Subcellular localization of a high affinity binding site for D-myo-inositol 1,4,5-trisphosphate from Chenopodium rubrum

It is now generally accepted that a phosphoinositide cycle is involved in t he transduction of a variety of signals in plant cells. In animal cells, th e binding of D-myo-inositol 1,4,5-trisphosphate (InsP(3)) to a receptor loc ated on the endoplasmic reticulum (ER) triggers an efflux of calcium releas e from the ER. Sites that bind InsP(3) with high affinity and specificity h ave also been described in plant cells, but their precise intracellular loc ations have not been conclusively identified. Ln contrast to animal cells, it has been suggested that in plants the vacuole is the major intracellular store of calcium involved in signal induced calcium release. The aim of th is work was to determine the intracellular localization of InsP(3)-binding sites obtained from 3-week-old Chenopodium rubrum leaves. Microsomal membra nes were fractionated by sucrose density gradient centrifugation in the pre sence and absence of Mg2+ and alternatively by free-flow electrophoresis. A n ER-enriched fraction was also prepared. The following enzymes were employ ed as specific membrane markers: antimycin A-insensitive NADH-cytochrome c reductase for ER, cytochrome c oxidase for mitochondrial membrane, pyrophos phatase for tonoplast, and 1,3-beta-D-glucansynthase for plasma membrane. L n all membrane separations, InsP(3)-binding sites were concentrated in the fractions that were enriched with ER membranes. These data clearly demonstr ate that the previously characterized InsP(3)-binding site from C. rubrum i s localized on the ER. This finding supports previous suggestions of an alt ernative non-vacuolar InsP(3)-sensitive calcium store in plant cells.