TY - JOUR
T1 - Cargo Release from Myosin V Requires the Convergence of Parallel Pathways that Phosphorylate and Ubiquitylate the Cargo Adaptor
AU - Wong, Sara
AU - Hepowit, Nathaniel L.
AU - Port, Sarah A.
AU - Yau, Richard G.
AU - Peng, Yutian
AU - Azad, Nadia
AU - Habib, Alim
AU - Harpaz, Nofar
AU - Schuldiner, Maya
AU - Hughson, Frederick M.
AU - MacGurn, Jason A.
AU - Weisman, Lois S.
N1 - We thank members of the Weisman lab for their insightful comments. We thank Dr. Natsuko Jin and Noah Steinfeld for the yck3Δ mutant strains and Dr. Yui Jin for the pRS413-mCherry-Myo2 plasmid and Myo2-TAP strain. We thank Dr. Scott D. Emr for providing the HOPS mutant strains. We thank Dr. Christian Ungermann for providing Vps41 mutant strains. We thank Dr. Raymond J. Deshaies for providing the Cdc48 mutant strain. We thank Dr. Mark Hochstrasser for providing the proteasome mutant strains. This work was supported by the National Institutes of Health grant R01 GM062261 to L.S.W. and a University of Michigan-Israel Partnership for Research pilot grant to M.S. and L.S.W. S.W. was supported in part by the National Institutes of Health grant T32 GM007315 , National Institutes of Health Predoctoral Fellowship F31 AR073677 , and the University of Michigan Rackham Predoctoral Fellowship. This work was supported in part by the National Institutes of Health grant R01GM071574 to F.M.H. and by the Deutsche Forschungsgemeinschaft fellowship PO2195/1-1 to S.A.P. Author contributions - S.W. designed the experiments, conducted the experiments, and wrote the paper. N.H. and J.A.M. conducted the SILAC experiments. S.A.P. and F.M.H. constructed the recombinant Vps41 and designed the purification method. R.G.Y. conducted experiments using the phospho-specific antibodies. Y.P. conducted experiments testing the Yck3 deletion mutant. N.A. conducted experiments testing the Vps39 deletion mutant. A.H. conducted experiments testing Vps41 binding to Vac17. N.H. and M.S. conducted experiments with the Vps41 deletion mutant. L.S.W. designed the experiments and wrote the paper.
PY - 2020/11/16
Y1 - 2020/11/16
N2 - Cellular function requires molecular motors to transport cargoes to their correct intracellular locations. The regulated assembly and disassembly of motor-adaptor complexes ensures that cargoes are loaded at their origin and unloaded at their destination. In Saccharomyces cerevisiae, early in the cell cycle, a portion of the vacuole is transported into the emerging bud. This transport requires a myosin V motor, Myo2, which attaches to the vacuole via Vac17, the vacuole-specific adaptor protein. Vac17 also binds to Vac8, a vacuolar membrane protein. Once the vacuole is brought to the bud cortex via the Myo2-Vac17-Vac8 complex, Vac17 is degraded and the vacuole is released from Myo2. However, mechanisms governing dissociation of the Myo2-Vac17-Vac8 complex are not well understood. Ubiquitylation of the Vac17 adaptor at the bud cortex provides spatial regulation of vacuole release. Here, we report that ubiquitylation alone is not sufficient for cargo release. We find that a parallel pathway, which initiates on the vacuole, converges with ubiquitylation to release the vacuole from Myo2. Specifically, we show that Yck3 and Vps41, independent of their known roles in homotypic fusion and protein sorting (HOPS)-mediated vesicle tethering, are required for the phosphorylation of Vac17 in its Myo2 binding domain. These phosphorylation events allow ubiquitylated Vac17 to be released from Myo2 and Vac8. Our data suggest that Vps41 is regulating the phosphorylation of Vac17 via Yck3, a casein kinase I, and likely another unknown kinase. That parallel pathways are required to release the vacuole from Myo2 suggests that multiple signals are integrated to terminate organelle inheritance.
AB - Cellular function requires molecular motors to transport cargoes to their correct intracellular locations. The regulated assembly and disassembly of motor-adaptor complexes ensures that cargoes are loaded at their origin and unloaded at their destination. In Saccharomyces cerevisiae, early in the cell cycle, a portion of the vacuole is transported into the emerging bud. This transport requires a myosin V motor, Myo2, which attaches to the vacuole via Vac17, the vacuole-specific adaptor protein. Vac17 also binds to Vac8, a vacuolar membrane protein. Once the vacuole is brought to the bud cortex via the Myo2-Vac17-Vac8 complex, Vac17 is degraded and the vacuole is released from Myo2. However, mechanisms governing dissociation of the Myo2-Vac17-Vac8 complex are not well understood. Ubiquitylation of the Vac17 adaptor at the bud cortex provides spatial regulation of vacuole release. Here, we report that ubiquitylation alone is not sufficient for cargo release. We find that a parallel pathway, which initiates on the vacuole, converges with ubiquitylation to release the vacuole from Myo2. Specifically, we show that Yck3 and Vps41, independent of their known roles in homotypic fusion and protein sorting (HOPS)-mediated vesicle tethering, are required for the phosphorylation of Vac17 in its Myo2 binding domain. These phosphorylation events allow ubiquitylated Vac17 to be released from Myo2 and Vac8. Our data suggest that Vps41 is regulating the phosphorylation of Vac17 via Yck3, a casein kinase I, and likely another unknown kinase. That parallel pathways are required to release the vacuole from Myo2 suggests that multiple signals are integrated to terminate organelle inheritance.
UR - http://www.scopus.com/inward/record.url?scp=85092615583&partnerID=8YFLogxK
U2 - 10.1016/j.cub.2020.08.062
DO - 10.1016/j.cub.2020.08.062
M3 - مقالة
C2 - 32916113
SN - 0960-9822
VL - 30
SP - 4399
EP - 4412
JO - Current Biology
JF - Current Biology
IS - 22
ER -