HIGH-LEVEL EXPRESSION OF NONACETYLATABLE ALPHA-TUBULIN IN CHLAMYDOMONAS-REINHARDTII

Following the discovery of acetylated alpha-tubulin in the flagella of Chlamydomonas, many studies have documented the presence of acetylate d alpha-tubulin in a variety of evolutionarily divergent organisms. Wh ile this posttranslational modification may define an isoform with a u nique function, the primary effect of alpha-tubulin acetylation remain s unknown. To study the function of alpha-tubulin acetylation, we have transformed Chlamydomonas, an organism in which almost all of the fla gellar tubulin and a subset of the cytoplasmic microtubules are acetyl ated, with an alpha1-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 d istinguish mutagenized alpha-tubulin from that produced by the two end ogenous alpha-tubulin genes, mutant alpha-tubulin was tagged with an e pitope from influenza virus hemagglutinin. Utilizing the constitutive Chlamydomonas rubisco small subunit S2 promoter, we have obtained in s elected clones high levels of nonacetylatable alpha-tubulin expression approximating 50-70% of the total flagellar alpha-tubulin. Immunofluo rescence and immunoblot analysis of transformed cells indicated that n onacetylatable alpha-tubulin could assemble, along with endogenous alp ha-tubulin, into both cytoplasmic and flagellar microtubules. However, no gross phenotypic effects were observed, suggesting that the effect of alpha-tubulin acetylation is subtle.