Localization of the Kar 3 Kinesin Heavy Chain-related Protein Requires the Cikl Interacting Protein

The Kar3 protein (Kar3p), a protein related to kinesin heavy chain, and the Cikl protein (Ciklp) appear to participate in the same cellular processes in S. cerevisiae. Phenotypic analysis of mutants indicates that both CIK1 and KAR3 participate in spindle formation and karyogamy. In addition, the expression of both genes is induced by pheromone treatment. In vegetatively growing ceils, both Cikl::/3-gal and Kar3::B-gal fusions localize to the spindle pole body (SPB), and after pheromone treatment both fusion proteins localize to the spindle pole body and cytoplasmic microtubules. The dependence of Ciklp and Kar3p localization upon one another was investigated by indirect immunofluorescence of fusion proteins in pheromone-treated cells. The Ciklp::/~-gal fusion does not localize to the SPB or microtubules in a kar3A strain, and the Kar3p::B-gal fusion protein does not localize to microtubu! e-associated structures in a ciklA strain. Thus, these proteins appear to be interdependent for localization to the SPB and microtubules. Analysis by both the two-hybrid system and coimmunoprecipitation experiments indicates that Ciklp and Kar3p interact, suggesting that they are part of the same protein complex. These data indicate that interaction between a putative kinesin heavy chain-related protein and another protein can determine the localization of motor activity and thereby affect the functional specificity of the motor complex. " ~ ~ ' I C R O T U B U L E S participate in a wide variety of | ~ k / | cellular processes, including mitosis, organdie . Jk I ' . L transport, and cell motility. These processes are mediated by microtubule motors, often kinesin-related proteins (vale et al., 1985a; Zhang et al., 1990; Enos and Morris, 1990; Meluh and Rose, 1990; McDonald et al., 1990; Walker et al., 1990; Roof et al., 1992a; Hoyt et al., 1992; Hagan and Yanagida, 1990; Hagan and Yanagida, 1992). Kinesin was originally identified as a tetrameric complex comprised of two heavy chain subunits and two light chains (vale et al., 1985a). The heavy chain contains a motor domain that mediates translocation of the motor along microtubules (Yang et al., 1989); the function of the light chain is unknown. Recently, a large number of kinesin heavy chain-related molecules have been found and characterized (vale et al., 1985a; Zhang et al., 1990; Enos and Morris, 1990; Meluh and Rose, 1990; McDonald et al., 1990; Walker et al., 1990; Roof et al., 1992a; Hoyt et al., 1992; Hagan and Yanagida, 1990; Hagan and Yanagida, 1992). These proteins often contain a central coiled-coil domain flanked by a common motor domain and a nonconserved globular domain. Despite the similarities among the different members of the kinesin famAddress all correspondence to M. Snyder, Department of Biology, Yale University, P.O. Box 208103, New Haven, CT 06511-8103. ily, the various motor proteins participate in many different functions (Vale et al., 1985b; McDonald et al., 1990; Zhang et al., 1990; Saxton et al., 1991; Saunders and Hoyt, 1992; O'Connell et al., 1993; Goldstein, 1990). The specificity of function has been speculated to be conferred in part by the nonhomologous region of these proteins (Yang et al., 1989; Meluh and Rose, 1990; Enos and Morris, 1990; Vale and Goldstein, 1990). Through its interaction with light chains or other proteins, this region may determine the localization of the microtubule motor, affect its activity, and/or specify the type of cargo carried (Vale et al., 1985a; Cyr et al., 1991; Wedaman et al., 1993). So far no direct evidence supports this hypothesis. The yeast Saccharomyces cerevisiae is an excellent system for the genetic analysis of microtubule-dependent processes such as chromosome segregation during mitosis and nuclear fusion (karyogamy) during conjugation. Molecular characterization of several mutants defective in these processes has uncovered a family of genes encoding kinesin-related proteins. These genes include KIP1 and CIN8 which participate in the establishment and maintenance of the mitotic apparatus and KAR3 which also functions in chromosome segregation and is required for karyogamy (Hoyt et al., 1992; Roof et al., 1992b; Meluh and Rose, 1990). Genetic screens that enrich for mutants defective in microtubule processes have identified other genes whose products interact with microtubules but share no or little sequence similarity to previously © The Rockefeller University Press, 0021-9525/94/02/507/13 $2.00 The Journal of Cell Biology, Volume 124, Number 4, February 1994 507-519 507 on N ovem er 9, 2017 jcb.rress. org D ow nladed fom characterized proteins (Berlin et al . , 1990; Page and Snyder, 1992). Such genes could encode proteins that function via cooperation with kinesin-like motors. Mutations in such a gene would be expected to result in similar phenotypes as those exhibited in cells mutant for the corresponding kinesin-related gene. Given this assumption, the Cikl gene product is a candidate for interacting with the Kar3 kinesin-