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

Abstract

Phosphoinositides are ubiquitous membrane components of various intracellular compartments, which regulate many diverse cellular functions including membrane trafficking events, secretion, actin cytoskeletal organization, cellular proliferation, and inhibition of apoptosis (reviewed in Refs. 1-4). Many of these functions are mediated by binding and recruiting signaling proteins which contain specific phosphoinositidebinding domains such as SH2 domains, pleckstrin homology domains, FYVE domains, C2 domains or polybasic domains, thereby localizing these effector proteins to specific membranes (reviewed in Refs. 3 and 4).Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P 2 ) 1 serves as a precursor to second messenger molecules such as inositol (1,4,5)-trisphosphate and phosphatidylinositol (3,4,5)-trisphosphate, but also independent of further modification regulates the actin cytoskeleton and membrane trafficking (1, 5). PtdIns(4,5)P 2 binds to actin-binding proteins such as profilin and gelsolin (6) and displaces capping proteins from actin filaments, allowing polymerization and formation of actin stress fibers (7-9). PtdIns(4,5)P 2 also plays a role in regulating vesicle budding and in the recruitment and activation of proteins involved in the coating of vesicles (2).Cellular levels of PtdIns(4,5)P 2 are regulated by a series of lipid phosphorylation and dephosphorylation reactions mediated by specific lipid kinases and phosphatases. Inositol polyphosphate 5-phosphatases (5-phosphatases) regulate cellular PtdIns(4,5)P 2 levels by hydrolyzing the 5-position phosphate from the inositol ring forming phosphatidylinositol 4-phosphate (PtdIns(4)P) (10, 11). The budding yeast Saccharomyces cerevisiae has four 5-phosphatase genes, INP51, INP52, INP53, and INP54. Inp51p, Inp52p, and Inp53p each comprise an N-terminal SacI domain, a central 5-phosphatase domain, and a C-terminal proline-rich region (12, 13). These enzymes share significant sequence homology with the mammalian homologue synaptojanin, which regulates the recycling of synaptic vesicles in nerve terminals (14). Synaptojanin, Inp52p, and Inp53p contain two catalytic domains, a central 5-phosphatase domain and an N-terminal SacI domain which hydrolyzes PtdIns(3,5)P 2 , PtdIns(4)P, and PtdIns(3)P forming PtdIns (15). Null mutation of any two SacI domain-containing 5-phosphatases results in plasma membrane invaginations and thickened cell walls, defects in polarization of the actin cytoskeleton, and impaired endocytosis (12, 13). However, double SacI domain-containing 5-phosphatase null mutants display normal secretion of invertase suggesting that Inp51p, Inp52p, and Inp53p do not play a role in regulating secretion (16). A triple SacI domain-containing 5-phosphatase null mutant is nonviable suggesting Inp54p cannot function to rescue the loss of these three 5-phosphatases.

Identification of a Novel Domain in Two Mammalian Inositol-polyphosphate 5-Phosphatases That Mediates Membrane Ruffle Localization

“…Yeast sac1 null mutants demonstrate accumulation of PtdIns(4)P, and trafficking from the ER to the Golgi is slowed (39). In a manner analogous to SKIP, the yeast PtdIns(4,5)P 2 5-phosphatase Inp54p localizes to the cytosolic surface of the ER, anchored to this site via its C terminus (43). Furthermore, inp54 null mutants demonstrate enhanced secretion of reporter proteins from the ER, consistent with the contention that PtdIns(4,5)P 2 may play a positive role in the regulation of secretory transport from this compartment (43).…”

“…In a manner analogous to SKIP, the yeast PtdIns(4,5)P 2 5-phosphatase Inp54p localizes to the cytosolic surface of the ER, anchored to this site via its C terminus (43). Furthermore, inp54 null mutants demonstrate enhanced secretion of reporter proteins from the ER, consistent with the contention that PtdIns(4,5)P 2 may play a positive role in the regulation of secretory transport from this compartment (43). The localization of SKIP to the ER may play a role analogous to Inp54p in regulating PtdIns(4,5)P 2 levels and thereby vesicular trafficking from this compartment.…”

Phosphoinositides in Constitutive Membrane Traffic

“…It localizes to the ER, and its deletion increases the rate of secretion of a reporter, indicating that it functions directly or indirectly in secretion (389). In mammals there are a number of other 5-phosphatases, most of which have been investigated for roles in signal transduction or apoptosis, but not membrane traffic.…”

“…The COPII coat responsible for membrane protein export from the ER preferentially binds to acidic phospholipids but does not show a preference for specific PIPs (221). Several PI kinases and phosphatases are located on the ER, but the exact role that they play in secretion, if any, is not known (224,389,392). Membrane traffic between the ER and Golgi is inhibited when PtdOH production by PLD is blocked (21,322), but it is not clear if this is an effect on the ER or the Golgi.…”

Compartmentalization of Phosphatidylinositol 4,5-Bisphosphate Signaling Evidenced Using Targeted Phosphatases

“…A useful approach to measuring PIP 2 functions in cells while avoiding chemical treatments is to alter membrane PIP 2 levels using membrane-targeted PIP 2 -specific phosphatases (1). One example has been targeting the catalytic domain of the yeast PIP 2 -specific phosphatase Inp54p to the plasma membrane using the membrane-anchoring signal of the Src family kinase Lyn (Lyn-Inp54p) (8,28). Expression of membrane-anchored Inp54p has demonstrated a role for PIP 2 in maintaining interactions between the plasma membrane and underlying actin cytoskeleton (8).…”