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Eukaryotic Cell doi:10.1128/EC.00197-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A Sfi1p-like Centrin-Binding Protein mediates centrin based Ca²⁺-dependent contractility in Paramecium

CNRS, Centre de Génétique Moléculaire, UPR 2167, Gif-sur-Yvette, F-91198; Univ Paris-Sud, Orsay, F-91405; Université Pierre et Marie Curie- Paris 6, Paris, F-75005; Laboratoire de Spectrométrie de Masse, Institut de Chimie des Substances Naturelles, UPR 2301, CNRS, F-91198 Gif-sur-Yvette, France

^* To whom correspondence should be addressed. Email: klotz{at}cgm.cnrs-gif.fr.

	Abstract

The characterization and structural analyses of Sfi1p, a S.cerevisae centrin-binding protein essential for the spindle pole body duplication, have suggested molecular models to account for centrin mediated, Ca²⁺-dependant, contractility processes (Li, S., A. M. Sandercock, P. Conduit, C. V. Robinson, R. L. Williams, and J. V. Kilmartin. 2006. J. Cell Biol. 173:867-877). Such processes can be analyzed in Paramecium which harbours a large Ca²⁺-dependant contractile cytoskeletal network, the infraciliary lattice (ICL). Previous biochemical and genetical studies had shown that the ICL is composed of diverse centrin isoforms and a high molecular mass centrin-associated protein, whose reduced size in the mutant "démaillé" (dem1) correlates with a defective organization of the ICL. Using peptide sequences obtained from the high molecular mass protein to probe the Paramecium genome sequence, we characterized the PtCENBP1 gene which encodes a 460 kDa protein. PtCenBP1p displays 6 almost perfect repeats of 427 aa and harbours 89 potential centrin-binding sites with a consensus motif [LLX₁₁F/LX₂WK/R], similar to the centrin-binding sites of ScSfi1p. The smaller (260kDa) protein encoded by the PtCenBP1-dem1 mutant allele comprises only 2 repeats of 427 aa and 46 centrin-binding sites. By RNAi and GFP-fusion experiments, we show that PtCenBP1p forms the backbone of the ICL and plays an essential role in its assembly and contractility. This study provides the first in vivo demonstration of the role of Sfi1p-like proteins in centrin-mediated Ca²⁺-dependant contractile processes.