Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Jan 21;156(2):271-85.
doi: 10.1083/jcb.200109077. Epub 2002 Jan 21.

A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway

Edina Harsay  1 , Randy Schekman
Affiliations

A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway

Edina Harsay et al. J Cell Biol. .

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.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
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.
Figure 2.
Figure 2.
Gradient fractionation of vps-ts sec6-4 mutants. Strains were grown at 24°C in SD with required amino acids for maintaining plasmids (vps4-ts sec6, EHY348; pep12-ts sec6, EHY413) or in YPD (vps27-ts sec6, EHY374; vps4Δ sec6, EHY327). Strains grown in SD were shifted to YPD at 24°C for 2 h before 30- or 60-min shifts to 37°C in prewarmed YPD, pH 4.5. (Bottom right) A single culture of vps4Δ sec6 cells was split in half; one half was maintained at 24°C and the other half shifted to 32°C (semi-permissive for sec6-4) for 1h. Cells were fractionated as for Fig. 1, and gradient fractions were assayed for invertase and exoglucanase activities.
Figure 3.
Figure 3.
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.
Figure 3.
Figure 3.
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.
Figure 4.
Figure 4.
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.
Figure 5.
Figure 5.
Light but not dense invertase-containing vesicles can be immunoisolated from vps4-ts sec6-4 cells. (A) 20–55% Percoll gradients from the fractionation of a vps4-ts sec6-4 strain (EHY348) contain both light and dense invertase-containing vesicles (as in Nycodenz gradiens; Fig. 2) and light invertase-containing vesicles cofractionate with ATPase (Pma1p) activity. Cells in the gradient shown were shifted to 37°C for 30 min, including ∼5 min warm-up time. (B) Membranes (equal volumes) from invertase peak fractions (#6 or #16 as indicated) from the gradient in A were incubated with undersaturating Dynabeads M500 coated with either monoclonal antibody #17 or with affinity-purified anti-Pma1p polyconal antibodies. The percent invertase bound to the beads is shown above the bars.
Figure 6.
Figure 6.
Thin section EM of vesicles immunoisolated with anti-Pma1p monoclonal antibody #17 bound to Dynabeads protein G. A vps4Δ sec6-4 strain (EHY327) was fractionated as in the legend to Fig. 5, and membranes from the light invertase peak fraction were immunoisolated using undersaturating beads. Bar, 200 nm.
Figure 7.
Figure 7.
Nycodenz gradient fractionation (performed as in the legend to Figs. 1 and 2) of a chc1-ts sec6-4 strain (EHY242). The gradient profiles of invertase and exoglucanase show a missorting of these exocytic cargo molecules, indicating that clathrin plays a role in invertase transport, as was observed for vps sec6-4 strains (Figs. 1 and 2).
Figure 8.
Figure 8.
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.
Figure 8.
Figure 8.
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.
Figure 9.
Figure 9.
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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ausubel, F.M., R. Brent, R. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1987. Current Protocols in Molecular Biology. John Wiley & Sons Inc., New York.
    1. Babst, M., T.K. Sato, L.M. Banta, and S.D. Emr. 1997. Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p. EMBO J. 16:1820–1831. - PMC - PubMed
    1. Becherer, K.A., S.E. Rieder, S.D. Emr, and E.W. Jones. 1996. Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast. Mol. Biol. Cell. 7:579–594. - PMC - PubMed
    1. Bleekemolen, J.E., M. Stein, K. von Figura, J.W. Slot, and H.J. Geuze. 1988. The two mannose 6-phosphate receptors have almost identical subcellular distributions in U937 monocytes. Eur. J. Cell Biol. 47:366–372. - PubMed
    1. Brada, D., and R. Schekman. 1988. Coincident localization of secretory and plasma membrane proteins in organelles of the yeast secretory pathway. J. Bacteriol. 170:2775–2783. - PMC - PubMed

Publication types

MeSH terms