Tissue Distribution and Intracellular Localisation of the 75‐kDa Inositol Polyphosphate 5‐Phosphatase

Abstract

The 75-kDa inositol polyphosphate 5-phosphatase (75-kDa 5-phosphatase) hydrolyses several important mediators of intracellular calcium homeostasis, including inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Northern analysis of various human tissues revealed the 75-kDa 5-phosphatase has a ubiquitous expression, where differential splicing may occur in specific tissues. Prominent expression of a 4.4-kb transcript was noted in human lung, thymus, testes and placenta, and a 4.6-kb transcript was observed in heart, brain, kidney, ovary and colon. Determination of the intracellular location of the enzyme by indirect immunofluorescence, demonstrated that the 75-kDa 5-phosphatase was associated with mitochondrial and cytosolic cellular compartments. Immunoprecipitation of the total cell homogenate of human lung carcinoma cells (A549) with anti-(recombinant 75-kDa 5-phosphatase) antibodies revealed that the 75-kDa 5-phosphatase is the major PtdIns(4,5)P2 5-phosphatase in this cell line. Analysis of PtdIns(4,5)P2 5-phosphatase activity in subcellular fractions of A549 cells revealed peak 75-kDa 5-phosphatase enzyme activity in the cytosolic and mitochondrial enriched fractions. Immunoblot analysis further confirmed the mitochondrial location of the enzyme. This study demonstrates the tissue distribution and intracellular location of the 75-kDa 5-phosphatase and reveals a novel location for an enzyme involved in phosphatidylinositol turnover.