Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

Abstract

The alga Polytomella contains several organelles composed of microtubules, including 4 flagella and hundreds of cytoskeletal microtubules. Brown and co-workers showed (1976) that the flagella could be removed and the cytoskeletons dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in Polytomella cytoplasm, a 35S-labeled cytoplasmic fraction was subjected to 2 cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled Polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated Polytomella flagella by 2 dimensional gel electrophoresis showed that, whereas the .beta.-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the 2 .alpha.-tubulins had distinctly different isoelectric points. As a 2nd method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant .alpha.-tubulin of this in vitro-assembled cytoskeletal tubulin corresponded to the major cytoplasmic .alpha.-tubulin obtained by coassembly of labeled Polytomella cytoplasmic extract with brain tubulin and was quite distinct from the .alpha.-tubulin of purified flagella. Two different microtubule-containing organelles from the same cell are composed of distinct tubulins.