Mutant kinesin-2 motor subunits increase chromosome loss

Abstract

The Chlamydomonas anterograde intraflagellar transport motor, kinesin-2, is isolated as a heterotrimeric complex containing two motor subunits and a nonmotor subunit known as kinesin-associated polypeptide or KAP. One of the two motor subunits is encoded by the FLA10 gene. The sequence of the second motor subunit was obtained by mass spectrometry and sequencing. It shows 46.9% identity with the Fla10 motor subunit and the gene maps to linkage group XII/XIII near RPL9. The temperature-sensitive flagellar assembly mutants fla1 and fla8 are linked to this kinesin-2 motor subunit. In each strain, a unique single point mutation gives rise to a unique single amino acid substitution within the motor domain. The fla8 strain is named fla8-1 and the fla1 strain is named fla8-2. The fla8 and fla10 alleles show a chromosome loss phenotype. To analyze this chromosome loss phenotype, intragenic revertants of fla8-1, fla8-2, and fla10-14 were generated. The analysis of the mutants and the revertants demonstrates the importance of a pocket in the amino terminus of these motor subunits for both motor activity and for a novel, dominant effect on the fidelity of chromosome segregation.

Figure 1.

The FLA8 gene encodes a kinesin-2 motor protein. (A) The FLA8 gene consists of 12 exons separated by 11 introns with the size of each shown. (B) The Fla8 protein is an amino-terminal kinesin predicted to form a 267-amino acid coiled coil domain broken by a 30-amino acid hinge (H). (C) Coomassie Blue-stained SDS-PAGE (7.5% acrylamide) of sucrose density gradient purified kinesin-2 used for MALDI-TOF mass spectrometry analysis. The subunits of 96, 90, and 86 kDa correspond to KAP, Fla10, and Fla8, respectively. TUB, tubulin. (D) Alignment of Fla10 and Fla8 protein sequences. Tryptic peptides identified by mass spectrometry are underlined. Amino acids that are changed in the fla8-1, fla8-2, or fla10-14 alleles are indicated by shading.

Figure 2.

Flagellar loss of fla8 and fla10 mutant alleles when shifted to the restrictive temperature. Compared with wild-type cells (red diamond), the fla10-1 (blue square), fla10-14 (blue circle), fla8-1 (pink square), and fla8-2 (pink triangle) alleles begin to lose their flagella after 3 h at 32°C, whereas fla10-2 (blue triangle) and fla10-14; fla3 (orange triangle) are aflagellate at all temperatures. The fla10-14 intragenic revertants, R16 (green square), R27 (green diamond), R10 (green circle), R8 (green asterisk), and R13 (green triangle) are identical to wild-type cells at the permissive and restrictive temperature.

Figure 3.

Determining chromosome loss using markers on linkage group III and VI. An increased frequency of chromosome loss results in cells that are cycloheximide-resistant or mating-type plus- and 5-MAA–resistant and requires acetate for growth. Cells that have undergone mitotic recombination will be cyclohexamide resistant and mating-type minus and 5-MAA resistant and do not require acetate for growth.

Figure 4.

Effect of UV irradiation on mitotic segregation of linkage group VI after various exposure times (in minutes) to UV irradiation. Mitotic recombination and chromosome loss of linkage group VI (diamond), chromosome loss of linkage group VI (square), and mitotic recombination of the left arm of linkage group VI (triangle).

Figure 5.

Location of amino acids altered in the fla8 and fla10 mutant and revertant alleles as modeled onto the crystal structure of KIF1A with AMPPNP (green) bound (PDB 1VFV). The P-loop is shown in purple. (A and B) Fla10 E₂₄ and L₁₉₆ correspond to KIF1A E₁₉ and M₁₉₆, respectively, and are shown in red. Revertant fla10-14 amino acids K₂₅, N₃₂₉, and G₃₃₁ correspond to KIF1A M₂₀, A₃₃₀, and S₃₃₂, respectively, and are shown in yellow. (C–F) Fla8 amino acids E₂₁ and F₅₅ correspond to KIF1A E₁₉ and F₅₂, respectively, and are shown in red. Revertant fla8-1 and fla8-2 amino acids N₄₂, N₅₇, H₁₀₀, N₃₁₂, M₃₁₃, and G₃₁₄ correspond to KIF1A V₃₈, Y₅₄, Y₁₀₅, A₃₃₀, L₃₃₁, and S₃₃₂, respectively, and are shown in yellow. (G) Alignment of Fla8, Fla10, and KIF1A in the regions of interest. Red shading indicates mutations in one of the kinesin-2 motor subunits, yellow shading indicates amino acids that are altered in the revertants, and purple shading indicates the P-loop residues.