Identification of an organelle-specific myosin V receptor

Class V myosins are widely distributed among diverse organisms and move cargo along actin filaments. Some myosin Vs move multiple types of cargo, where the timing of movement and the destinations of selected cargoes are unique. Here, we report the discovery of an organelle-specific myosin V receptor. Vac17p, a novel protein, is a component of the vacuole-specific receptor for Myo2p, a Saccharomyces cerevisiae myosin V. Vac17p interacts with the Myo2p cargo-binding domain, but not with vacuole inheritance-defective myo2 mutants that have single amino acid changes within this region. Moreover, a region of the Myo2p tail required specifically for secretory vesicle transport is neither required for vacuole inheritance nor for Vac17p–Myo2p interactions. Vac17p is localized on the vacuole membrane, and vacuole-associated Myo2p increases in proportion with an increase in Vac17p. Furthermore, Vac17p is not required for movement of other cargo moved by Myo2p. These findings demonstrate that Vac17p is a component of a vacuole-specific receptor for Myo2p. Organelle-specific receptors such as Vac17p provide a mechanism whereby a single type of myosin V can move diverse cargoes to distinct destinations at different times

Osmotic stress–induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p

Phosphatidylinositol 3,5-bisphosphate (PtdIns[3,5]P2) was first identified as a nonabundant phospholipid whose levels increase in response to osmotic stress. In yeast, Fab1p catalyzes formation of PtdIns(3,5)P2 via phosphorylation of PtdIns(3)P. We have identified Vac14p, a novel vacuolar protein that regulates PtdIns(3,5)P2 synthesis by modulating Fab1p activity in both the absence and presence of osmotic stress. We find that PtdIns(3)P levels are also elevated in response to osmotic stress, yet, only the elevation of PtdIns(3,5)P2 levels are regulated by Vac14p. Under basal conditions the levels of PtdIns(3,5)P2 are 18–28-fold lower than the levels of PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P2. After a 10 min exposure to hyperosmotic stress the levels of PtdIns(3,5)P2 rise 20-fold, bringing it to a cellular concentration that is similar to the other phosphoinositides. This suggests that PtdIns(3,5)P2 plays a major role in osmotic stress, perhaps via regulation of vacuolar volume. In fact, during hyperosmotic stress the vacuole morphology of wild-type cells changes dramatically, to smaller, more highly fragmented vacuoles, whereas mutants unable to synthesize PtdIns(3,5)P2 continue to maintain a single large vacuole. These findings demonstrate that Vac14p regulates the levels of PtdIns(3,5)P2 and provide insight into why PtdIns(3,5)P2 levels rise in response to osmotic stress

Vac8p, an Armadillo Repeat Protein, Coordinates Vacuole Inheritance With Multiple Vacuolar Processes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74978/1/j.1600-0854.2006.00458.x.pd

Phosphoinositide 5-Phosphatase Fig4p Is Required for both Acute Rise and Subsequent Fall in Stress-Induced Phosphatidylinositol 3,5-Bisphosphate Levels

Phosphoinositide lipids regulate complex events via the recruitment of proteins to a specialized region of the membrane at a specific time. Precise control of both the synthesis and turnover of phosphoinositide lipids is integral to membrane trafficking, signal transduction, and cytoskeletal rearrangements. Little is known about the acute regulation of the levels of these signaling lipids. When Saccharomyces cerevisiae cells are treated with hyperosmotic medium the levels of phosphatidylinositol 3,5-bisphosphate (PI3,5P(2)) increase 20-fold. Here we show that this 20-fold increase is rapid and occurs within 5 min. Surprisingly, these elevated levels are transient. Fifteen minutes following hyperosmotic shock they decrease at a rapid rate, even though the cells remain in hyperosmotic medium. In parallel with the rapid increase in the levels of PI3,5P(2), vacuole volume decreases rapidly. Furthermore, concomitant with a return to basal levels of PI3,5P(2) vacuole volume is restored. We show that Fig4p, consistent with its proposed role as a PI3,5P(2) 5-phosphatase, is required in vivo for this rapid return to basal levels of PI3,5P(2). Surprisingly, we find that Fig4p is also required for the hyperosmotic shock-induced increase in PI3,5P(2) levels. These findings demonstrate that following hyperosmotic shock, large, transient changes occur in the levels of PI3,5P(2) and further suggest that Fig4p is important in regulating both the acute rise and subsequent fall in PI3,5P(2) levels

Apg13p and Vac8p Are Part of a Complex of Phosphoproteins That Are Required for Cytoplasm to Vacuole Targeting

We have been studying protein components that function in the cytoplasm to vacuole targeting (Cvt) pathway and the overlapping process of macroautophagy. The Vac8 and Apg13 proteins are required for the import of aminopeptidase I (API) through the Cvt pathway. We have identified a protein-protein interaction between Vac8p and Apg13p by both two-hybrid and co-immunoprecipitation analysis. Subcellular fractionation of API indicates that Vac8p and Apg13p are involved in the vesicle formation step of the Cvt pathway. Kinetic analysis of the Cvt pathway and autophagy indicates that, although Vac8p is essential for Cvt transport, it is less important for autophagy. In vivo phosphorylation experiments demonstrate that both Vac8p and Apg13p are phosphorylated proteins, and Apg13p phosphorylation is regulated by changing nutrient conditions. Although Apg13p interacts with the serine/threonine kinase Apg1p, this protein is not required for phosphorylation of either Vac8p or Apg13p. Subcellular fractionation experiments indicate that Apg13p and a fraction of Apg1p are membrane-associated. Vac8p and Apg13p may be part of a larger protein complex that includes Apg1p and additional interacting proteins. Together, these components may form a protein complex that regulates the conversion between Cvt transport and autophagy in response to changing nutrient conditions.