Domains within the GARP subunit Vps54 confer separate functions in complex assembly and early endosome recognition

Abstract

Tethering complexes contribute to the specificity of membrane fusion by recognizing organelle features on both donor and acceptor membranes. The Golgi-associated retrograde protein (GARP) complex is required for retrograde traffic from both early and late endosomes to the trans-Golgi network (TGN), presenting a paradox as to how a single complex can interact specifically with vesicles from multiple upstream compartments. We have found that a subunit of the GARP complex, Vps54, can be separated into N- and C-terminal regions that have different functions. Whereas the N-terminus of Vps54 is important for GARP complex assembly and stability, a conserved C-terminal domain mediates localization to an early endocytic compartment. Mutation of this C-terminal domain has no effect on retrograde transport from late endosomes. However, a specific defect in retrieval of Snc1 from early endosomes is observed when recycling from late endosomes to the Golgi is blocked. These data suggest that separate domains recruit tethering complexes to different upstream compartments to regulate individual trafficking pathways.

Figure 1.

The N-terminus of Vps54 is important for GARP complex assembly and stability. Plasmids encoding HA-tagged full-length Vps54 (FL; pCC9), its N-terminal region (N; pCC10), or C-terminal region (C; pCC6) were expressed in vps54Δ strains containing integrated myc-tagged Vps51 (CCY7, lanes 1-4), Vps52 (CCY5, lanes 5-8), or Vps53 (CCY6, lanes 9-12). (A) Whole cell lysates were subjected to immunoprecipitation under nondenaturing conditions using HA antibodies and copurifying HA- and myc-tagged GARP subunits were detected by Western blot. (B) Steady state levels of GARP subunits in the strains described above were determined by anti-myc Western blotting of whole cell lysates. Protein abundance was determined by densitometry and expressed as a percentage of levels in cells expressing full-length Vps54. Note that the N-terminal region of Vps54 largely stabilizes the other GARP subunits.

Figure 2.

C-terminally truncated Vps54 is functional in late endosome to TGN transport, but defective in sorting from early endosomes. (A) GARP is required for retrograde traffic from both early and late endosomes. Vacuolar localization of CPY requires retrograde traffic from late endosomes (LE) as represented by the dotted line, whereas plasma membrane localization of Snc1 requires retrograde traffic from early endosomes (EE) as represented by a dashed line. Vps5, a subunit of the retromer, is specifically required for LE-to-TGN transport. (B) Vps54-N partially rescues the CPY sorting defect of vps54Δ. Secreted CPY was detected by colony overlay assay from 1/1000 OD₆₀₀ units of vps54Δ cells (BY54Δ) transformed with plasmids to express wild-type Vps54 (pCC9), Vps54-N (pCC10), Vps54-C (pCC6), or vector alone (pVT102L). (C) Overexpressing the N-terminal region of Vps54 partially restores localization of Snc1 to the plasma membrane. Snc1-GFP expressed from a CEN plasmid (pCS07) in the strains described in B was visualized by fluorescence microscopy. (D) The ability of Vps54-N to localize Snc1-GFP to the plasma membrane requires late endosome-to-TGN transport. Snc1 was coexpressed with truncated forms of Vps54 in vps5Δ vps54Δ double mutants (NQY116) using plasmids described above.

Figure 3.

Differential localization of N- and C-terminal domains of Vps54. (A) Vps54-N, but not Vps54-C, directs the localization of Vps52-GFP to the TGN. The vps54Δ strain (CSY51), which contains an integrated copy of Vps52-GFP, was transformed with plasmids for the expression of HA-tagged full-length (pCC9), N-terminal (pCC10) or C-terminal (pCC6) forms of Vps54 and cells were viewed by fluorescence microscopy. (B) Vps54-C localized to a polarized structure. vps54Δ cells (BY54Δ) expressing the Vps54 plasmids described in A, as well as wild-type cells (BY4741) expressing Vps54-C (pCC6), were fixed, labeled with a monoclonal anti-HA antibody followed by an FITC-conjugated secondary antibody, and observed by fluorescence microscopy.

Figure 4.

The C-terminal domain of Vps54 localizes to sites of polarized growth throughout the cell cycle. (A-C) HA-tagged Vps54-C (pCC6) was visualized by immunofluorescence microscopy in fixed vps54Δ cells (BY54Δ). (A) Representative images of unbudded cells or cells with small, medium, or large buds show polarization of Vps54-C through the cell cycle. (B) Localization of Vps54-C to sites of polarized growth was abolished by treatment with the actin inhibitor latrunculin B. (C) Treating cells with the mating pheromone alpha-factor induced G1 arrest and the relocalization of Vps54-C to shmoo tips. Treatment with the microtubule inhibitor nocodazole induced G2/M arrest but did not disrupt the cortical localization of Vps54-C. Cell cycle arrest was confirmed by FACS analysis of DNA content (shown on the right). Peaks corresponding to 1n, 2n, and 4n DNA content are indicated.

Figure 5.

