The precise control of intracellular free Ca2+-concentration ([Ca2+]i) is a prerequisite for the function of Ca2+ as a second messenger in cells. Ca2+-ATPases of sarco (endo) plasmic reticulum (SERCAs) and plasma membranes (PMCA) play crucial roles in this control and maintenance of the [Ca2+]i. A cDNA that seems to encode a third distinct class, secretory pathway Ca2+-ATPases {SPCA} has been identified and mammalian Golgi and brain synaptic vesicles contain a Ca2+-ATPase with distinct characteristics from those of pMCAs and SERCAs. We propose to obtain a detailed understanding of the functional integrity and genetic diversity of SPCA by expressing the enzyme transiently and stably in mammalian tissue culture cells and comparing its properties with those of the Ca2+-pump of the brain synaptic vesicles and Golgi membranes in secretory cells. The corresponding cDNAs of genes encoding any additional SPCA isoforms that may exist in mammalian genome will be isolated and characterized by utilizing PCR, northern and Southern blot techniques and the tissue distribution of their mRNAs will be assessed. These will provide insight into the role of intravesicular Ca2+ in the control of membrane budding and fusion during vesicle trafficking and hence Information regarding diseases that result from ineffective functioning of secretory tissues.
