The Function of Yeast Adenylyl Cyclase-Associated Proteins in Cell Growth Regulation

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In budding yeast adenylyl cyclase is a downstream effector of Ras proteins. We found adenylyl cyclase forms a complex with two cyclase-associated proteins ; 70-kDa CAP and 50-kDa p50, and analyzed their structures and functions.1. CAP is a bifunctional protein ; its N-terminal region is required for association with adenylyl cyclase, while its C-terminal region is homologous to mammalian actin-binding protein ASP-56 and presumably involved in cytoskeletal regulation. Gene-disruption and mutational studies showed that CAP is not essential for interaction of cyclase with Ras proteins. We assigned a CAP-binding site of cyclase to its C-terminal domain and created mutants which lost the ability to bind CAP.Yeast cells whose chromosomal cyclase genes were replaced by the CAP-nonbinding mutants posessed cyclase activity fully responsive to Ras in vitro. However, in the activated RAS2^<Vall9> background they did not exhibit abnormal phenotypes and exaggerated cAMP response to glucouse, which … More were characteristic of RAS2^<Vall9> cells carrying wild-type cyclase. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the cAMP response. These results suggested that the association with CAP, although not involved in response to wild-type Ras, is required for the exaggerated response of cyclase to activated Ras. Thus, we found a way to suppress the exaggerated action of activated Ras without affecting that of wild-type Ras. Mechanisms underlying this are currently under investigation.2. p50 binds to leucine-rich repeats domain of adenylyl cyclase, a Ras-binding site. p50 was purified, and N-terminal amino acid sequence determaned. The result indicated p50 was identical with a ribosomal large subunit protein, L3. The function of L3 is presently unknown except for involvement in tricodermin-resistance. We are preparing antibody against L3 for analysis of its function. We speculate that the association of L3 with cyclase may represent a link between Ras pathway and protein synthesis machinery. Less