Piper et al., 1997). Vps1p is required for the normal transport of both CPY and ALP to the vacuole, but Pep12p, Vps4p, and Vps27p are not required for ALP transport. Proteins required for the transport of ALP but not of CPY include Vps41p (Cowles et al., 1997b) and components of the AP-3 adaptin complex (Cowles et al., 1997a; Stepp et al., 1997). We fractionated an apl6Δ sec6-4 mutant, which lacks the β-subunit of the AP-3 complex, to determine whether blocking the ALP pathway to the vacuole affects invertase transport. As shown in Fig. 9 A, this mutant sorted invertase properly into dense vesicles. The slightly lowered level of exoglucanase activity in the light vesicles was reproducible in two experiments; however, these vesicles contained abundant ATPase activity, so the light vesicles were formed properly (unpublished data). Similar results for invertase sorting were obtained for vps41-ts sec6-4 (Fig. 9 B) and vps41Δ sec6-4 (unpublished data), indicating that blocking the ALP pathway does not have a significant effect on the exocytic pathway. Although the sorting of CPY is not affected by a vps41-ts mutation after short shifts to a restrictive temperature, CPY missorting is severe in vps41Δ (Cowles et al., 1997b; Radisky et al., 1997), whereas invertase sorting is only slightly affected. Such differences in missorting are also noted among vacuolar hydrolases that traffic by the CPY (endosome-mediated) pathway. In vps41Δ, the secretion of the soluble vacuolar hydrolase, PrA, is much less severe than that of CPY (Radisky et al., 1997).

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