An Sfi1p-like centrin-binding protein mediates centrin-based Ca2+ -dependent contractility in Paramecium tetraurelia.
The previous characterization and structural analyses of Sfi1p, a Saccharomyces cerevisiae centrin-binding protein essential for spindle pole body duplication, have suggested molecular models to account for centrin-mediated, Ca2+-dependent contractility processes (S. Li, A. M. Sandercock, P. Conduit, C. V. Robinson, R. L. Williams, and J. V. Kilmartin, J. Cell Biol. 173:867-877, 2006). Such processes can be analyzed by using Paramecium tetraurelia, which harbors a large Ca2+ -dependent contractile cytoskeletal network, the infraciliary lattice (ICL). Previous biochemical and genetic studies have shown that the ICL is composed of diverse centrin isoforms and a high-molecular-mass centrin-associated protein, whose reduced size in the démaillé (dem1) mutant correlates with defective organization of the ICL. Using sequences derived 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 six almost perfect repeats of ca. 427 amino acids (aa) and harbors 89 potential centrin-binding sites with the consensus motif LLX11F/LX2WK/R, similar to the centrin-binding sites of ScSfi1p. The smaller (260-kDa) protein encoded by the dem1 mutant PtCenBP1 allele comprises only two repeats of 427 aa and 46 centrin-binding sites. By using RNA interference and green fluorescent protein fusion experiments, we showed 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+-dependent contractile processes.
Centre de Génétique Moléculaire, UPR 2167, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France.
Delphine
D
Gogendeau
Janine
J
Beisson
Nicole Garreau
NG
de Loubresse
Jean-Pierre
JP
Le Caer
Françoise
F
Ruiz
Jean
J
Cohen
Linda
L
Sperling
France
F
Koll
Catherine
C
Klotz
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects*; D002923:Cilia / Q000187:drug effects / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016805:Paramecium tetraurelia / Q000166:cytology* / Q000187:drug effects* / Q000648:ultrastructure; D010641:Phenotype; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
D018797:Cell Cycle Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C121820:SFI1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium