Cloning and characterization of the kinesin-related protein, Krp1p, in Schizosaccharomyces pombe

Abstract

Kinesin have been cloned in many organisms. They played important roles in the transport of cell organelles, polarized growth, and secretion. We report here the identification of a kinesin-related protein in Schizosaccharomyces pombe, which was named kinesin-related protein (Krp1p). The primer sequences were driven from the highly conserved area of the kinesin genes in other organisms. We cloned kinesin genes from S. pombe using the PCR technique. Sequence analysis revealed that krp1+ has a 1,665 bp open-reading frame (ORF) that encoded a protein that consisted of 554 amino acids with a molecular weight of 61,900. It is homologous to the proteins that belong to the kinesin heavy chain (KHC) superfamily [Gen-Bank accession No. AF156966 (genomic DNA) and AF247188 (mRNA)]. To characterize Krp1p, the gene was disrupted and overexpressed in S. pombe. Cells that contained a krp1+ null allele were viable. Overexpression of Krp1p resulted in the inhibition of mitotic growth; cells became elongated, branched, and formed aberrant septa. To identify proteins that interact with Krp1p, the yeast two-hybrid system was used. As a result, the novel protein, designated kinesin associated protein (Kap1p), was identified and showed structural homology to the proteins of the myosin family (Gen-Bank accession No. AF351206). The data from the overexpression and two-hybrid study of Krp1p may provide information that Krp1p can have roles in cytokinesis with myosin.