SAXO proteins mediate tubulin turnover in axonemal microtubules of Chlamydomonas

ABSTRACT Cilia and eukaryotic flagella are microtubule-based organelles that are crucial for cell motility and signaling. SAXO proteins (denoted for ‘stabilizers of axonemal microtubules’) are found exclusively in flagellated or ciliated organisms, but their physiological functions remain unclear. We investigated four SAXO proteins (SAXO1–SAXO4) in Chlamydomonas reinhardtii, identified via bioinformatics. All localize to cilia but differ in axonemal binding and spatial distribution. Single SAXO knockouts had no effect, whereas double mutants (saxo1/2, saxo1/3 and saxo2/3) showed shorter cilia. This phenotype intensified in the saxo1/2/3 triple mutant but not further in the quadruple mutant. Ciliary beating remained normal in saxo1/2/3 mutants, even under mechanical stress, indicating that SAXO1–SAXO3 are not essential for ciliary rigidity. Biochemical and proteomic analyses revealed no significant changes in the ciliary proteome or in tubulin acetylation, tyrosination and glutamylation within cilia. However, dikaryon assays with labeled tubulin showed that there was increased axonemal tubulin turnover in saxo1/2/3 mutant. Our findings underscore a crucial role of SAXO proteins in stabilizing axonemal microtubules by reducing tubulin turnover, thereby regulating ciliary length and assembly, and provide new insights into their function in cilia.