Jcb_201411010 1..7

The spatial organization of eukaryotic cells depends on a regulated cytoskeleton and motor proteins that use it as tracks for cargo transport and delivery. The major motor proteins implicated in cargo transport are the kinesins and dyneins that use microtubule tracks and unconventional myosins, most notably myosin Vs, which use actin filaments. The delivery cycle of transporting motors must be precisely regulated both spatially and temporally to allow for proper cargo capture and dissociation. Here we examine the in vivo consequences of defects in myosin V motor regulation. Myosin Vs are highly conserved motor proteins that transport specific cargoes in fungal, plant (called myosin XI), and animal cells (Hammer and Sellers, 2012). In mammalian cells, myosin V motors have been implicated in transporting melanosomes (Wu et al., 1998), recycling endosomes (Lapierre et al., 2001), and the endoplasmic reticulum (Wagner et al., 2011). To perform these functions, all myosin V motors consist of two heavy chains with N-terminal motors dimerizing along a coiledcoil stalk, six associated light chains decorating each forcetransducing lever arm, and two cargo-binding globular tail domains at the C terminus (Hammer and Sellers, 2012). Biochemical and structural data has suggested that mammalian myosin Vs undergo an autoinhibitory interaction between the head domain and globular tail domain. The existence of the folded inhibited state of mammalian myosin V is supported by sedimentation analysis (Krementsov et al., 2004; Li et al., 2004; Wang et al., 2004), cryoelectron microscopy reconstruction (Liu et al., 2006; Thirumurugan et al., 2006), and ATPase activity assays (Li et al., 2008). Activation of the motor occurs after cargo receptor binding for MyoVa in vitro (Li et al., 2005; Sckolnick et al., 2013), as is the case for yeast myosin V in vivo (Donovan and Bretscher, 2012). Notably, the residues predicted to form the ionic bond between domains have been identified (Li et al., 2008; Nascimento et al., 2013). These include mouse MyoVa residues D121/D134/D136 in the head domain and K1706/K1779 in the tail domain. However, despite a decade of work characterizing this interaction biochemically, to our knowledge no one has defined how misregulation of myosin V affects motor functions such as cargo capture and recycling in vivo. Budding yeast is an especially amenable system in which to probe the function of myosin V motors because of the absence of microtubule-based transport systems (Pruyne et al., 2004). In addition, spatially segregated areas of cargo capture in the mother cell and delivery to the daughter cell enable easy phenotypic analysis. The budding yeast myosin V motor Myo2p is responsible for transporting a wide array of organelles and Cell organization requires regulated cargo transport along cytoskeletal elements. Myosin V motors are among the most conserved organelle motors and have been well characterized in both yeast and mammalian systems. Biochemical data for mammalian myosin V suggest that a head-to-tail autoinhibitory interaction is a primary means of regulation, but the in vivo significance of this interaction has not been studied. Here we generated and characterized mutations in the yeast myosin V Myo2p to reveal that it is regulated by a head-to-tail interaction and that loss of regulation renders the myosin V constitutively active. We show that an unregulated motor is very deleterious for growth, resulting in severe defects in Myo2mediated transport processes, including secretory vesicle transport, mitochondrial inheritance, and nuclear orientation. All of the defects associated with motor misregulation could be rescued by artificially restoring regulation. Thus, spatial and temporal regulation of myosin V in vivo by a head-to-tail interaction is critical for the normal delivery functions of the motor. Head-to-tail regulation is critical for the in vivo function of myosin V