Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase.
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.
Brett
Christopher L
CL
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Plemel
Rachael L
RL
Lobingier
Braden T
BT
Lobinger
Braden T
BT
Vignali
Marissa
M
Fields
Stanley
S
Merz
Alexey J
AJ
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D047389:Casein Kinase I / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004843:Epistasis, Genetic; D008561:Membrane Fusion / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*
D033942:Adaptor Proteins, Vesicular Transport; C439608:Gyp7 protein, S cerevisiae; D008565:Membrane Proteins; D021122:Protein Subunits; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C107706:VAM3 protein, S cerevisiae; C406817:VAM6 protein, S cerevisiae; C083878:VPS41 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D020703:ras GTPase-Activating Proteins; D047389:Casein Kinase I; C479667:YCK3 protein, S cerevisiae; C079005:YPT7 protein, S cerevisiae; D020691:rab GTP-Binding Proteins