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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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Missorted CPY peaks with secretory vesicles in vps sec6 mutants but not with vacuolar, ER, or Golgi markers. (A) Western blots of gradient fractions from sec6-4 cells incubated at 37°C for 60 min show that secretory vesicles (Sec4p peak in fraction #8) do not cofractionate with the TGN/early endosome markers Kex2p and Tlg1p or with late endosomes (Pep12p), vacuoles (ALP), or ER (Sec61p). Sec4p and Tlg1p were detected in a single gradient from EHY227 cells; the other proteins were detected for EHY432 (sec6-4 cells with a plasmid expressing HA-tagged Kex2p). Cells were fractionated as in the legend to Fig. 1. (B) Immunoblots of gradient fractions indicate that missorted CPY cofractionates with the light secretory vesicle markers Pma1p and Bgl2p in the vps1Δ sec6-4 (EHY225) and vps4Δ sec6-4 (EHY478) mutants, whereas in vps10Δ sec6-4 (EHY282) CPY is in dense vesicles. In VPS SEC cells (EHY376 shifted to 37°C), Kex2p peaks at a density intermediate between light and dense secretory vesicles (Kex2p is unstable in mutants shifted to restrictive temperature), suggesting that invertase and CPY are not in Kex2p compartments in vps sec6 mutants. Cells were grown and fractionated as in the legend Fig. 1, except that for the vps10Δ sec6-4 and vps4Δ sec6-4 gradients, cells were shifted to 37°C for 40 min rather than 60 min, which greatly reduced the proteolysis of CPY; invertase profiles were similar for the two shift times. Lane numbers correspond with gradient fraction numbers. Unnumbered lanes are CPY processing standards: sec18-1 after a temperature shift contains ER (p1) and vacuole (m) forms, whereas pep4Δ contains the Golgi (p2) form.
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fig3: Missorted CPY peaks with secretory vesicles in vps sec6 mutants but not with vacuolar, ER, or Golgi markers. (A) Western blots of gradient fractions from sec6-4 cells incubated at 37°C for 60 min show that secretory vesicles (Sec4p peak in fraction #8) do not cofractionate with the TGN/early endosome markers Kex2p and Tlg1p or with late endosomes (Pep12p), vacuoles (ALP), or ER (Sec61p). Sec4p and Tlg1p were detected in a single gradient from EHY227 cells; the other proteins were detected for EHY432 (sec6-4 cells with a plasmid expressing HA-tagged Kex2p). Cells were fractionated as in the legend to Fig. 1. (B) Immunoblots of gradient fractions indicate that missorted CPY cofractionates with the light secretory vesicle markers Pma1p and Bgl2p in the vps1Δ sec6-4 (EHY225) and vps4Δ sec6-4 (EHY478) mutants, whereas in vps10Δ sec6-4 (EHY282) CPY is in dense vesicles. In VPS SEC cells (EHY376 shifted to 37°C), Kex2p peaks at a density intermediate between light and dense secretory vesicles (Kex2p is unstable in mutants shifted to restrictive temperature), suggesting that invertase and CPY are not in Kex2p compartments in vps sec6 mutants. Cells were grown and fractionated as in the legend Fig. 1, except that for the vps10Δ sec6-4 and vps4Δ sec6-4 gradients, cells were shifted to 37°C for 40 min rather than 60 min, which greatly reduced the proteolysis of CPY; invertase profiles were similar for the two shift times. Lane numbers correspond with gradient fraction numbers. Unnumbered lanes are CPY processing standards: sec18-1 after a temperature shift contains ER (p1) and vacuole (m) forms, whereas pep4Δ contains the Golgi (p2) form.

Mentions: Exocytic vesicles that accumulate in sec6-4 cells did not cofractionate (copeak) with other organelles (Fig. 3 A; Harsay and Bretscher, 1995), although Sec4p on exocytic vesicles was only slightly denser than the early endosome/Golgi syntaxin Tlg1p, so we cannot exclude the possibility that Tlg1p is present on light-density vesicles. The fractionation of the TGN/endosomal marker Kex2p was difficult to assess, since Kex2p is known to become unstable in cells shifted to 37°C (Wilcox et al., 1992), and this instability was exacerbated in sec6-4 cells (Fig. 3 A). In wild-type cells, Kex2p peaks at the top of the gradient and in intermediate-density fractions (Fig. 3 B) that correspond to the density of accumulated invertase in vps27 sec6-4 cells (Figs. 1 and 2). Kex2p is believed to cycle between a late Golgi compartment and endosomes (Cereghino et al., 1995; Bryant and Stevens, 1997), but the identities and relative densities of Golgi and endosomal compartments in density gradients are unclear (Singer-Krüger et al., 1993; Holthuis et al., 1998a). The late endosomal syntaxin, Pep12p, the ER membrane protein, Sec61p, and the vacuolar membrane protein, alkaline phosphatase (ALP), clearly do not copeak with exocytic vesicles (Fig. 3 A). Similar results were obtained for vps sec6 cells (unpublished data; we did not examine Kex2p and Tlg1p in double mutants).


