Targeted deletion of Minpp1 provides new insight into the activity of multiple inositol polyphosphate phosphatase in vivo

Multiple inositol polyphosphate phosphatase (Minpp1) metabolizes inositol 1 ,3,4,5,6-pentakisphosphate (InsP(5)) and inositol hexakisphosphate (InsP(6) ) with high affinity in vitro. However, Minpp1 is compartmentalized in the endoplasmic reticulum (ER) lumen, where access of enzyme to these predomina ntly cytosolic substrates in vivo has not previously been demonstrated. To gain insight into the physiological activity of Minpp1, Minpp1-deficient mi ce were generated by homologous recombination. Tissue extracts from Minpp1- deficient mice lacked detectable Minpp1 mRNA expression and Minpp1 enzyme a ctivity. Unexpectedly, Minpp1-deficient mice were viable, fertile, and with out obvious defects. Although Minpp1 expression is upregulated during chond rocyte hypertrophy, normal chondrocyte differentiation and bone development were observed in Minpp1-deficient mice. Biochemical analyses demonstrate t hat InsP(5) and InsP(6) are in vivo substrates for ER-based Minpp1, as leve ls of these polyphosphates in Minpp1-deficient embryonic fibroblasts were 3 0 to 45% higher than in wild-type cells. This increase was reversed by rein troducing exogenous Minpp1 into the ER. Thus, ER-based Minpp1 plays a signi ficant role in the maintenance of steady state levels of InsP(5) and InsP(6 ). These polyphosphates could be reduced below their natural levels by aber rant expression in the cytosol of a truncated Minpp1 lacking its ER-targeti ng N terminus. This was accompanied by slowed cellular proliferation, indic ating that maintenance of cellular InsP(5) and InsP(6) is essential to norm al cell growth. Yet, depletion of cellular inositol polyphosphates during e rythropoiesis emerges as an additional physiological activity of Minpp1; lo ss of this enzyme activity in erythrocytes from Minpp1-deficient mice was a ccompanied by upregulation of a novel, substitutive inositol polyphosphate phosphatase.