Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase

Abstract

Rab guanosine triphosphatases (GTPases) are pivotal regulators of membrane identity and dynamics, but the in vivo pathways that control Rab signaling are poorly defined. Here, we show that the GTPase-activating protein Gyp7 inactivates the yeast vacuole Rab Ypt7 in vivo. To efficiently terminate Ypt7 signaling, Gyp7 requires downstream assistance from an inhibitory casein kinase I, Yck3. Yck3 mediates phosphorylation of at least two Ypt7 signaling targets: a tether, the Vps-C/homotypic fusion and vacuole protein sorting (HOPS) subunit Vps41, and a SNARE, Vam3. Phosphorylation of both substrates is opposed by Ypt7-guanosine triphosphate (GTP). We further demonstrate that Ypt7 binds not one but two Vps-C/HOPS subunits: Vps39, a putative Ypt7 nucleotide exchange factor, and Vps41. Gyp7-stimulated GTP hydrolysis on Ypt7 therefore appears to trigger both passive termination of Ypt7 signaling and active kinase-mediated inhibition of Ypt7's downstream targets. We propose that signal propagation through the Ypt7 pathway is controlled by integrated feedback and feed-forward loops. In this model, Yck3 enforces a requirement for the activated Rab in docking and fusion.

Figure 1.

Gyp7 regulates vacuole morphology in vivo and in vitro. (a) BY4742 wild-type S. cerevisiae–harboring plasmids expressing each of eight GYP paralogues, one GYP-like gene (Gyl1), or two catalytically inactive Gyp7 mutants (GYP7-ΔTBC or GYP7-R458K) behind a tetracycline-repressible promotor was grown in SC medium with (repressed) or without (expressed) 5 μM doxycycline. The cells were stained with the endocytic tracer FM4-64 as described in Materials and methods, and imaged. (b) BY4742 wild-type (GYP7) and gyp7Δ cells with or without a 2-μ plasmid overexpressing GYP7 (+GYP7) were stained with FM4-64, exposed to hypotonic (water), isotonic (SC), or hypertonic (SC + 0.4 M NaCl) media for either 5 min (hypotonic) or 30 min (isotonic and hypertonic). Vacuole morphology was scored as either round or multilobed according to LaGrassa and Ungermann (2005). 2,325 cells were analyzed. (c) Vacuoles isolated from BJ3505 and DKY2168 wild-type, gyp7Δ-, or GYP7-overexpressing yeast cells were incubated for 90 min in the presence of ATP and increasing concentrations of sorbitol, and homotypic vacuole fusion was measured. Similar results for gyp7Δ vacuoles were reported previously (Brett and Merz, 2008). n ≥ 4 for all experiments shown. Error bars indicate mean ± SEM. Statistical significance is denoted by asterisks. *, P ≤ 0.05; ***, P ≤ 0.001. Bars, 2 μm.

Figure 2.

Gyp7 prevents vacuole fusion by inactivating Ypt7. (a and b) Homotypic fusion was measured with vacuoles isolated from BJ3505 and DKY2168 wild-type (wt), gyp7Δ, YPT7_Q68L, or GYP7-overexpressing cells (+GYP7 2μ). Vacuoles were incubated with ATP for 90 min in the presence of increasing concentrations of rGdi1 (a) or rGyp1_TBC (b). The mean fusion values were fit to sigmoidal dose–response curves (r² > 0.98). IC₅₀ values for the fits are shown in parentheses. n ≥ 4 for all experiments shown. Error bars indicate mean ± SEM. (c) Wild-type DKY6128 cells containing chromosomal copies of wild-type YPT7 or the GTP hydrolysis-deficient allele YPT7_Q68L in the presence or absence of GYP7 expressed from a high copy 2-μm plasmid were grown in SC medium, stained with FM4-64, and imaged. Bar, 2 μm.

Figure 3.

Native Gyp7 inactivates Ypt7p in vivo. (a) Vacuoles isolated from BJ3505 wild type (GYP7) or gyp7Δ were incubated with ATP for 40 min with 9.5 μM rGdi1 absent or present during the last 10 min. Vacuoles were sedimented, and membrane association of Vps41, Vam3, and Ypt7 was assessed by immunoblotting. The white line indicates that intervening lanes have been spliced out. (b) Vacuoles isolated from BJ3505 wild-type (GYP7, top) or gyp7Δ cells (bottom) were pretreated with or without 200 μM GTPγS for 10 min at 27°C and incubated for 40 min with ATP in the presence or absence of 3.6 μM rGyp1_TBC at four concentrations of sorbitol (standard conditions). During the last 10 min of incubation, 9.5 μM rGdi1 was added. Vacuoles were immediately sedimented, and the membrane distribution of Ypt7 was assessed by immunoblotting. One third of the total isolated supernatant (S) and one fifth of the pellet (P) were analyzed by immunoblotting.