Vps54 C-terminus colocalizes with early endocytic markers but not markers of late Golgi or secretory vesicles. (A) Snc1-GFP localizes to a punctate polarized structure that colocalizes with the HA-tagged C-terminus of Vps54. (B) Vps54-C does not colocalize with the late-Golgi marker Sec7-GFP in NQY111 cells. (C) Vps54-C and Sec4 colocalize at the bud tip but not in the mother cell. Treatment with latrunculin B disperses polarized staining patterns and abolishes the colocalization of Vps54-C with Sec4 (D) but not that of Snc1-GFP and Vps54-C (E). (F) Vps54-C colocalizes with the styryl dye FM4-64 after a short chase. vps54Δ cells (BY54Δ) expressing GFP-tagged Vps54-C (pCC12) were allowed to take up FM4-64 for 5 min at 30°C after labeling the plasma membrane for 15 min on ice and viewed by fluorescence microscopy of unfixed cells. (G) Vps54-C partially but consistently colocalizes with Rcy1-GFP (arrows). vps54Δ cells expressing GFP-tagged Rcy1 from the GAL1 promoter (pJMG95) and coexpressing HA-tagged Vps54-C from a plasmid (pCC6) were visualized by double label indirect immunofluorescence microscopy after 8 h of galactose induction. In A-C, E, and G, epitope tags were visualized by double label indirect immunofluorescence microscopy using anti-HA and anti-GFP antibodies. In C and D, the localization of Sec4 was visualized using a mAb directed against Sec4.

Figure 6.

Morphology of Vps54-C-expressing cells by electron microscopy. (A-C) Clusters of irregular 100-200-nm organelles in the bud and in the mother cell near the mother-bud junction seen in representative cells from a vps54Δ strain (LCY200) expressing Vps54-C (pCC6) but not vector alone are indicated with an asterisk. (D) Enlarged view of structures that are boxed in C. (E) Immunoelectron microscopy of the same strain double-labeled with anti-HA antibodies conjugated to 5-nm gold particles, and anti-actin particles conjugated to 10-nm gold particles. HA staining is found on clustered membrane profiles (closed arrowheads) and on the limiting membrane of vesicular profiles (arrows). Scale bars, 0.20 μm.

Figure 7.

Conserved residues in the C-terminus of Vps54 are required for localization to the polarized, early endocytic structure. (A) Alignment of Vps54 from S. cerevisiae with homologues in Schizosaccharomyces pombe (O14093), Drosophila melanogaster (Q9VLC0), Caenorhabditis elegans (Q22639), and human (NP_057600) showing evolutionarily conserved regions in the C-terminal domain. Gray shading shows conservation across 50% of sequences, whereas black shading indicates more highly conserved or invariant residues. Arrows indicate residues that were mutated to create Vps54^EW or Vps54^EWNS. Sequence alignment was generated with T-coffee (Notredame et al., 2000) (B) vps54Δ strains (BY54Δ) expressing either Vps54-C (pCC5), truncated forms that each contain one of the conserved regions in the C-terminus (Vps54-C^593-735, pCC13; Vps54-C^724-889, pCC4), or Vps54-C harboring point mutations (Vps54-C^EW, pCC19) from a galactose-inducible promoter were grown to log phase in galactose, fixed, and processed for immunofluorescence microscopy.

Figure 8.

Point mutations in the C-terminal Vps54 domain abolish sorting from early endosomes without affecting late endosome to TGN transport. (A) The early endosome-sorting defect of Vps54^EWNS is apparent in cells deficient in late endosome recycling. Vps54 (pLC104) and the indicated mutant forms (Vps54^EW, pCC14; Vps54^NS, pCC15; Vps54^EWNS, pCC16; Vps54-N, pCC10) were coexpressed with Snc1-GFP (pCS07) in vps54Δ vps5Δ (NQY116) double mutant strains or in a vps54Δ single mutant (BY54Δ). Snc1-GFP was visualized by fluorescence microscopy of fixed cells. Neither the E689A W691A nor N805A S806A mutations alone were sufficient to destroy the early endosome sorting function of Vps54, but plasma membrane localization of Snc1 was abolished in vps5Δ strains expressing the Vps54 quadruple mutant. (B) The Vps54^EWNS mutant remains functional for late endosome-to-TGN transport. CPY secretion was determined by a colony overlay assay for vps54Δ strains (BY54Δ) expressing wild-type and mutant forms of Vps54 as described in A.

Figure 9.

Vps54 is not required for recycling of Ste3Δ365-GFP. (A) vps54Δ Ste3Δ365-GFP (NQY113) cells expressing full-length Vps54 (pCC9), empty vector, or Vps54-C (pCC6) were visualized by fluorescence microscopy before treatment with a-factor (t = 0), immediately after a 45-min incubation with a-factor (t = 45 min, a-factor) and 30 min after removing a-factor from the media (t = 30-min wash). Ste3Δ365-GFP is recycled from endosomes back to the plasma membrane in both wild-type (Vps54) and vps54Δ cells after the 30-min wash at 30°C. Expression of Vps54-C does not impair Ste3Δ365-GFP recycling or affect its intracellular localization.

Figure 10.

Model of Vps54 function in retrograde transport from early endosomes. (A) In wild-type cells, distinct domains within the GARP complex target the complex to retrograde vesicles budding from early and late endosomal membranes. GARP subsequently directs the tethering of these vesicles to the TGN through interactions with Rab and SNARE proteins (not illustrated). (B) Loss of the Vps54-C domain (dark gray appendage) specifically blocks GARP complex recruitment to early endosomes, resulting in the rerouting of Snc1 to late endosomes (light gray arrow), where it is retrieved via the retrograde pathway used by Vps10 (gray arrow). According to this model, a separate, as-yet-unidentified domain (light gray appendage) recruits GARP to late endosomes.