Fission yeast pkl1 is a kinesin-related protein involved in mitotic spindle function.
We have used anti-peptide antibodies raised against highly conserved regions of the kinesin motor domain to identify kinesin-related proteins in the fission yeast Schizosaccharomyces pombe. Here we report the identification of a new kinesin-related protein, which we have named pkl1. Sequence homology and domain organization place pkl1 in the Kar3/ncd subfamily of kinesin-related proteins. Bacterially expressed pkl1 fusion proteins display microtubule-stimulated ATPase activity, nucleotide-sensitive binding, and bundling of microtubules. Immunofluorescence studies with affinity-purified antibodies indicate that the pkl1 protein localizes to the nucleus and the mitotic spindle. Pkl1 null mutants are viable but have increased sensitivity to microtubule-disrupting drugs. Disruption of pkl1+ suppresses mutations in another kinesin-related protein, cut7, which is known to act in the spindle. Overexpression of pkl1 to very high levels causes a similar phenotype to that seen in cut7 mutants: V-shaped and star-shaped microtubule structures are observed, which we interpret to be spindles with unseparated spindle poles. These observations suggest that pkl1 and cut7 provide opposing forces in the spindle. We propose that pkl1 functions as a microtubule-dependent motor that is involved in microtubule organization in the mitotic spindle.
Department of Molecular and Cell Biology, University of California, Berkeley 94720-3200, USA.
A L
AL
Pidoux
M
M
LeDizet
W Z
WZ
Cande
D000595:Amino Acid Sequence; D001483:Base Sequence; D019084:Fluorescent Antibody Technique, Indirect; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D016547:Kinesins / Q000737:chemistry*; D008870:Microtubules / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000502:physiology*; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure
C104159:Cut7 protein, S pombe; D005656:Fungal Proteins; D009711:Nucleotides; C104160:PKL1 protein, S pombe; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D016547:Kinesins