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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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Vesicles immunoisolated from vps1Δ sec6-4 cells using anti-Pma1p monoclonal antibodies contain invertase and Bgl2p. (A) EHY225 (vps1Δ sec6-4) and EHY227 (sec6-4) cells were shifted to 37°C for 40 min and fractionated on 20–55% Percoll step gradients as described in Materials and methods; fractions were collected from the top and assayed for enzyme activities. (B) Two different anti-Pma1p monoclonal antibodies (Ab #15 and Ab #17) bound to Dynabeads protein G were used to immunoisolate Pma1p-containing membranes from the invertase peak fraction (#6) from a vps1 sec6 Percoll gradient fractionation. Either 25 or 50 μl beads were used as indicated. All incubations contained the same amount of membrane with invertase activity shown as “Total.” In antibody competition experiments, peptides corresponding to the mapped monoclonal epitopes (pep15, pep17) were preincubated with the beads and included in the immunoisolation reactions. (C) Western blot to detect Bgl2p in the immunoisolation reactions described in B.
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fig4: Vesicles immunoisolated from vps1Δ sec6-4 cells using anti-Pma1p monoclonal antibodies contain invertase and Bgl2p. (A) EHY225 (vps1Δ sec6-4) and EHY227 (sec6-4) cells were shifted to 37°C for 40 min and fractionated on 20–55% Percoll step gradients as described in Materials and methods; fractions were collected from the top and assayed for enzyme activities. (B) Two different anti-Pma1p monoclonal antibodies (Ab #15 and Ab #17) bound to Dynabeads protein G were used to immunoisolate Pma1p-containing membranes from the invertase peak fraction (#6) from a vps1 sec6 Percoll gradient fractionation. Either 25 or 50 μl beads were used as indicated. All incubations contained the same amount of membrane with invertase activity shown as “Total.” In antibody competition experiments, peptides corresponding to the mapped monoclonal epitopes (pep15, pep17) were preincubated with the beads and included in the immunoisolation reactions. (C) Western blot to detect Bgl2p in the immunoisolation reactions described in B.

Mentions: The cofractionation of Pma1p and missorted invertase in vps sec6-4 mutants suggests that the proteins may be packaged into a common carrier. However, it is also possible that invertase is missorted into a different class of vesicles with fractionation properties very similar to that of Pma1p-transporting vesicles. To distinguish between these two possibilities, we immunoisolated Pma1p-transporting vesicles from vps sec6-4 mutants and assessed whether these vesicles contain invertase (Figs. 4 and 5) . Immunoisolations were performed with membranes fractionated on Percoll step gradients. The purpose of gradient fractionation was to separate light and dense secretory vesicles and to remove soluble invertase released from organelles and float vesicles away from proteases that reduce immunoisolation efficiency. A Percoll gradient was used rather than Nycodenz in order to maintain osmotic conditions during the gradient fractionation and immunoisolation procedures. We found that the invertase vesicles are particularly sensitive to osmotic changes. The low viscosity of Percoll also enabled organelles to reach equilibrium density after a 1-h centrifugation so that immunoisolation of fragile vesicles could be performed more quickly. Fig. 4 A shows the Percoll gradient fractionation profile of invertase from sec6-4 and vps1Δ sec6-4 cells. Using two different anti-Pma1p monoclonal antibodies bound to magnetic beads (see Materials and methods), we could isolate close to 60% of the invertase present in vps1 sec6 invertase peak fractions (Fig. 4 B). Very similar results were obtained for the light vesicle cargo protein Bgl2p (Fig. 4 C). The fraction of invertase and Bgl2p not isolated may correspond to leakage from the vesicles or their presence in cofractionating organelles with lower amounts of Pma1p. To demonstrate the specificity of the immunoisolation procedure, we used peptides corresponding to the mapped epitopes of each of the antibodies (Serrano et al., 1993) in competition experiments (Fig. 4, B and C). In each case, the corresponding peptide competed specifically with the monoclonal antibody, with the peptide for antibody #17 being a more effective competitor (most likely due to a closer resemblance to the corresponding sequence in the folded native protein).


