An Actin Monomer Binding Activity Localizes to the Carboxyl-terminal Half of the Saccharomyces cerevisiae Cyclase-associated Protein

doi:10.1074/jbc.270.10.5680

An Actin Monomer Binding Activity Localizes to the Carboxyl-terminal Half of the Saccharomyces cerevisiae Cyclase-associated Protein (*)

From the (1)Department of Pharmacology and the Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and the
(2)Department of Biological Sciences, Columbia University, New York, New York 10027

^¶ Beckman Young investigator. To whom correspondence should be addressed.

Abstract

The Saccharomyces cerevisiae adenylyl cyclase complex contains at least two subunits, a 200-kDa catalytic subunit and a 70-kDa cyclase-associated protein, CAP (also called Srv2p). Genetic studies suggested two roles for CAP, one as a positive regulator of cAMP levels in yeast and a second role as a cytoskeletal regulator. We present evidence showing that CAP sequesters monomeric actin (K in the range of 0.5-5 μM), decreasing actin incorporation into actin filaments. Anti-CAP monoclonal antibodies co-immunoprecipitate a protein with a molecular size of about 46 kDa. When CAP was purified from yeast using an anti-CAP monoclonal antibody column, the 46-kDa protein co-purified with a stoichiometry of about 1:1 with CAP. Western blots identified the 46-kDa protein as yeast actin. CAP also bound to muscle actin in vitro in immunoprecipitation assays and falling ball viscometry assays. Experiments with pyrene-labeled actin demonstrated that CAP sequesters actin monomers. The actin monomer binding activity is localized to the carboxyl-terminal half of CAP. Together, these data suggest that yeast CAP regulates the yeast cytoskeleton by sequestering actin monomers.

Footnotes

↵^§ Contributed equally to this manuscript.
↵* This work was supported by National Institutes of Health R01-GM-48241. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CAP

cyclase-associated protein

MES

2-(N-morpholino)ethanesulfonic acid.
↵²N. L. Freeman, T. Lila, K. Mintzer, Z. Chen, A. J. Pahk, R. Ren, D. G. Drubin, and J. Field, unpublished observations.
- Received November 30, 1994.