The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins.

Abstract

In the yeast Saccharomyces cerevisiae, adenylyl cyclase is regulated by RAS proteins. We show here that the yeast adenylyl cyclase forms at least two high-molecular-weight complexes, one with the RAS proteindependent adenyWl cyclase activity and the other with the Mn2+-dependent activity, which are separable by their size difference. The 70-kDa adenylyl cyclase-associated protein (CAP) existed in the former complex but not in the latter. Missense (6,8,9,21,29,36,37). Genetic and biochemical studies demonstrated that the yeast RAS proteins are essential regulatory elements of adenylyl cyclase (6, 39). The S. cerevisiae adenylyl cyclase, a product of the CYRI gene (25), consists of 2,026 amino acid residues that comprise at least four domains: the amino-terminal, the middle repetitive, the catalytic, and the carboxy-terminal domains (20,43). The catalytic domain, located between amino acid positions 1646 and 1890, retains the Mn2+-dependent adenylyl cyclase activity, but the remaining portion appears to be required for activation of the Mg2e-dependent activity by GTP-bound forms of RAS proteins (7,17,20,34,43). The middle repetitive region is composed of a repetition of 23-amino-acid leucine-rich motifs which have homology to the leucine-rich repeats family proteins of yeasts, mammals, and Drosophila melanogaster (16,18,24,26,28,30,35,38). Studies using deletion and insertion mutagenesis showed that the leucine-rich repeats are required for interaction of adenylyl cyclase with RAS proteins (7,34 (13,15). We report here the characterization of the RAS protein-responsive adenylyl cyclase. We show that CAP does not appear to be essential for interaction of adenylyl cyclase with RAS proteins. In addition, we used site-specific mutagenesis to examine structural elements in the leucine-rich repeats which are essential for the interaction with RAS proteins. MATERIALS AND METHODSCell strains, growth media, and transformation. S. cerevisiae strains TK35-1 (MATct leu2 his3 trpl ura3 cyrl-2 ras2::LEU2) and SP1 (AM Ta leu2 his3 trpl ura3 ade8 canl) have been described elsewhere (21,34

Effect of Association with Adenylyl Cyclase-Associated Protein on the Interaction of Yeast Adenylyl Cyclase with Ras Protein

“…However, the function of CAP on the Ras-adenylyl cyclase pathway resides solely in its N-terminal region and is separable from the functions of the other regions as reported previously (11,22). Although the N-terminal function is required for the exaggerated cAMP response of yeast cells in the RAS2 background, we observed that association with CAP has no effect on the in vitro activation of adenylyl cyclase by the unmodified Ras proteins, which were expressed and purified from E. coli (43,44). Here, we found that the purified GST-fusion protein of adenylyl cyclase, vastly overexpressed in yeast, became insensitive to the stimulatory effect of farnesylation of Ras and, at the same time, exhibited very limited association with CAP.…”

“…The polyclonal antisera for the full-length CAP and for GST were described previously (43,44). SDS-PAGE and Western immunoblot analysis were performed as described elsewhere (26,39 a Strains SP1, TK36-1, TM5-1, and NS1-1 were described previously (21,29,37).…”

“…The resulting transformants were grown to the density of 10 7 cells/ml, harvested by centrifugation, disrupted by shaking with glass beads in buffer C (50 mM 2-(N-morpholino)ethanesulfonic acid [pH 6.2], 0.1 mM MgCl 2 , 0.1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 1 mM ␤-mercaptoethanol) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and a crude membrane fraction was prepared as described previously (37,47). GST-CYR1(x-x) protein was solubilized from the crude membrane fraction with buffer C containing 1% Lubrol PX, 0.5 M NaCl, 1 mM dithiothreitol, and 1 mM PMSF as described before (25,43). After centrifugation at 100,000 ϫ g for 1 h, the supernatant was incubated with glutathione-Sepharose resin (Pharmacia) in buffer A (20 mM Tris-HCl [pH 7.4], 40 mM NaCl, 5 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, 0.1% Lubrol PX) by continuous mixing for 4 h at 4ЊC.…”

“…However, the mechanism by which CAP is involved in the Ras-adenylyl cyclase interaction was unknown. By using an in vitro reconstituted system, we have shown that CAP is not essential for activation of adenylyl cyclase by purified unmodified Ras proteins produced in Escherichia coli (43,44).…”

Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

“…However, its role in normal RAS/adenylyl cyclase pathway signaling has yet to be elucidated (Fedor-Chaiken et al, 1990;Wang et al, 1992Wang et al, , 1993. cAMP-dependent protein kinase-mediated signals are required for growth and cell cycle progression past the unbudded (G1) stage in S. cerevisiae, and recent reports have suggested that cAMP-mediated events can also delay the timing of bud emergence by exerting a negative effect on G1 cyclin synthesis (Broach, 1991;Baroni et al, 1994;Tokiwa et al, 1994).…”