schenckii. Conclusion We have shown the presence of a new G protein α subunit in S. schenckii, SSG-2. The cDNA sequence Selleckchem GSK1838705A of the ssg-2 gene encoded a 355 amino acid Gα subunit of 40.90 kDa containing the 5 consensus domains present in all Gα subunits. The genomic sequence has four introns, whose positions are conserved in the other fungal homologues of this gene. Yeast two-hybrid analysis using the complete amino acid sequence of SSG-2 identified a PLA2 homologue as an interacting partner of this G protein subunit. This 846 amino acid protein was encoded by an intronless
gene. The 92.62 kDa protein encoded by this gene contained all the domains and amino acid residues that characterize cytosolic phospholipase A2. PLA2 and other phospholipases in fungi have very diverse roles not only as virulence factors but also in membrane homeostasis and signal transduction. Inhibitor studies showed that this PLA2 homologue and its interaction with SSG-2 were necessary
for the re-entry of S. MI-503 supplier schenckii yeast cells into the budding cycle suggesting a role for this important virulence factor in the control of dimorphism in this fungus and for the expression of the yeast form. The effects of PLA2 on the yeast cell cycle could be viewed as resulting from the generation of lipid messenger molecules or from membrane remodelling that affects the G1->S transition and G protein activity. The relationship reported here between these two proteins, SSG-2 and SSPLA2, constitutes Cyclosporin A molecular weight the first report of the interaction of a fungal phospholipase and a G protein subunit and the possible involvement of G protein in fungal virulence and morphogenesis. Methods Strains and culture conditions S. schenckii (ATCC 58251) was used for all experiments. The yeast form of this fungus was obtained as described . S. cerevisiae strains AH109 and Y187 were supplied with the MATCHMAKER Two-Hybrid System 3 (Clontech Laboratories Inc., Palo Alto, CA). Nucleic acids isolation DNA and RNA
Farnesyltransferase were obtained from S. schenckii yeast cells as described previously using the methods of Sherman , and Chomczynski & Sacchi , respectively. Poly A+ RNA was obtained from total RNA using the mRNA Purification Kit from Amersham Biosciences (Piscataway, NJ, USA). Sequencing the ssg-2 gene Polymerase chain Reaction and Rapid amplification of cDNA ends (RACE) S. schenckii DNA (100 ng) was used as template for polymerase chain reaction (PCR) with primers (100–200 ng) targeted to conserved motifs in Gα subunits. The primers used were: GESGKST (fw) 5′ ggtgc(c/t)ggtga(a/g)tc(a/c)gg(a/t)aa(a/g)tc 3′; KWIHCF (rev) 5′ aagcag tgaatccacttc 3′; TQATDT (rev) 5′gtatcggtagcttgggtc 3′; MGACMS (fw) 5′ atggg ggcttgcatgagt 3′ and KDSGIL (rev) 5′ taggataccggaatctttg 3′.