Essential kinesins: characterization of Caenorhabditis elegans KLP-15.
Kinesins form a superfamily of molecular motors involved in cell division and intracellular transport. Twenty kinesins have been found in the Caenorhabditis elegans genome, and four of these belong to the kinesin-14 subfamily, i.e., kinesins with C-terminal motor domains. Three of these kinesin-14s, KLP-15, KLP-16, and KLP-17, form a distinct subgroup in which KLP-15 and KLP-16 are more than 90% identical and appear to be related by a relatively recent gene duplication. They are essential for meiotic spindle organization and chromosome segregation, and are mostly expressed in the germline. With 587 amino acids each, they are among the smallest kinesins known. Using bacterially expressed KLP-15 constructs with different length extensions preceding the motor domain, we have determined in vitro the following characteristic properties: ATPase activity, microtubule binding, oligomeric state, microtubule gliding activity, and direction of movement. The constructs exhibit a monomer-dimer equilibrium that depends on the length of the predicted alpha-helical coiled-coil region preceding the motor domain. The longest construct with the complete coiled-coil domain is a stable dimer, and the shortest construct with only seven amino acids preceding the motor domain is a monomer. In microtubule gliding assays, the monomer is immobile whereas the fully dimeric KLP-15 construct supports gliding at 2.3 microm/min and moves toward microtubule minus ends, like other members of the kinesin-14 subfamily studied to date.
Robin
Gautier
G
Institut de Biologie Structurale, UMR 5075 CEA/CNRS/UJF, 41 rue Jules Horowitz, 38027 Grenoble Cedex 01, France.
DeBonis
Salvatore
S
Dornier
Aurélie
A
Cappello
Giovanni
G
Ebel
Christine
C
Wade
Richard H
RH
Thierry-Mieg
Danielle
D
Kozielski
Frank
F
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D005822:Genetic Vectors; D016547:Kinesins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D014461:Ultracentrifugation
D029742:Caenorhabditis elegans Proteins; C407129:KLP-15 protein, C elegans; D008869:Microtubule-Associated Proteins; D020409:Molecular Motor Proteins; D010446:Peptide Fragments; D000251:Adenosine Triphosphatases; D016547:Kinesins