Transcription, biochemistry and localization of nematode annexins.
The transcription of three annexin genes in the nematode, Caenorhabditis elegans, was detected by reverse transcriptase/polymerase chain reaction amplification of messenger RNAs. The highest level of expression was from the nex-1 gene, with lower levels detected for the nex-2 and nex-3 genes. The expression of nex-1 was reduced in the Dauer larval stage relative to the other annexins, correlating with the absence of the spermathecal valves, a major site of nex-1 protein localization. Recombinant nex-1 protein was expressed in yeast, isolated by calcium-dependent binding to acidic phospholipids, and its membrane binding and aggregating activities characterized using bovine chromaffin granules as a representative intracellular substrate. Binding to granule membranes was promoted by calcium with half-maximal binding seen at 630 microM calcium. Chromaffin granule aggregation was similarly promoted by the nex-1 protein at 630 microM calcium. This low sensitivity to calcium suggests the annexin can only be activated in vivo near the plasma membrane or other sources of calcium. Sequences including the nex-1 promoter were fused to the gene for green fluorescent protein and this construct was introduced into nematodes by microinjection. Examination of transgenic offspring revealed specific nex-1 promoter activity in the pharynx, the hypodermal cells, the vulva, and the spermathecal valve, locations in which the annexin may function in collagen secretion/deposition and membrane-membrane interactions. A sensitive anti-nex-1 antibody labelled with rhodamine was injected into body cavities of the nematode but did not detect extracellular nex-1 protein. Therefore, this annexin is apparently cytosolic and may function on the cytoplasmic side of the plasma membrane of the spermathecal valve to chaperon the folding of this membrane during the opening and closing of the valve.
Daigle
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Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
Creutz
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CE
D000818:Animals; D017302:Annexins / Q000032:analysis / Q000235:genetics*; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000032:analysis / Q000235:genetics*; D002417:Cattle; D049452:Green Fluorescent Proteins; D015801:Helminth Proteins / Q000032:analysis / Q000235:genetics*; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D017302:Annexins; D029742:Caenorhabditis elegans Proteins; D015801:Helminth Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; C098890:Nex-1 protein, C elegans; C519288:Nex-2 protein, C elegans; C519289:Nex-3 protein, C elegans; D020033:Protein Isoforms; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins