IDENTIFICATION, LOCALIZATION, AND FUNCTIONAL IMPLICATIONS OF AN ABUNDANT NEMATODE ANNEXIN

Cultures of the nematode C. elegans were examined for the presence of calcium-dependent, phospholipid-binding proteins of the annexin class. A single protein of apparent mass on SDS-polyacrylamide gels of 32 kD was isolated from soluble extracts of nematode cultures on the basis of its ability to bind to phospholipids in a calcium-dependent manner. After verification of the protein as an annexin by peptide sequencing , an antiserum to the protein was prepared and used to isolate a corre sponding cDNA from an expression library in phage lambda gt11. The enc oded protein, herein referred to as the nex-1 annexin, has a mass of 3 5 kD and is 36-42% identical in sequence to 10 known mammalian annexin s. Several unique modifications were found in the portions of the sequ ence corresponding to calcium-binding sites. Possible phosphorylation sites in the NH2-terminal domain of the nematode annexin correspond to those of mammalian annexins. The gene for this annexin (nex-1) was ph ysically mapped to chromosome III in the vicinity of the dpy-17 geneti c marker. Two other annexin genes (nex-2 and nex-3) were also identifi ed in chromosome III sequences reported by the nematode genomic sequen cing project (Sulston, J., Z. Du, K. Thomas, R. Wilson, L. Hillier, R. Staden, N. Halloran, P. Green, J. Thierry-Mieg, L. Qiu, et al. 1992. Nature (Lond.). 356:37-41). The nex-1 annexin was localized in the nem atode by immunofluorescence and by electron microscopy using immunogol d labeling. The protein is associated with membrane systems of the sec retory gland cells of the pharynx, with sites of cuticle formation in the grinder in the pharynx, with yolk granules in oocytes, with the ut erine wall and vulva, and with membrane systems in the spermathecal va lve. The presence of the annexin in association with the membranes of the spermathecal valve suggests a novel function of the protein in the folding and unfolding of these membranes as eggs pass through the val ve. The localizations also indicate roles for the annexin correspondin g to those proposed in mammalian systems in membrane trafficking, coll agen deposition, and extracellular matrix formation.