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

Abstract

The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sac1 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 membrane morphology. We have cloned and characterized the Sac1-containing 5-phosphatases Inp52p and Inp53p. Purified recombinant Inp52p lacking the Sac1 domain hydrolyzed phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] and PtdIns(3,5)P 2 . Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP). In resting cells recombinant GFP-tagged 5-phosphatases were expressed diffusely throughout the cell but were excluded from the nucleus. Following hyperosmotic stress the GFPtagged 5-phosphatases rapidly and transiently associated with actin patches, independent of actin, in both the mother and daughter cells of budding yeast as demonstrated by colocalization with rhodamine phalloidin. Both the Sac1 domain and proline-rich domains were able to independently mediate translocation of Inp52p to actin patches, following hyperosmotic stress, while the Inp53p proline-rich domain alone was sufficient for stressmediated localization. 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 hyperosmotic 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 invaginations. This may provide a mechanism for regulating actin polymerization and cell growth as an acute adaptive response to hyperosmotic stress.The actin cytoskeleton plays a fundamental role in regulating cytokinesis and organelle transport. In the budding yeast Saccharomyces cerevisiae genetic and morphological evidence indicates that actin regulates cell growth. In yeast filamentous actin is found in two morphologically identified forms, cables and patches (1, 24). Actin cables are found mainly in the mother cell and extend along the axis of growth, which is asymmetrical to the emerging daughter cell. Cables are involved in regulating organelle inheritance and vesicle targeting. Actin patches are associated with plasma membrane invaginations and are motile structures that move along the plasma membrane in response to osmotic stress (6, 33, 47). It has been speculated that actin patches may be necessary machinery for maintaining secretion or endocytosis. Actin cables and patches may form part of an integrated system, as their distributions often change simultaneously (23). However, the signaling mechanisms regulating the assembly and movement of actin cables and patches in response to osmotic and other stimuli are not well understood.The phosphoinositides are ubiquitous components of eukaryotic membranes and are critic...

The Yeast Inositol Polyphosphate 5-Phosphatase Inp54p Localizes to the Endoplasmic Reticulum via a C-terminal Hydrophobic Anchoring Tail

“…However, this has yet to be shown. Recently our laboratory has demonstrated that Inp52p and Inp53p are localized diffusely throughout the cell excluding the nucleus and translocate to cortical actin patches following hyperosmotic stress (21). The intracellular localization of Inp51p has not been delineated, as the enzyme is extensively proteolyzed when expressed as a recombinant protein (21,55).…”

“…Transformants were selected by their ability to grow on complete minimal media lacking leucine, and were subsequently screened for homologous integration of the LEU2 marker by PCR. Null mutations of the SacI domain-containing 5-phosphatases has been described previously (21).…”

“…The PCR product was cloned into the BamHI site of pRS416-GFP (21) in-frame with the C-terminal GFP. The construct was then transformed into an inp54 null mutant strain using the S. cerevisiae EasyComp Transformation kit (Invitrogen) and the resulting transformants selected on complete minimal media lacking uracil.…”

Mechanosensitivity of GIRK Channels Is Mediated by Protein Kinase C-dependent Channel-Phosphatidylinositol 4,5-Bisphosphate Interaction

“…In this case, PKC will make a positive-feedback circuit by enhancing the PIP 2 hydrolysis, and play a role of signal amplifier. PKC activation can also reduce the membrane PIP 2 content by regulation of the phosphatidylinositol transfer protein (35), and regulation of activity and location of lipid phosphatases and/or lipid kinases (21,22). The molecular identity of lipid kinases and phosphatases regulating the PIP 2 metabolism remain controversial.…”

Phosphoinositides in Constitutive Membrane Traffic

“…INS51 has genetic interactions with PAN1, a protein required for endocytosis and regulation of actin (380). Inp52p and Inp53p have both 5-phosphatase activity as well as Sac domain phosphatase activity and are involved in regulating actin patches at the plasma membrane (PM) (260,338). Disruption of INP53, but not INP51 or INP52, causes sorting defects at the TGN and increases the rate of transport of reporter proteins to the vacuole (19,125).…”