Transcription, biochemistry and localization of nematode annexins

The transcription of three annexin genes in the nematode, Caenorhabditis el egans, was detected by reverse transcriptase/polymerase chain reaction ampl ification of messenger RNAs, The highest level of expression was from the n ex-1 gene, with lower levels detected for the nex-2 and nex-3 genes. The ex pression of nex-1 was reduced in the Dauer larval stage relative to the oth er annexins, correlating with the absence of the spermathecal valves, a maj or site of nex-1 protein localization. Recombinant nex-1 protein was expres sed in yeast, isolated by calcium-dependent binding to acidic phospholipids , and its membrane binding and aggregating activities characterized using b ovine chromaffin granules as a representative intracellular substrate. Bind ing to granule membranes was promoted by calcium with half-maximal binding seen at 630 mu M calcium. Chromaffin granule aggregation was similarly prom oted by the nex-1 protein at 630 mu M calcium. This low sensitivity to calc ium suggests the annexin can only be activated in vivo near the plasma memb rane or other sources of calcium, Sequences including the nex-1 promoter we re fused to the gene for green fluorescent protein and this construct was i ntroduced into nematodes by microinjection, Examination of transgenic offsp ring revealed specific nex-1 promoter activity in the pharynx, the hypoderm al cells, the vulva, and the spermathecal valve, locations in which the ann exin may function in collagen secretion/deposition and membrane-membrane in teractions. A sensitive anti-nex-1 antibody labelled with rhodamine was inj ected into body cavities of the nematode but did not detect extracellular n ex-1 protein. Therefore, this annexin is apparently cytosolic and may funct ion on the cytoplasmic side of the plasma membrane of the spermathecal valv e to chaperon the folding of this membrane during the opening and closing o f the valve.