Figure 4.

Yck3 functions downstream of Ypt7 on the vacuole. Vacuoles isolated from BY4742 pep4Δ and pho8Δ wild- type cells (YCK3) or yck3Δ cells were incubated with either ATP (a) or rVam7 (b) for 90 min in the presence of increasing concentrations of rGdi1. Fusion activity was measured and is shown as a fraction of the activity observed in the absence of rGdi1. Datasets were fit to sigmoidal dose–response curves (r² > 0.97); the IC₅₀ values for the fits are shown in parentheses. Error bars indicate mean ± SEM. (c) BY4742 single or double mutant cells with the indicated genotypes were grown without (left) or with (right) the GYP7 overexpression plasmid pYADH1-GYP7-His6 and stained with FM4-64 under isotonic conditions. Vacuole morphology was scored, and the fraction of cells with fragmented or multilobed vacuoles is indicated for each strain. 3,836 cells were analyzed. n ≥ 4 for all experiments shown. Bar, 3 μm.

Figure 5.

Ypt7-GTP opposes Yck3-mediated phosphorylation of Ypt7 targets. (a) Vacuoles isolated from BJ3505 gyp7Δ cells were subjected to the same analysis as in Fig. 3 b, and the membrane distribution of Vps41 was assessed by immunoblotting. (b) Vacuoles isolated from BY4742 pep4Δ wild-type (YCK3) or yck3Δ cells were incubated for 70 min with or without ATP in the presence of increasing concentrations of sorbitol. Only standard conditions (200 mM sorbitol) are shown for YCK3 vacuoles. Vacuoles were immediately sedimented, and membrane pellets were assayed for Vps41, Vam3, and Ypt7 by immunoblotting. (c) BY4742 pep4Δ vacuoles were incubated for 70 min with or without ATP in the presence or absence of three concentrations of CIP. Vacuoles were sedimented, and immunoblot analysis was performed on the membrane pellets to assess Vps41 and Vam3. (d) Vacuoles isolated from BY4742 pep4Δ wild-type (YCK3) or yck3Δ cells were incubated for 70 min with ATP in the presence or absence of 9.5 μM rGdi1 or 3.6 μM rGyp1_TBC at four concentrations of sorbitol. Vacuoles were sedimented, and immunoblot analysis was performed on the membrane pellets to assess Vps41 and Vam3. One fifth of the total membrane pellet was used for these analyses. Note the collapse of high M_r forms (*) of Vps41 and Vam3 to the low M_r form (o) when GTPγS is present, sorbitol is decreased, YCK3 is deleted, CIP is added, or rGdi1 is absent.

Figure 6.

A second Ypt7-binding site in Vps-C/HOPS subunit Vps41. (a) Two-hybrid interaction tests were performed by mating cells harboring the indicated bait constructs against a miniarray of prey constructs as indicated, selecting for diploid cells, and replica plating the diploid cells to selective medium lacking histidine and containing 3-aminotriazole to increase stringency (see Materials and methods). The prey miniarray contains all 11 yeast Rab GTPases as well as mutant forms of Vps21 and Ypt7. The wild-type Ypt7 prey is indicated by a dashed circle. The Ypt7 or Vps21 mutants shown affect nucleotide binding; e.g., Ypt7-D129N and -I41S are predicted to have reduced affinity for both GDP and GTP. 2K and 6K mutants contain Arg substitutions at Lys residues reported to be ubiquitylated in native Ypt7 (Kleijnen et al., 2007). (b) Pull-down experiments were performed using eight different GST-Rab fusions or GST alone with cell lysates from BJ3505 Vps39-GFP cells. The amount of Vps41, Vps39-GFP, or Vps33 bound to the GST-Rab resin or found in lysate (load) was determined by immunoblotting. GST-Ypt11 is similar in size to Vps33, interfering with the migration of Vps33 in the Ypt11 lane. (c) Pull-down experiments were performed with either GST-Ypt7 or GST-Sec4 and lysates from BY4742 wild-type (wt), vps39Δ (39Δ), or vps41Δ (41Δ) cells in the presence of 200 μM GDP or GTPγS. Vps41 or Vps33 in the load and eluate fractions was detected by immunoblotting. (d) Pull-down experiments were performed with either GST-Ypt7 or GST-Sec4 resins and 200-nM recombinant full-length Vps41 (rVps41) in the presence of 200 μM GDP or GTPγS. Input and bound proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue.

Figure 7.

Feed-forward inhibition of Ypt7 signaling. (a) General scheme. (b) Implementation at the yeast vacuole. Lines terminating in arrows indicate positive regulation, and lines terminating in bars indicate inhibition. Vps39 and Vps41 are subunits of the Vps-C/HOPS protein complex, which also contains Vps11, 16, 18, and 33.