Characterization of Green Alga, Yeast, and Human Centrins

doi:10.1074/jbc.271.37.22453

Characterization of Green Alga, Yeast, and Human Centrins

SPECIFIC SUBDOMAIN FEATURES DETERMINE FUNCTIONAL DIVERSITY*

From the ^‡ Max-Planck-Institut für Biochemie, Genzentrum, Am Klopferspitz 18a, 82152 Martinsried and the
^¶ Universität zu Köln, Botanisches Institut, Gyrhofstrasse 15, 50931 Köln, Federal Republic of Germany

^∥ To whom correspondence should be addressed. Tel.: 49-89-85783969; Fax: 49-89-85783810; E-mail: eschiebel{at}genmic.mpg.de.

Abstract

Centrins are a subfamily within the superfamily of Ca²⁺-modulated proteins that play a fundamental role in centrosome duplication and contraction of centrin-based fiber systems. We examined the individual molecular properties of yeast, green alga, and human centrins. Circular dichroism spectroscopy revealed a divergent influence of Ca²⁺ binding on the α-helical content of these proteins. Ca²⁺-free centrins were elongated in shape as determined by size exclusion chromatography. The presence of Ca²⁺ and binding peptide resulted in more spherical shaped centrins. In contrast to yeast calmodulin, centrins formed multimers in the Ca²⁺-bound state. This oligomerization was significantly reduced in the absence of Ca²⁺ and in the presence of binding peptide. The Ca²⁺-dependent polymerization of the green alga Scherffelia dubia centrin (SdCen) resulted in a filamentous network. This molecular property was mainly dependent on the amino-terminal subdomain and the peptide-binding site of SdCen. Finally, we analyzed whether SdCen and Cdc31p-SdCen hybrid proteins functionally substitute for the Saccharomyces cerevisiae centrin Cdc31p. Only hybrid proteins containing the amino-terminal subdomain or the third EF-hand of SdCen and the other subdomains from Cdc31p were functional in vivo.

Footnotes

↵^§ Contributed equally to this work.
↵* This work was supported by Deutsche Forschungsgemeinschaft Grants Schi-295/2-1 and Me-658/9-3. 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.
↵¹ The abbreviations used are:

SPB

spindle pole body

CrCen

C. reinhardtii centrin

SdCen

S. dubia centrin

HsCen1

H. sapiens centrin

HsCen2

H. sapiens caltractin

ScCaM

S. cerevisiae calmodulin

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

DTT

1,4-dithiothreitol

MOPS

4-morpholinepropanesulfonic acid

AMM

apparent molecular mass.
↵²B. M. Geier, H. Wiech, and E. Schiebel, submitted for publication.
- Received April 16, 1996.
- Revision received June 29, 1996.