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.

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.…”

“…Overexpression of Cla4p inhibits the movement of mitochondria, vacuole and peroxisomes, whereas overexpression of Slt2p delays ER inheritance (Fig. 6E) (Bartholomew and Hardy, 2009;Du et al, 2006). Deletion of Ptc1p or Nbp2p leads to similar phenotypes (Fig.…”

“…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. Loss of Ptc1p results in the mislocalization of the motor proteins responsible for the movements of organelles as well as the stability of adaptor proteins linking organelles to their respective motors (Bartholomew and Hardy, 2009;Jin et al, 2009). Rather than signalling via organelle-specific Ptc1p substrates, we propose a general influence of Ptc1p on these processes via associations with the kinases Bck1p, Ste20p and Cla4p.…”

“…Although Hog1p can be expected to phosphorylate many target proteins, most of the phenotypic characteristics of nbp2 or ptc1 yeast strains cannot be accounted for by the increased basal Hog1p activity alone. For example, the Nbp2p-Ptc1p-dependent delivery of the vacuole and ER to daughter cells depends on the kinases Cla4p and Slt2p, respectively, and the increased sensitivity to cell wall stress was genetically traced to the MAPKK Mkk1p/2p (Bartholomew and Hardy, 2009;Du et al, 2006;Ohkuni et al, 2003).…”

Sharing the cell's bounty – organelle inheritance in yeast

“…Direct phosphorylation of Vac17p by Cdk1p has been implicated in the increased affinity of Vac17p for Myo2p (Peng and Weisman, 2008). The p21-activated kinases (PAKs) Cla4p and Ste20p have been implicated in terminating vacuole inheritance (Bartholomew and Hardy, 2009), but whether phosphorylation of Vac17p is required for its degradation and whether Vac17p is a direct target of PAKs remained unclear. A recent study has shown that different stages of vacuole inheritance are controlled by distinct Vac17p phosphorylation events.…”