Global analysis of yeast endosomal transport identifies the vps55/68 sorting complex

Abstract

Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process.

Figure 1.

Genome-scale identification of protein-sorting mutants. (A) Yeast knockout strains were plated as 1536 arrays on solid agar (left) or on agar covered with a nitrocellulose filter. Secreted CPY retained on the filter was visualized by Western blotting (right). (B) Top 20 CPY-secreting mutants. Genome-wide screens for CPY missorting were carried out in duplicate on three independent deletion collections, and a median endosomal-sorting index was computed based on densitometry of digital images. (C) Ranked secretion values for the 2000 top CPY-secreting mutants. The top 279 strains were chosen for further analysis. (D) Pie chart showing the subcellular localization of proteins corresponding to the 279 mutants based on a genome-wide GFP fusion study (Huh et al., 2003). Vacuole, endosome, “punctate composite,” Golgi, and ER localizations were enriched in the mutant set compared with the proteome.

Figure 2.

Unbiased prediction of protein complexes based on phenotypic profiling. Miniarrays containing the top CPY-secreting strains were tested for phenotypes including CPY sorting, pro-alpha factor processing, sensitivity to chemical inhibitors, and chitin content. A graph theoretic approach was used to compute p values that identify significant clusters. (A) Enlargement of region of heat map enriched for significant clusters. Clusters corresponding to known protein complexes are indicated. (B) Clusters of three or more genes that have a significance score (p) ≤ 0.05 and a sorting index (SI) >13% are illustrated. The protein complex represented by the greatest number of genes in a given cluster is indicated; known subunits of that complex are shown in blue. Subunits of additional complexes in the same cluster are also color-coded. The relative contribution of each complex to the process of endosomal sorting was estimated by calculating the median sorting index for all elements of that cluster.

Figure 3.

(A–D) Vps55p and Vps68p colocalize at endosomes and the vacuole-limiting membrane. The following strains were fixed and processed for double-label fluorescence microscopy. (A) Vps68-HA and Vps55-myc were coexpressed from plasmids in vps55 vps68 (BWY4) mutants and labeled with anti-myc mAb and anti-HA rabbit antiserum. (B) myc-tagged Vps21p was coexpressed with Vps68-HA from plasmids in vps68 cells (BY4741-6279) and double-labeled with anti-HA mAb and anti-myc rabbit antiserum. (C) Plasmid-expressed Vps68-HA was colocalized with Sec7-GFP in BWY10 cells labeled with rabbit anti-HA antiserum. (D) Plasmid-borne Vps68-HA was expressed in vps68 (BY4741-6279) cells double-labeled with an antiserum to endogenous Vph1 and anti-HA mAb. (E) Vps55p and Vps68p form a complex. Proteins were precipitated with anti-HA antiserum from extracts prepared from vps55 (BY4741-6842) cells expressing AU1-tagged Vps55, vps68 (BY4741-6279) cells expressing HA-tagged Vps68p, or vps55 vps68 (BWY4) cells expressing both tagged proteins, as indicated, and resolved by SDS-PAGE. Lysates and immunoprecipitates (IP) were analyzed by Western blotting with anti-HA and anti-AU1 mAbs. (F) Vps55p and Vps68p stabilize each other. vps55 (BY4741-6842; lane 1), vps68 (BY4741-6279; lane 4), or vps55 vps68 (BWY4; lanes 2, 3, and 5) strains containing plasmids for the expression of HA-tagged Vps55p and/or HA-tagged Vps68p were analyzed by Western blotting with anti-HA mAbs. Presence or absence of endogenous, untagged Vps68p or Vps55p is indicated. (G) The Vps55/68 complex contains a single copy of Vps55p. Detergent extracts were prepared from vps55 (BY4741-6842) mutant cells containing plasmids for the expression of AU1- and/or HA-tagged Vps55p (lanes 1–3 and 5–7), as indicated, and from vps55 vps68 (BWY4) expressing both AU1-tagged Vps55p and HA-tagged Vps68p (lanes 4 and 8). Proteins were immunoprecipitated with anti-HA antiserum and protein G-Sepharose, resolved by SDS-PAGE, and analyzed by Western blotting with anti-HA and anti-AU1 mAbs.

Figure 4.

