Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identi fied in plants. In contrast to the most closely related pumps in animals, p lasma membrane-type Ca2+-ATPases, members of this new subfamily are disting uished by a calmodulin-regulated autoinhibitor located at the N-terminal in stead of a C-terminal end. In addition, at least some isoforms appear to re side in non-plasma membrane locations. To begin delineating their functions , we investigated the subcellular localization of isoform ACA2p (Arabidopsi s Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density su crose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluor escence protein at its C terminus (ACA2-GFPp) and expressed in transgenic A rabidopsis. We collected fluorescence images from live root cells using con focal and computational optical-sectioning microscopy. ACA2-GFPp appeared a s a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, w hich is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmod ulin-regulated pumps identified in plants or animals.