Coiled-coil Interaction of N-terminal 36 Residues of Cyclase-associated Protein with Adenylyl Cyclase Is Sufficient for Its Function in Saccharomyces cerevisiae Ras Pathway

doi:10.1074/jbc.273.43.28019

Coiled-coil Interaction of N-terminal 36 Residues of Cyclase-associated Protein with Adenylyl Cyclase Is Sufficient for Its Function in Saccharomyces cerevisiae Ras Pathway*

From the Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

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 conferRAS2 ^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 αXXαXXX (where α 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.

Footnotes

↵* This investigation was supported by Grants-in-Aid for Scientific Research on Priority Areas and for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Yamanouchi Foundation for Research on Metabolic Disease.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed. Tel.: 81-78-341-7451 (ext. 3230); Fax: 81-78-341-3837; E-mail: kataoka{at}kobe-u.ac.jp.
Abbreviations:

CAP

adenylyl cyclase-associated protein

CYR1

adenylyl cyclase

GST

glutathioneS-transferase

PCR

polymerase chain reaction

GBT

GAL4 DNA-binding domain

GAD

GAL4 transactivation domain

MES

2-(N-morpholino)ethanesulfonic acid.
- Received June 30, 1998.
- Revision received August 4, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.