Protein phosphorylation and membrane association of sucrose synthase in developing tomato fruit

Abstract

Calcium-dependent protein kinase (CDPK) activities were detected both in the soluble and the membrane fraction of various tomato (Lycopersicon esculentum Mill.) organs, using a synthetic peptide mimicking the serine 11 phosphorylation site of a tomato sucrose synthase (SS, EC 2.4.1.13) isoform as substrate. The levels of membrane and soluble Ser-CDPK activities were differentially regulated during fruit development. The membrane Ser-CDPK activity was maximal in young fruit but decreased as the fruit developed, suggesting a specific role during fruit growth. Using an in gel assay with purified tomato SS as substrate, we showed that partially purified soluble and membrane Ser-CDPK preparations both contained a SS-kinase polypeptide of 55 kDa. The membrane and soluble Ser-CDPK activities were largely inactivated in the absence of calcium or when MgCl(2) was replaced by MnCl(2). Both soluble and membrane Ser-CDPK activities were very sensitive to staurosporine. Using Fe(III)-immobilized metal chromatography to determine the apparent phosphorylation status of the enzyme in vivo, we showed that soluble SS was largely dephosphorylated in fruits fed EGTA or staurosporine, compared to fruits fed water or sucrose. Moreover, the level of SS increased by about two-fold in the membrane fraction of fruits fed the Ser-CDPK inhibitors, compared to the control. The level of SS protein in the membrane and soluble fractions of tomato fruit was developmentally regulated, the membrane form being specifically detected in actively growing fruits. Together, our results suggest that a mechanism involving protein phosphorylation/dephosphorylation and/or calcium would in part control the association of SS isoforms with membranes in developing tomato fruit.