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)

Vesicles immunoisolated from vps1Δ sec6-4 cells using anti-Pma1p monoclonal antibodies contain invertase and Bgl2p. (A) EHY225 (vps1Δ sec6-4) and EHY227 (sec6-4) cells were shifted to 37°C for 40 min and fractionated on 20–55% Percoll step gradients as described in Materials and methods; fractions were collected from the top and assayed for enzyme activities. (B) Two different anti-Pma1p monoclonal antibodies (Ab #15 and Ab #17) bound to Dynabeads protein G were used to immunoisolate Pma1p-containing membranes from the invertase peak fraction (#6) from a vps1 sec6 Percoll gradient fractionation. Either 25 or 50 μl beads were used as indicated. All incubations contained the same amount of membrane with invertase activity shown as “Total.” In antibody competition experiments, peptides corresponding to the mapped monoclonal epitopes (pep15, pep17) were preincubated with the beads and included in the immunoisolation reactions. (C) Western blot to detect Bgl2p in the immunoisolation reactions described in B.
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fig4: Vesicles immunoisolated from vps1Δ sec6-4 cells using anti-Pma1p monoclonal antibodies contain invertase and Bgl2p. (A) EHY225 (vps1Δ sec6-4) and EHY227 (sec6-4) cells were shifted to 37°C for 40 min and fractionated on 20–55% Percoll step gradients as described in Materials and methods; fractions were collected from the top and assayed for enzyme activities. (B) Two different anti-Pma1p monoclonal antibodies (Ab #15 and Ab #17) bound to Dynabeads protein G were used to immunoisolate Pma1p-containing membranes from the invertase peak fraction (#6) from a vps1 sec6 Percoll gradient fractionation. Either 25 or 50 μl beads were used as indicated. All incubations contained the same amount of membrane with invertase activity shown as “Total.” In antibody competition experiments, peptides corresponding to the mapped monoclonal epitopes (pep15, pep17) were preincubated with the beads and included in the immunoisolation reactions. (C) Western blot to detect Bgl2p in the immunoisolation reactions described in B.
Mentions: The cofractionation of Pma1p and missorted invertase in vps sec6-4 mutants suggests that the proteins may be packaged into a common carrier. However, it is also possible that invertase is missorted into a different class of vesicles with fractionation properties very similar to that of Pma1p-transporting vesicles. To distinguish between these two possibilities, we immunoisolated Pma1p-transporting vesicles from vps sec6-4 mutants and assessed whether these vesicles contain invertase (Figs. 4 and 5) . Immunoisolations were performed with membranes fractionated on Percoll step gradients. The purpose of gradient fractionation was to separate light and dense secretory vesicles and to remove soluble invertase released from organelles and float vesicles away from proteases that reduce immunoisolation efficiency. A Percoll gradient was used rather than Nycodenz in order to maintain osmotic conditions during the gradient fractionation and immunoisolation procedures. We found that the invertase vesicles are particularly sensitive to osmotic changes. The low viscosity of Percoll also enabled organelles to reach equilibrium density after a 1-h centrifugation so that immunoisolation of fragile vesicles could be performed more quickly. Fig. 4 A shows the Percoll gradient fractionation profile of invertase from sec6-4 and vps1Δ sec6-4 cells. Using two different anti-Pma1p monoclonal antibodies bound to magnetic beads (see Materials and methods), we could isolate close to 60% of the invertase present in vps1 sec6 invertase peak fractions (Fig. 4 B). Very similar results were obtained for the light vesicle cargo protein Bgl2p (Fig. 4 C). The fraction of invertase and Bgl2p not isolated may correspond to leakage from the vesicles or their presence in cofractionating organelles with lower amounts of Pma1p. To demonstrate the specificity of the immunoisolation procedure, we used peptides corresponding to the mapped epitopes of each of the antibodies (Serrano et al., 1993) in competition experiments (Fig. 4, B and C). In each case, the corresponding peptide competed specifically with the monoclonal antibody, with the peptide for antibody #17 being a more effective competitor (most likely due to a closer resemblance to the corresponding sequence in the folded native protein).

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