The Kar3p Kinesin-related Protein Forms a Novel Heterodimeric Structure with Its Associated Protein Cik1p

Abstract

Proteins that physically associate with members of the kinesin superfamily are critical for the functional diversity observed for these microtubule motor proteins. However, quaternary structures of complexes between kinesins and kinesin-associated proteins are poorly defined. We have analyzed the nature of the interaction between the Kar3 motor protein, a minus-end-directed kinesin from yeast, and its associated protein Cik1. Extraction experiments demonstrate that Kar3p and Cik1p are tightly associated. Mapping of the interaction domains of the two proteins by two-hybrid analyses indicates that Kar3p and Cik1p associate in a highly specific manner along the lengths of their respective coiled-coil domains. Sucrose gradient velocity centrifugation and gel filtration experiments were used to determine the size of the Kar3-Cik1 complex from both mating pheromone-treated cells and vegetatively growing cells. These experiments predict a size for this complex that is consistent with that of a heterodimer containing one Kar3p subunit and one Cik1p subunit. Finally, immunoprecipitation of epitope-tagged and untagged proteins confirms that only one subunit of Kar3p and Cik1p are present in the Kar3-Cik1 complex. These findings demonstrate that the Kar3-Cik1 complex has a novel heterodimeric structure not observed previously for kinesin complexes.

Kinesin Kar3Cik1 ATPase Pathway for Microtubule Cross-linking

“…The truncated KAR3 gene was cloned into plasmid pET24d and when expressed yields amino acid residues Met-Ala-Lys 268 -Lys 729 with a predicted M r ϭ 52,833. The truncated version of CIK1 (encoding Lys 252 -Asp 594 ) was cloned into plasmid pET15b and when expressed yields residues MGSSH 6 SSGGLVPRGSHMet-Lys 252 -Asp 594 with pre-dicted M r ϭ 43,059. Both plasmids were transformed and coexpressed in Escherichia coli BL21-CodonPlus(DE3)-RIL, yielding a Kar3Cik1 heterodimer that formed through native coiled-coil interactions and with the predicted M r ϭ 95,892.…”

“…However, KAR3 when expressed dimerizes with either Cik1 or Vik1, both non-nucleotide binding proteins, to generate two functionally distinct kinesin-14 motors in vivo (3)(4)(5)(6). Kar3Cik1 during mating promotes MT 2 shortening for karyogamy, the nuclear fusion event (3)(4)(5)(6)(7)(8)(9)(10).…”

“…However, KAR3 when expressed dimerizes with either Cik1 or Vik1, both non-nucleotide binding proteins, to generate two functionally distinct kinesin-14 motors in vivo (3)(4)(5)(6). Kar3Cik1 during mating promotes MT 2 shortening for karyogamy, the nuclear fusion event (3)(4)(5)(6)(7)(8)(9)(10). In contrast, during mitosis Kar3Cik1 cross-links anti-parallel ipMTs (11), and its inward force stabilizes the spindle by counter-balancing the outward forces generated by kinesin-5 Cin8 and Kip1 (11)(12)(13)(14)(15)(16)(17)(18).…”

Drosophila Ncd reveals an evolutionarily conserved powerstroke mechanism for homodimeric and heterodimeric kinesin-14s

“…The kinesin-14s exhibit C-terminal motor domains connected by an N-terminal continuous coiled-coil stalk with an N-terminal ATP-independent MT binding site (7,(18)(19)(20)(21)(22). And although most of the kinesin-14s are homodimeric, some yeast species, including Saccharomyces cerevisiae and Candida glabrata, contain heterodimeric kinesin-14s (13,14,19). The conventional hypothesis for homodimeric kinesin-14 force generation proposed that only one motor head interacts with the MT and only one ATP turnover is required to complete the powerstroke (Fig.…”

“…Sequence analysis indicates that all members of the kinesin-14 subfamily are dimeric, yet the structural organization of kinesin-14 motors differs from the N-terminal processive kinesins. The kinesin-14s exhibit C-terminal motor domains connected by an N-terminal continuous coiled-coil stalk with an N-terminal ATP-independent MT binding site (7,(18)(19)(20)(21)(22). And although most of the kinesin-14s are homodimeric, some yeast species, including Saccharomyces cerevisiae and Candida glabrata, contain heterodimeric kinesin-14s (13,14,19).…”