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)

Missorted CPY peaks with secretory vesicles in vps sec6 mutants but not with vacuolar, ER, or Golgi markers. (A) Western blots of gradient fractions from sec6-4 cells incubated at 37°C for 60 min show that secretory vesicles (Sec4p peak in fraction #8) do not cofractionate with the TGN/early endosome markers Kex2p and Tlg1p or with late endosomes (Pep12p), vacuoles (ALP), or ER (Sec61p). Sec4p and Tlg1p were detected in a single gradient from EHY227 cells; the other proteins were detected for EHY432 (sec6-4 cells with a plasmid expressing HA-tagged Kex2p). Cells were fractionated as in the legend to Fig. 1. (B) Immunoblots of gradient fractions indicate that missorted CPY cofractionates with the light secretory vesicle markers Pma1p and Bgl2p in the vps1Δ sec6-4 (EHY225) and vps4Δ sec6-4 (EHY478) mutants, whereas in vps10Δ sec6-4 (EHY282) CPY is in dense vesicles. In VPS SEC cells (EHY376 shifted to 37°C), Kex2p peaks at a density intermediate between light and dense secretory vesicles (Kex2p is unstable in mutants shifted to restrictive temperature), suggesting that invertase and CPY are not in Kex2p compartments in vps sec6 mutants. Cells were grown and fractionated as in the legend Fig. 1, except that for the vps10Δ sec6-4 and vps4Δ sec6-4 gradients, cells were shifted to 37°C for 40 min rather than 60 min, which greatly reduced the proteolysis of CPY; invertase profiles were similar for the two shift times. Lane numbers correspond with gradient fraction numbers. Unnumbered lanes are CPY processing standards: sec18-1 after a temperature shift contains ER (p1) and vacuole (m) forms, whereas pep4Δ contains the Golgi (p2) form.
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fig3: Missorted CPY peaks with secretory vesicles in vps sec6 mutants but not with vacuolar, ER, or Golgi markers. (A) Western blots of gradient fractions from sec6-4 cells incubated at 37°C for 60 min show that secretory vesicles (Sec4p peak in fraction #8) do not cofractionate with the TGN/early endosome markers Kex2p and Tlg1p or with late endosomes (Pep12p), vacuoles (ALP), or ER (Sec61p). Sec4p and Tlg1p were detected in a single gradient from EHY227 cells; the other proteins were detected for EHY432 (sec6-4 cells with a plasmid expressing HA-tagged Kex2p). Cells were fractionated as in the legend to Fig. 1. (B) Immunoblots of gradient fractions indicate that missorted CPY cofractionates with the light secretory vesicle markers Pma1p and Bgl2p in the vps1Δ sec6-4 (EHY225) and vps4Δ sec6-4 (EHY478) mutants, whereas in vps10Δ sec6-4 (EHY282) CPY is in dense vesicles. In VPS SEC cells (EHY376 shifted to 37°C), Kex2p peaks at a density intermediate between light and dense secretory vesicles (Kex2p is unstable in mutants shifted to restrictive temperature), suggesting that invertase and CPY are not in Kex2p compartments in vps sec6 mutants. Cells were grown and fractionated as in the legend Fig. 1, except that for the vps10Δ sec6-4 and vps4Δ sec6-4 gradients, cells were shifted to 37°C for 40 min rather than 60 min, which greatly reduced the proteolysis of CPY; invertase profiles were similar for the two shift times. Lane numbers correspond with gradient fraction numbers. Unnumbered lanes are CPY processing standards: sec18-1 after a temperature shift contains ER (p1) and vacuole (m) forms, whereas pep4Δ contains the Golgi (p2) form.
Mentions: Exocytic vesicles that accumulate in sec6-4 cells did not cofractionate (copeak) with other organelles (Fig. 3 A; Harsay and Bretscher, 1995), although Sec4p on exocytic vesicles was only slightly denser than the early endosome/Golgi syntaxin Tlg1p, so we cannot exclude the possibility that Tlg1p is present on light-density vesicles. The fractionation of the TGN/endosomal marker Kex2p was difficult to assess, since Kex2p is known to become unstable in cells shifted to 37°C (Wilcox et al., 1992), and this instability was exacerbated in sec6-4 cells (Fig. 3 A). In wild-type cells, Kex2p peaks at the top of the gradient and in intermediate-density fractions (Fig. 3 B) that correspond to the density of accumulated invertase in vps27 sec6-4 cells (Figs. 1 and 2). Kex2p is believed to cycle between a late Golgi compartment and endosomes (Cereghino et al., 1995; Bryant and Stevens, 1997), but the identities and relative densities of Golgi and endosomal compartments in density gradients are unclear (Singer-Krüger et al., 1993; Holthuis et al., 1998a). The late endosomal syntaxin, Pep12p, the ER membrane protein, Sec61p, and the vacuolar membrane protein, alkaline phosphatase (ALP), clearly do not copeak with exocytic vesicles (Fig. 3 A). Similar results were obtained for vps sec6 cells (unpublished data; we did not examine Kex2p and Tlg1p in double mutants).

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