CLONING AND CHARACTERIZATION OF THE SEC18 GENE FROM CANDIDA-ALBICANS

The SEC18 gene product is required for protein transport at different stages in the Saccharomyces cerevisiae secretory pathway. The homologo us SEC18 gene from Candida albicans has been cloned by complementation of a sec18-1 S. cerevisiae thermosensitive mutant using a C albicans genomic library in YRp7. Sequence analysis of the gene revealed a 2382 -bp open reading frame which coded for a protein of 88 926 kDa. By an in vitro transcription-translation coupled reaction of the C albicans SEC18 gene, a protein of approximately 85 kDa was obtained. Hydrophobi city analysis of the protein did not show any predicted signal sequenc e nor transmembrane anchor domain. These results and the fact that gly cosylation was absent in the protein indicated that C albicans Sec18p did not enter in the secretory pathway. The alignment of the amino aci d sequence revealed that the SEC18 gene from C albicans was homologous to the SEC18 from S. cerevisiae (50% amino acid identity) and to the gene that coded the N-ethylmaleimide-sensitive factor (NSF) protein (4 3% amino acid identity). Moreover, the C albicans Sec18p also showed t he putative ATP binding site present in S. cerevisiae Sec18p and in NS F.