IN THE BUDDING YEAST KLUYVEROMYCES-MARXIANUS, ADENYLATE-CYCLASE IS REGULATED BY RAS PROTEIN(S) IN-VITRO

The presence of adenylate cyclase activity was first demonstrated in m embrane fractions from the budding yeast Khuyveromyces marxianus. The enzyme showed a Mn2+- and Mg2+-dependent activity, with optimal pH at around 6 as observed in other yeast species. As in Saccharomyces cerev isiae, where adenylate cyclase is regulated by RAS1 and RAS2, we detec ted a guanyl nucleotide-dependent activity. Interestingly Y13-259 mono clonal antibody, raised against mammalian p21(Ha-ras), inhibited Mg2plus GTP-gamma-S-dependent cAMP production, suggesting that the GTP bi nding proteins invoked in adenylate cyclase regulation could be Ras pr oteins. The same antibody recognized on Western blot and immunoprecipi tated a 40 kDa polypeptide from K. marxianus crude membranes. This pol ypeptide was not detected by an anti-RAS2 polyclonal antibody raised a gainst S. cerevisiae RAS2 protein, suggesting that Ras proteins from t he two species could be structurally different.