A SINGLE-GENE MAY ENCODE DIFFERENTIALLY LOCALIZED CA2-ATPASES IN TOMATO()

Previously, a partial-length cDNA and a complete genomic clone encodin g a putative sarcoplasmic reticulum-type Ca2+-ATPase (LCA, Lycopersico n Ca2+-ATPase)were isolated from tomato. To determine the subcellular localization of this Ca2+-ATPase, specific polyclonal antibodies raise d against a fusion protein encoding a portion of the LCA polypeptide w ere generated. Based on hybridization of the LCA cDNA and of the nucle otide sequence encoding the fusion protein to genomic DNA, it appears that LCA and the fusion protein domain are encoded by a single gene in tomato. Antibodies raised against the LCA domain fusion protein react ed specifically with two polypeptides of 116 and 120 kD that are local ized in the vacuolar and plasma membranes, respectively. The distribut ion of vanadate-sensitive ATP-dependent Ca2+ transport activities in s ucrose gradients coincided with the distribution of the immunodetected proteins. The ATP-dependent Ca2+ transport activities associated with tonoplast and plasma membrane fractions shared similar properties, be cause both fractions were inhibited by vanadate but insensitive to car bonyl cyanide m-chlorophenylhydrazone, nitrate, and calmodulin. Moreov er, antibodies raised against the LCA domain fusion protein inhibited ATP-dependent Ca2+ uptake activity associated with both the tonoplast and plasma membrane fractions. These data suggest that a single gene ( LCA) may encode two P-type Ca2+-ATPase isoforms that are differentiall y localized in the tonoplast and plasma membrane of tomato roots.