Identification, Characterization, and Intracellular Distribution of Cofilin in Dictyostelium discoideum

doi:10.1074/jbc.270.18.10923

Identification, Characterization, and Intracellular Distribution of Cofilin in Dictyostelium discoideum(*)

From the (1) Department of Cell Biology and Department of Molecular Biology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113 and the
(2) Department of Pure and Applied Science, College of Arts and Science, University of Tokyo, Komaba, Tokyo 153, Japan

^§ To whom correspondence should be addressed:
Dept. of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113, Japan
. Tel.: 81-3-3823-2101; Fax: 81-3-5685-2932.

Abstract

We identified and purified an actin monomer-binding protein of apparent molecular weight of 15,000 from Dictyostelium discoideum. The 15-kDa protein depolymerized actin filaments in a pH-dependent manner. The protein also had an activity to decrease apparent viscosity of actin solutions in a dose-dependent manner. This activity was inhibited by phosphatidyl inositides. Molecular cloning of genes encoding this protein revealed that the protein is 42% identical in its primary sequence to yeast cofilin. We concluded that the 15-kDa protein is cofilin of this organism. D. discoideum cells contain two cofilin genes (DCOF1 and DCOF2) whose nucleotide sequences were entirely identical in their exsons while the promoter and intron regions were different. Promoter assay experiments revealed that D COF1 is expressed both in vegetative and differentiating cells and that D COF2 is not expressed under any conditions examined. Gene disruption experiments suggested that D COF1 might be essential for the proliferation of D. discoideum cells whereas the disruption of D COF2 was proven not to alter any phenotypes. Indirect immunofluorescence microscopic observations showed that cofilin is distributed diffusely throughout cytoplasm in vegetative cells. In flattened cells under starvation stress, cofilin localized at dramatically reorganizing actin-cytoskeletons in ruffling membranes of the leading edge, but not at rigid actin meshwork in focal adhesion plaques. These results suggest that cofilin may be involved in dynamic reorganization of membranous actin cytoskeletons.

Footnotes

↵* 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:

PI

phosphatidylinositol

CBB

Coomassie Brilliant Blue; IP inositol 1,4,5-triphosphate

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

PC

phosphatidylcholine

PCR

polymerase chain reaction

PIP

phosphatidylinositol monophosphate

PIP₂

phosphatidylinositol bisphosphate

PS

phosphatidylserine

RT

reverse transcriptase

MES

4-morphoinepropanesulfonic acid

bp

base pair(s)

kbp

kilobase pair(s).
↵² H. Awzawa, K. Sutoh, S. Tsubuki, S. Kawashima, A. Ishii, and I. Yahara, unpublished results.
↵³ K. Iida, unpublished data.