IDENTIFICATION OF CANDIDA-ALBICANS ALS2 AND ALS4 AND LOCALIZATION OF ALS PROTEINS TO THE FUNGAL CELL-SURFACE

Additional genes in the growing ALS family of Candida albicans were is olated by PCR screening of a genomic fosmid library with primers desig ned from the consensus tandem-repeat sequence of ALS1, This procedure yielded fosmids encoding ALS2 and ALS4. ALS2 and ALS4 conformed to the three-domain structure of ALS genes, which consists of a central doma in of tandemly repeated copies of a 108-bp motif, an upstream domain o f highly conserved sequences, and a domain of divergent sequences 3 ' of the tandem repeats. Alignment of five predicted Als protein sequenc es indicated conservation of N- and C-terminal hydrophobic regions whi ch have the hallmarks of secretory signal sequences and glycosylphosph atidylinositol addition sites, respectively. Heterologous expression o f an N-terminal fragment of Als1p in Saccharomyces cerevisiae demonstr ated function of the putative signal sequence with cleavage following Ala17, This signal sequence cleavage site was conserved in the four ot her Als proteins analyzed, suggesting identical processing of each pro tein. Primary-structure features of the five Als proteins suggested a cell-surface localization, which was confirmed by indirect immunofluor escence with an anti-Als antiserum. Staining was observed on mother ye asts and germ tubes, although the intensity of staining on the mother yeast decreased with elongation of the germ tube. Similar to other ALS genes, ALS2 and ALS4 were differentially regulated. ALS4 expression w as correlated with the growth phase of the culture; ALS2 expression wa s not observed under many different in vitro growth conditions. The da ta presented here demonstrate that ALS genes encode cell-surface prote ins and support the conclusion that the size and number of Als protein s on the C. albicans cell surface vary,vith strain and growth conditio ns.