JCB_201607020 1557..1566

Myosin V motors transport organelles to their correct intracellular locations. Transport initiates when myosin V binds cargo-specific adapters and attaches to organelles (Weisman, 2006; Westermann, 2014; Knoblach and Rachubinski, 2016). Upon arrival at their correct locations, organelles detach from myosin V, thereby terminating transport. The molecular mechanisms that signal the arrival of organelles at their correct locations remain unclear. At the beginning of the cell cycle in Saccharomyces cerevisiae, the myosin V motor Myo2 binds the vacuole adapter Vac17 and transports a portion of the mother vacuole into the bud (Ishikawa et al., 2003; Peng and Weisman, 2008; Eves et al., 2012). In the bud, the vacuole is released from Myo2, and Vac17 is degraded (Tang et al., 2003). Degradation of Vac17 requires phosphorylated Vac17-T240, which recruits the E3 ubiquitin ligase Dma1. Dma1 then ubiquitylates Vac17, targeting Vac17 for degradation by the proteasome (Yau et al., 2014). Late in the cell cycle, Myo2 transports secretory vesicles, but not the vacuole, to the mother-bud neck (Govindan et al., 1995; Pruyne et al., 1998; Schott et al., 1999). In mutants defective in the degradation of Vac17, the vacuole remains attached to Myo2 and is inappropriately transported to the mother-bud neck late in the cell cycle (Tang et al., 2003; Yau et al., 2014). In yeast, the p21-activated kinases (PAKs) Cla4 and Ste20 localize to the bud cortex and regulate several cell cycle–related processes, including actin cytoskeleton polarization, septin ring assembly, and cytokinesis (Cvrcková et al., 1995; Peter et al., 1996; Kumar et al., 2009; Boyce and Andrianopoulos, 2011). Moreover, it was proposed that PAKs indirectly regulate vacuole inheritance via Lte1, a Cla4-activated guanine nucleotide exchange factor (Bartholomew and Hardy, 2009). In this study, we report that PAKs directly phosphorylate Vac17 to signal the arrival of the vacuole at its correct location. We show that before the termination of vacuole inheritance, the vacuole extends to the bud cortex, where Vac17 colocalizes with Cla4. In contrast to a previous study, we find that neither degradation of Vac17 nor termination of vacuole transport require Lte1 (Bartholomew and Hardy, 2009). Instead, Cla4 directly phosphorylates Vac17-S222. This phosphorylation event is required for Dma1-dependent ubiquitylation and degradation of Vac17 as well as the release of the vacuole from Myo2. Moreover, in a Vac17 mutant that cannot be phosphorylated at S222, the vacuole remains attached to the bud cortex, which suggests that this is the intracellular location where Cla4 phosphorylates Vac17. Collectively, these studies suggest that at the bud cortex, Cla4 initiates a signaling cascade to regulate the ubiquitylation of Vac17 and complete the termination of vacuole transport, thereby ensuring that the vacuole is deposited at the correct intracellular location. Correct positioning of organelles is essential to eukaryotic cells. Molecular motors transport organelles to their proper destinations, yet little is known about the pathways that define these destinations. In Saccharomyces cerevisiae, the myosin V motor Myo2 binds the vacuole-specific adapter Vac17 to attach to the vacuole/lysosome and initiate transport. After arrival in the bud, Myo2 releases the vacuole, and Vac17 is degraded. However, the mechanisms that spatially regulate this release were not established. In this study, we report that the bud cortex is a landmark that signals a successful delivery of the vacuole to the bud. We demonstrate that upon arrival at the bud cortex, Vac17 is phosphorylated by Cla4. Cla4-dependent phosphorylation is required for the ubiquitylation and subsequent degradation of Vac17 and the release of the vacuole from Myo2. Our study reveals a critical step in the spatial regulation of myosin V–dependent organelle transport and may reveal common mechanisms for how molecular motors accurately deposit cargoes at the correct locations. Spatial regulation of organelle release from myosin V transport by p21-activated kinases