PTC1Is Required for Vacuole Inheritance and Promotes the Association of the Myosin-V Vacuole-specific Receptor Complex

Abstract

Organelle inheritance occurs during cell division. In Saccharomyces cerevisiae, inheritance of the vacuole, and the distribution of mitochondria and cortical endoplasmic reticulum are regulated by Ptc1p, a type 2C protein phosphatase. Here we show that PTC1/VAC10 controls the distribution of additional cargoes moved by a myosin-V motor. These include peroxisomes, secretory vesicles, cargoes of Myo2p, and ASH1 mRNA, a cargo of Myo4p. We find that Ptc1p is required for the proper distribution of both Myo2p and Myo4p. Surprisingly, PTC1 is also required to maintain the steady-state levels of organelle-specific receptors, including Vac17p, Inp2p, and Mmr1p, which attach Myo2p to the vacuole, peroxisomes, and mitochondria, respectively. Furthermore, Vac17p fused to the cargo-binding domain of Myo2p suppressed the vacuole inheritance defect in ptc1⌬ cells. These findings suggest that PTC1 promotes the association of myosin-V with its organelle-specific adaptor proteins. Moreover, these observations suggest that despite the existence of organelle-specific receptors, there is a higher order regulation that coordinates the movement of diverse cellular components. INTRODUCTIONDuring each cell cycle, cytoplasmic organelles are actively distributed between dividing cells to maintain organelle copy number and volume. The yeast Saccharomyces cerevisiae is an excellent model system for studying the spatial and temporal control of organelle inheritance. In yeast, several organelles are transmitted from mother to daughter cells. These include the vacuole, mitochondria, the endoplasmic reticulum (ER), late-Golgi elements and peroxisomes (reviewed in Weisman, 2006).Early in the cell cycle, a portion of each organelle is transported into the emerging bud. The polarized transport of most organelles from the mother to the bud is an active process that requires the actin cytoskeleton, myosin-V motors, and receptor proteins, which physically connect the motor to organelle cargoes (Beach et al., 2000;Yin et al., 2000;Boldogh et al., 2001;Barr, 2002;Bretscher, 2003;Du et al., 2004;Fagarasanu et al., 2006bFagarasanu et al., , 2007Weisman, 2006). Thus, formation of a complex between the motor and receptor protein is important for polarized organelle transport.Similar to yeast, in vertebrates, myosin-V motors move cargoes along the actin cytoskeleton. The best studied cargo of vertebrate myosin-V are melanosomes, which are moved in melanocytes by myosin-Va. Melanosomes attach to myosin-Va through Rab27a and melanophilin (Fukuda and Kuroda, 2002;Wu et al., 2002). Rab27a, through geranylgeranylation, attaches to the melanosome membrane, and melanophilin connects myosin-Va and Rab27a. Myosin-V-based intracellular movement has been analyzed in many other eukaryotes, including frogs, fish, mammals, and plants; plant myosin-XI is the functional homologue of yeast and vertebrate myosin-V ( Kinkema and Schiefelbein, 1994;Provance and Mercer, 1999;Tuma and Gelfand, 1999;Desnos et al., 2007;Li and Nebenfuhr, 2007;Sheets et al., 2007;Shimmen, 2007).Ge...

A constraint network of interactions: protein–protein interaction analysis of the yeast type II phosphatase Ptc1p and its adaptor protein Nbp2p

“…Overexpression of Cla4p as well as deletion of PTC1 delays the delivery of vacuolar vesicles to the daughter cell (Bartholomew and Hardy, 2009;Jin et al, 2009). Our proof of an Nbp2p-mediated molecular link between Ptc1p and Cla4p provides a satisfying explanation for both observations.…”

“…Deletion of Ptc1p or Nbp2p leads to similar phenotypes (Fig. 6F) (Du et al, 2006;Jin et al, 2009;Roeder et al, 1998). Interrupting the link between Nbp2p and the PxxP kinases might therefore act as switch to slow down organelle migration to the daughter cell at the end of each cell cycle.…”

“…Owing to its interaction-induced proximity, Ptc1p might dephosphorylate either the Nbp2-bound kinases directly or their downstream targets to reduce the activities of the corresponding pathways (Fig. 8A,B) (Du et al, 2006;Jin et al, 2009;Lesage et al, 2004;Mapes and Ota, 2004;Ohkuni et al, 2003;Roeder et al, 1998). Thus, disruption of the link between Ptc1p and Nbp2p would activate all seven kinases at once.…”

“…Loss of either gene leads to a variety of phenotypes including osmo-sensitivity, decreased cell wall integrity, temperature sensitivity, the delay of organelle inheritance, and an increased sensitivity towards rapamycin and other stresses or chemicals (Du et al, 2006;Jin et al, 2009;Lesage et al, 2004;Mapes and Ota, 2004;Ohkuni et al, 2003;Roeder et al, 1998). This multifunctionality correlates with several genetic interactions that places both genes as hubs into similar but non-identical genetic interaction networks (Fiedler et al, 2009;Lesage et al, 2004;Lin et al, 2008;Pan et al, 2006;Tong et al, 2004).…”

Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1

“…These effects are independent of Hog1 dephosphorylation (Roeder et al 1998;Du et al 2006;Jin et al 2009). Moreover, lack of Ptc1 results in additional phenotypic traits, such as defects in transfer RNA (tRNA) splicing and growth in nonfermentable media (Robinson et al 1994) and, in haploid strains, cell separation defects and a random budding pattern at 37° ( Gonzalez et al 2006).…”