Cargo sorting in vps68 and vps55 mutants. (A) Secretion of newly synthesized CPY. Strains were labeled with [³⁵S]methionine for 10 min and chased for 60 min in the presence of 50 μg/ml unlabeled cysteine and methionine. CPY was immunoprecipitated from intracellular (I) and extracellular (E) fractions, analyzed by SDS-PAGE, and visualized by fluorography. Arrows indicate the position of Golgi-modified (p2) CPY and that of mature, vacuolar (m) CPY. (B) VPS68 acts downstream of the class E gene VPS27. Pulse-chase labeling and immunoprecipitation of Vps10p was carried out as described above using the following strains: wild-type (BLY1), vps27 (KEBY37), vps68 (MSY3), and vps27 vps68 (MSY11). Proteolytic cleavage of Vps10p in vps27 and vps27 vps68 double mutants generates a faster-migrating cleavage product (*). (C) Kinetics of ALP maturation are unchanged in vps68 mutants. ALP was immunoprecipitated from wild-type (RPY10) or vps68 cells (MSY2) that had been radiolabeled for 10 min with [³⁵S]methionine and chased for the times indicated. PEP4-dependent cleavage converts precursor (pro) forms of ALP into the mature (m) form. Asterisk (*) indicates a commonly observed degradation product. (D and E) Degradation of Ste3p is slowed in vps68 mutants. Ste3p was immunoprecipitated from wild-type (RPY10), vps68 (MSY2), or vps55 (LCY434) cultures that had been radiolabeled with [³⁵S]methionine for 10 min and chased with unlabeled cysteine and methionine for the times indicated. The total amount of Ste3 remaining at each time point was quantitated by densitometry. Representative images for wild-type, vps55, and vps68 strains are shown in D, and semilog plots are shown in E. Wild type, •; vps68, □; vps55, ◇.

Figure 5.

Endosomal accumulation of Ste3p and Sna3p in vps68 mutants. (A) Wild-type (wt; SF838-9D) and vps68 (MSY1) strains containing plasmids for the expression of Ste3-GFP and labeled with FM4-64 were viewed by double-label fluorescence microscopy of live cells. (B) Sna3-GFP was expressed from a plasmid in wild-type (SF838-9D), vps55 (BY4741-6842), vps68 (MSY1), and vps4 (BY4741-5588) strains. Lines (top panels) indicate the location of pixel intensity measurements (bottom panel). Note that Sna3-GFP is evenly distributed throughout the vacuolar lumen in wild-type, vps55, and vps68 strains, whereas pixel intensity is greatest at the vacuolar-limiting membrane in vps4 mutants. (C) Intracellular sorting of fluorescent lipids NBD-PC and FM4-64. NBD-PC is sorted to the vacuole lumen in wild-type, vps55, and vps68 mutants, but not in vps4 mutants. Strains are as described in A. Bar, 2 μm.

Figure 6.

Loss of VPS68 does not slow the endosome-to-cell surface recycling of Ste3Δ365p. (A) Ligand-dependent internalization of Ste3Δ365p. Wild type (NDY1181) and vps68 (BWY14) mutant cells were exposed to a-factor to induce endocytosis, and treated with protease to cleave surface-exposed Ste3Δ365 protein. Ste3Δ365p was detected in cell extracts by Western blotting with anti-HA mAb. (B) Cell surface recycling of internalized Ste3Δ365. Wild-type and vps68 strains expressing Ste3Δ365 were treated as described in A and incubated in fresh medium lacking a-factor. Aliquots removed at the times indicated were subjected to a second protease treatment. The time course with which the internal pool of Ste3Δ365 regains protease sensitivity provides a measure of cell surface recycling.

Figure 7.

VPS68 is required for two transport pathways out of the late endosome. Wild-type (SNY128) and vps68 (BWY12) strains containing the vps27Δ mutation and carrying pHY5 for the GAL1-driven expression of VPS27 were grown to log phase in raffinose medium. Aliquots removed 0, 45, 60, 75, and 90 min after the addition of galactose were processed for the localization of integrated, HA-tagged Vps10p and endogenous Vph1p by double-label immunofluorescence microscopy. (A) Representative images of cells fixed 0, 60, and 90 min after galactose addition. (B and C) Localization of Vph1p (B) and Vps10p (C) was scored for 100–200 cells at each time point. Dark gray bars, vacuole; black bars, class E compartment; light gray bars, dispersed/ambiguous. Bar, 2 μm.