Molecular dissection of the Cik1 N-terminus reveals a conserved KLTF motif. (A) Scheme of Cik1 mutagenesis screen. Cik1 mutants were encoded on centromeric plasmids and targeted to the nucleus by an SV40 NLS. After addition of auxin, the only remaining version of Cik1 in the cell was an assayed mutant. (B) Subsequent N-terminal truncations of Cik1 allowed to identify a novel functionally important region in the N-terminus. Second panel shows the precise mapping of a region between 54 and 74 aa of Cik1. (C) Sequence alignment of Cik1 tail regions from six budding yeast species illustrates a conserved KLTF peptide motif as the difference between Cik1^Δ69 and Cik1^Δ74. Residues were labeled with the ClustalW coloring scheme in Jalview. (D) The KLTF peptide motif defines the key difference between the two kinesin-associated proteins Cik1 and Vik1. Construction of Cik1-Vik1-Cik1 chimeras is shown schematically. Transplantation of the 69–101 aa region, including KLTF, of Cik1 to Vik1 allows the VCV4 (Vik1-Cik1-Vik1) chimera to fulfill Kinesin-14 mitotic functions.