Abstract
In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins. We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to confer RAS2(Val-19)-dependent heat shock sensitivity to yeast cells. The CAP-binding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus. Both of these regions contained tandem repetitions of a heptad motif alphaXXalphaXXX (where alpha represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats. Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAP-adenylyl cyclase interaction.
Highlights
The budding yeast Saccharomyces cerevisiae has two RAS genes, RAS1 and RAS2, whose protein products are structurally, functionally, and biochemically similar to mammalian Ras proto-oncoproteins
The N-terminal region of cyclase-associated protein (CAP) binds to the C-terminal region of adenylyl cyclase [19], and this association appears to be required for the proper in vivo response of adenylyl cyclase to Ras, because its loss by mutation of either CAP or adenylyl cyclase resulted in disappearance of the RAS2Val-19-dependent heat shock sensitivity and in a reduced cAMP response to glucose stimulation [19]
Mapping of the Mutual Binding Sites of CAP and Adenylyl Cyclase—Previous experiments had already mapped the Nterminal function of CAP to residues 1–168 [12] and the CAPbinding site of adenylyl cyclase to residues 1879 –2026 [19]
Summary
The budding yeast Saccharomyces cerevisiae has two RAS genes, RAS1 and RAS2, whose protein products are structurally, functionally, and biochemically similar to mammalian Ras proto-oncoproteins (for reviews, see Refs. 1 and 2). The N-terminal region of CAP binds to the C-terminal region of adenylyl cyclase [19], and this association appears to be required for the proper in vivo response of adenylyl cyclase to Ras, because its loss by mutation of either CAP or adenylyl cyclase resulted in disappearance of the RAS2Val-19-dependent heat shock sensitivity and in a reduced cAMP response to glucose stimulation [19]. This function resides solely in the CAP N-terminal region and is separable from the functions of the other regions as reported [12]. We have mapped a minimal region of CAP responsible for its N-terminal function and analyzed the molecular mechanism for its association with adenylyl cyclase
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
