Hansenula Polymorpha Vac8: A Vacuolar Membrane Protein Required for Vacuole Inheritance and Nucleus-Vacuole Junction Formation

Saccharomyces cerevisiae Vac8 is a vacuolar membrane protein, which functions in vacuole inheritance and fusion, nucleus-vacuole junctions, autophagy and the cytoplasm-to-vacuole-targeting pathway. Here, we analyzed Vac8 of the yeast Hansenula polymorpha. We show that HpVac8 localizes to the vacuolar membrane and concentrates in patches at nucleus-vacuole junctions. Analysis of a VAC8 deletion strain indicated that HpVac8 is required for vacuole inheritance and the formation of nuclear-vacuole junctions, but not for vacuole fusion. Previously, organelle proteomics resulted in the identification of Vac8 in peroxisomal fractions isolated from H. polymorpha and S. cerevisiae. However, deletion of H. polymorpha VAC8 had no effect on peroxisome biogenesis or peroxisome-vacuole contact sites.

PTC1 Is Required for Vacuole Inheritance and Promotes the Association of the Myosin-V Vacuole-specific Receptor Complex

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 ptc1Δ 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.

p21-Activated Kinases Cla4 and Ste20 Regulate Vacuole Inheritance in Saccharomyces cerevisiae

ABSTRACT Each time Saccharomyces cerevisiae cells divide they ensure that both the mother and daughter cell inherit a vacuole by actively transporting a portion of the vacuole into the bud. As the mother cell begins budding, a tubular and vesicular segregation structure forms that is transported into the bud by the myosin V motor Myo2, which is bound to the vacuole-specific myosin receptor, Vac17 (41, 59, 70, 79). Upon arriving in the bud the segregation structure is resolved to found the daughter vacuole. The mechanism that regulates segregation structure resolution in a spatially dependent manner is unknown. In addition to resolving the segregation structure, Vac17 is degraded specifically in the bud to provide directionality to vacuole inheritance. It has been proposed that bud-specific degradation of Vac17 is promoted by proteins localized to or activated solely in the bud (77). The p21-activated kinases (PAKs) Cla4 and Ste20 are localized to and activated in the bud. Here we report that Cla4 is localized to the segregation structure just prior to segregation structure resolution, and cells lacking PAK function fail to resolve the segregation structure. Overexpression of either Cla4 or Ste20 inhibited vacuole inheritance and this inhibition was suppressed by the expression of nondegradable VAC17. Finally, PAK activity was required for Vac17 degradation in late M phase and CLA4 overexpression promoted Vac17 degradation. We propose that Cla4 and Ste20 are bud-specific proteins that play roles in both segregation structure resolution and the degradation of Vac17.