Fission yeast pkl1 is a kinesin-related protein involved in mitotic spindle function.

Abstract

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.

Two Related Kinesins,klp5+andklp6+, Foster Microtubule Disassembly and Are Required for Meiosis in Fission Yeast

“…pombe has two KAR3 homologs, pkl1 ϩ and klp2 ϩ (Pidoux et al, 1996;Troxell et al, 2001). Neither of these kinesins is essential, either individually or together, but null mutants have altered sensitivities to TBZ and severe meiotic phenotypes.…”

“…The Schizosaccharomyces pombe genome encodes nine kinesin-related genes from seven subfamilies and one cytoplasmic dynein (Hagan and Yanagida, 1990;Pidoux et al, 1996;Yamamoto et al, 1999;Brazer et al, 2000;Browning et al, 2000;Troxell et al, 2001; pombe genome project). Among the kinesin subfamilies found in fission yeast, three have direct effects on microtubule dynamics and higher order microtubule organization (Goldstein and Philp, 1999).…”

pkl1+andklp2+: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis

“…Sequence from both ends of each insert showed that they encoded full-length copies of klp1 ϩ and klp2 ϩ , respectively. klp1 ϩ was identical to pkl1 ϩ , which has already been described (Pidoux et al, 1996), so this name will be used henceforth. pkl1 ϩ and klp2 ϩ were mapped to the left arm of chromosome I by probing a filter containing ordered arrays of cosmids (Hoheisel et al, 1993), with the use of the original PCR products and with the kind assistance of Dr. Elmar Maier (Imperial Cancer Research Fund, Genome Analysis Laboratory, London).…”

“…A complete disruption of the pkl1 ϩ gene has been made with the use of the genomic klp1 ϩ clone described above (Pidoux et al, 1996). A complete replacement of the klp2 ϩ by ura4 ϩ was made by a singlestep gene replacement protocol (Rothstein, 1991), with the use of a cassette containing two genomic regions flanking the either side of the klp2 ϩ gene and ura4 ϩ as the selectable marker.…”

“…Cut7p is a KLP of the BimC family that is essential for spindle formation in prophase and spindle elongation in anaphase B (Hagan and Yanagida, 1992). Pkl1p is a KLP of the KAR3 family that is not essential but localizes to the nucleus during interphase and to the spindle throughout mitosis; further, deletion of pkl1 ϩ is known to rescue some alleles of cut7 ts (Pidoux et al, 1996). S. pombe expresses one dynein heavy chain (Yamamoto et al, 1999), and although this motor is essential for nuclear migration during the sexual phase of the life cycle, it plays no detectable role in mitosis.…”