The yeast inositol polyphosphate 5-phosphatases Inp52p and Inp53p translocate to actin patches following hyperosmotic stress: Mechanism for regulating phosphatidylinositol 4,5-bisphosphate at plasma membrane invaginations

The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sad domain, followed by a 5 -phosphatase domain and a C-terminal proline-rich domain, Disruption of any two of these 5-phosphatases results in abnormal vacuolar and plasma membra ne morphology, We have cloned and characterized the Sad-containing 5-phosph atases Inp52p and Inp53p, Purified recombinant Inp52p lacking the Sad domai n hydrolyzed phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2] and Ptd Ins(3,5)P-2. Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP), In resting cells recombinan t GFP-tagged 5-phosphatases we re expressed diffusely throughout the cell b ut were excluded from the nucleus, Following hyperosmotic stress the GFP-ta gged 5-phosphatases rapidly and transiently associated viith actin patches, independent of actin, in both the mother and daughter cells of budding yea st as demonstrated by colocalization with rhodamine phalloidin. Both the Sa d domain and proline-rich domains were able to independently mediate transl ocation of Inp52p to actin patches, following hyperosmotic stress, while th e Inp53p proline-rich domain alone was sufficient for stress-mediated local ization, Overexpression of Inp52p or Inp53p, but not catalytically inactive Inp52p, which lacked PtdIns(4,5)P-2 5-phosphatase activity, resulted in a dramatic reduction in the repolarization time of actin patches following hy perosmotic stress, We propose that the osmotic-stress-induced translocation of Inp52p and Inp53p results in the localized regulation of PtdIns(3,5)P-2 and PtdIns(4,5)P-2 at actin patches and associated plasma membrane invagin ations, This may provide a mechanism for regulating actin polymerization an d cell growth as an acute adaptive response to hyperosmotic stress.