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. 2000 Feb;11(2):613-26.
doi: 10.1091/mbc.11.2.613.

VPS21 controls entry of endocytosed and biosynthetic proteins into the yeast prevacuolar compartment

S R Gerrard  1 , N J Bryant, T H Stevens
Affiliations
Free PMC article

VPS21 controls entry of endocytosed and biosynthetic proteins into the yeast prevacuolar compartment

S R Gerrard et al. Mol Biol Cell. 2000 Feb.
Free PMC article

Abstract

Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole. The class D VPS genes include VPS21, PEP12, and VPS45, which appear to encode components of a membrane fusion complex involved in Golgi-to-endosome transport. Vps21p is a member of the Rab family of small Ras-like GTPases and shows strong homology to the mammalian Rab5 protein, which is involved in endocytosis and the homotypic fusion of early endosomes. Although Rab5 and Vps21p appear homologous at the sequence level, it has not been clear if the functions of these two Rabs are similar. We find that Vps21p is an endosomal protein that is involved in the delivery of vacuolar and endocytosed proteins to the vacuole. Vacuolar and endocytosed proteins accumulate in distinct transport intermediates in cells that lack Vps21p function. Therefore, it appears that Vps21p is involved in two trafficking steps into the prevacuolar/late endosomal compartment.

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Figures

Figure 1
Figure 1
Vps21p and Pep12p localize to endosomal membranes. c-myc VPS21 pep4-3 (RPY57) and pep4-3 (SF838-9D) overexpressing VPS21 (pSRG92) were grown to midlog phase before fixation. Vps21p was labeled with mouse anti-c-myc antibodies and Alexa 594–conjugated secondary antibody. Pep12p was labeled with rabbit anti-Pep12p antibodies, followed by biotinylated secondary antibody and FITC-conjugated streptavidin. Staining was visualized by confocal microscopy.
Figure 2
Figure 2
vps21Δ and vps27Δ cells accumulate Ste3p and Vph1p in trafficking intermediates. pep4-3 (SF838-9D), vps27Δ pep4-3 (SGY73), and vps21Δ pep4-3 (SGY79) cells were grown to midlog phase at 25°C before fixation. Vph1p was labeled with rabbit anti-Vph1p antibodies and Alexa 594–conjugated secondary antibody. Ste3p was labeled with mouse anti-Ste3p antibodies, followed by biotinylated secondary antibody and FITC-conjugated streptavidin.
Figure 3
Figure 3
The Ste3p and Vph1p trafficking intermediates that accumulate in vps21Δ cells are partially separable by differential centrifugation. pep4-3 (SF838-9D) and vps21Δ pep4-3 (SGY79) strains were subjected to differential centrifugation to yield low-speed pellet (P13), high-speed pellet (P100), and S100 fractions, as described in MATERIALS AND METHODS. The amount of Ste3p, Vph1p, ALP, Pep12p, Vps21p, Vps10p, and phosphoglycerate kinase (PGK) in each of the fractions was assessed by immunoblot analysis.
Figure 4
Figure 4
Sucrose density gradient fractionation of P13 membranes from pep4-3 (SF838–9D) (A) and vps21Δ pep4-3 (SGY79) (B) cells. P13 membranes were prepared and applied to the top of a 40–60% linear sucrose density gradient. Samples were centrifuged for 18 h, and then 16 fractions were removed from the top of the gradient, as described in MATERIALS AND METHODS. The amount of Vph1p, ALP, Ste3p, Pep12p, and Vps21p in each of the fractions was assessed by immunoblot analysis.
Figure 5
Figure 5
VPS21 is required for proteolytic processing of Vps10p-Δ10* and CPY but not for proteolytic processing of ALP. Wild-type (RPY10) and vps21-T39K (SGY36) cells were incubated at 25°C or for 5 min at 37°C before the addition of [35S]Express. Cells were labeled for 10 min and then chased for the indicated times. Vps10p, Vps10p-Δ10*, CPY, and ALP were immunoprecipitated from cell lysates, as described in MATERIALS AND METHODS. The experimental procedure was repeated three times, and results of a representative experiment are shown.
Figure 6
Figure 6
VPS21 is required for the delivery of Vph1p but not for the delivery of ALP to the vacuole. The production of Vph1p was under the control of the galactose-inducible GAL1 promoter in both pep4-3 (NBY79) and vps21-T39K pep4-3 (SGY46) strains. Cells were grown to midlog phase at 25°C, galactose was added, and the cells were incubated for 30 min at either 25°C (A) or 37°C (B). Protein synthesis was terminated by the addition of cycloheximide, followed by incubation for an additional 45 min at 25°C (A) or for 15 min at 37°C (B) before fixation. Vph1p was labeled with rabbit anti-Vph1p antibodies and Texas Red–conjugated secondary antibody. ALP was labeled with mouse anti-ALP antibodies, followed by biotinylated secondary antibody and FITC-conjugated streptavidin.
Figure 7
Figure 7
VPS21 and VPS27 are required for the Pep4p protease–dependent degradation of Ste3p. Wild-type (RPY10) and vps21-T39K (SGY36) cells were incubated at 25°C or for 5 min at 37°C before the addition of [35S]Express. vps27Δ (SGY73) cells harbored the PEP4 plasmid (pTS18) and were incubated at 25°C before the addition of [35S]Express. Cells were labeled for 10 min and then chased for the indicated times. Ste3p was immunoprecipitated from cell lysates, as described in MATERIALS AND METHODS. The experimental procedure was repeated three times, and results of a representative experiment are shown.
Figure 8
Figure 8
VPS21 is epistatic to VPS27 for Vph1p trafficking. The production of Vph1p was under the control of the galactose-inducible GAL1 promoter in all strains. pep4-3 (NBY79), vps21-T39K pep4-3 (SGY46), vps27Δ pep4-3 (SGY77), and vps21-T39K vps27Δ pep4-3 (SGY78) strains were grown to midlog phase at 25°C, galactose was added, and the cells were incubated for 30 min at 37°C. Protein synthesis was terminated by the addition of cycloheximide, followed by incubation for an additional 15 min at 37°C before fixation. Vph1p and Vps10p were labeled with rabbit anti-Vph1p or rabbit anti-Vps10p antibodies, respectively, and Alexa 594–conjugated secondary antibody. DIC, differential interference contrast.
Figure 9
Figure 9
Transport pathways to the vacuole. The CPY pathway is taken by soluble vacuolar hydrolases and the membrane proteins Vps10p-Δ10* and Vph1p. This pathway is sensitive to mutation in VPS21 or VPS27. Vps21p controls Golgi-derived traffic into the PVC, and Vps27p controls traffic leaving the PVC for either the Golgi or the vacuole. The ALP pathway is not affected by mutation in VPS21 or VPS27. Endocytosed proteins, such as Ste3p, traffic via the plasma membrane and through the PVC en route to the vacuole. Vacuolar delivery of Ste3p is dependent on VPS21 and VPS27. The question marks refer to pathways for which there is little or no evidence in yeast.
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References

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