Acetylated a-Tubulin in Physarum: Immunological Characterization of the Isotype and Its Usage in Particular Microtubular Organelles

We have used monoclonal antibodies specific for acetylated and unacetylated a-tubulin to characterize the acetylated a-tubulin isotype of Physarum polycephalum, its expression in the life cycle, and its localization in particular microtubular organelles. We have used the monoclonal antibody 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) as the probe for acetylated ct-tubulin and have provided a biochemical characterization of the monoclonal antibody KMP-1 as a probe for unacetylated tubulin in Physarum. Concomitant use of these two probes has allowed us to characterize the acetylated a-tubulin of Physarum as the a3 isotype. We have detected this acetylated a3 tubulin isotype in both the flagellate and in the myxameba, but not in the plasmodium. In the flagellate, acetylated tubulin is present in both the flagellar axonemes and in an extensive array of cytoplasmic microtubules. The extensive arrangement of acetylated cytoplasmic microtubules and the flagellar axonemes are elaborated during the myxameba-flagellate transformation. In the myxameba, acetylated tubulin is not present in the cytoplasmic microtubules nor in the mitotic spindle microtubules, but is associated with the two centrioles of this cell. These findings, taken together with the apparent absence of acetylated a-tubulin in the ephemeral microtubules of the plasmodium suggest a natural correspondence between the presence of acetylated a-tubulin and microtubule organelles that are intrinsically stable or cross-linked. I N many organisms there is now excellent evidence for the existence of multi-gene families encoding both ctand [3-tubulin. The organization and number of the tubulin genes varies between organisms and it is clear that in many cases there is a complex pattern of differential expression that leads to the appearance of different tubulin isotypes in particular cell types or tissues (6, 25). There is also good evidence that expression of these tubulin multi-gene families can lead to the presence of multiple aor 13-tubulin polypeptides within individual cells. However, at least in the case of ¢t-tubulin, this is not the only method by which an individual cell can provide itself with a variety of tubulin isotypes. Two well-defined, posttranslational modifications have been described that produce alternative isotypes of a-tubulin. The ct-tubulin polypeptide can undergo both acetylation and detyrosination; in both cases the posttranslational modification appears reversible to produce, respectively, the original unacetylated and tyrosinated form of the polypeptide (1, 15-17, 30). The posttranslational acetylation of a-tubulin was discovered in the unicellular green alga Chlamydomonas reinhardtii. In this and other organisms such as trypanosomes (20, 28, 29), the major ct-tubulin detected in the flagellum possesses unique electrophoretic coordinates on a two-dimensional gel. This particular a-tubulin isotype (ct3) is formed via a posttranslational event from the tt-tubulin (al) encoded by the tubulin mRNA and the modification appears to occur either immediately before or immediately after the inclusion of a tubulin polypeptide into the microtubule. In Chlamydomonas, this posttranslational modification has been shown to involve the acetylation of the e-amino group of a lysine residue in the a-tubulin polypeptide (17). More recently, the enzymatic activity responsible for the acetylation reaction has been identified and characterized in isolated flagella of Chlamydomonas (10). The a-tubulin acetylase has been shown to have high specificity for a-tubulin, however the Chlamydomonas enzyme will acetylate the a-tubulin of both Chlamydomonas and mammalian brain. An important development in understanding the cellular distribution of acetylated tubulin has come with the recent description of a monoclonal antibody that specifically recognizes this form of ct-tubulin (24). The antibody was raised against the tubulin from the axonemes of sea urchin sperm flagella but has been shown to recognize acetylated a-tubulin in cilia and flagella from a variety of organisms. The antigen was not found in the soluble, cytoplasmic pool tubulin, confirming the earlier view of the time of production of the acetylated a-tubulin isotype during microtubule construction in vivo (15, 20). Modification of a-tubulin by acetylation leads to the production of a novel tubulin isotype, the true © The Rockefeller University Press, 0021-9525/87/01/41/9 $1.00 The Journal of Cell Biology, Volume 104, January 1987 41-49 41 on M ay 0, 2017 D ow nladed fom Published January 1, 1987