THE ANNEXINS OF C. ELEGANS: A NOVEL SPLICE VARIANT MAY BE SUBJECT TO CONTROL BY PHOSPHORYLATION C.E Creutz. S.E Snyder. and S N. Daiele Dept. of Pharmacology, University of Virginia, Charlottesville, VA 22908 The annexins are calcium-dependent, phospholipid binding proteins that may function in exocytosis, membrane structure and permeation, and signal transduction. In order to establish a system for genetic analysis of their hypothetical functions, we have been characterizing the annexins of the nematode, C. elegans. Annexins were isolated from postmicrosomal supernatants by calcium dependent binding to lipid vesicles. EGTA extracts of these vesicles appear to contain primarily a single protein of mass 32kDa when examined by SDS-PAGE. Peptides derived from this protein were sequenced and verified that the worm protein is an annexin. An antiserum was raised to the worm annexin and was found to react almost exclusively with a 32kDa band in Western blots of worm homogenates. The antiserum was used to isolate cDNAs for the protein from a lambda gtll library (kindly provided by A, Fire). The sequence of the worm annexin is 42Z identical to that of bovine annexin IV, the closest vertebrate homolog. The single annexin gene was physically mapped to the vicinity of
dpy-17 in LGIII. One of three cDNA clones isolated contained a 15 base splice insertion encoding five amino acids near the N-terminus. This insertion contained THR and TYR residues that align exactly with phosphorylation sites in mammalian annexins for protein kinase C and the src kinase.