High level expression of nonacetylatable α‐tubulin in Chlamydomonas reinhardtii

Abstract

Following the discovery of acetylated α-tubulin in the flagella of Chlamydomonas, many studies have documented the presence of acetylated α-tubulin in a variety of evolutionarily divergent organisms. While this posttranslational modification may define an isoform with a unique function, the primary effect of α-tubulin acetylation remains unknown. To study the function of α-tubulin acetylation, we have transformed Chlamydomoas, a organism in which almost all of the flagellar tubulin ad a subset of the cytoplasmic microtubules are acetylated, with a α1-tubulin gene whose product cannot be acetylated. Specifically, the codon for lysine 40, the lysine that is acetylated, has been replaced with the codons of nonacetylatable amino acids. To distinguish mutagenized α-tubulin from that produced by the two endogenous α-tubulin genes, mutant α-tubulin was tagged with an epitope from influenza virus hemagglutinin. Utilizing the constitutive Chlamydomonas rubisco small subunit S2 promoter, we have obtained in selected clones high levels of nonacetylatable α-tubulin expression approximating 50–70% of the total flagellar α-tubulin. Immunofluorescence and immunoblot analysis of transformed cells indicated that nonacetylatable α-tubulin could assemble, along with endogenous α-tubulin, into both cytoplasmic and flagellar microtubules. However, no gross phenotypic effects were observed, suggesting that the effect of α-tubulin acetylation is subtle.