The tubulin gene family of Paramecium: characterization and expression of the alpha PT1 and alpha PT2 genes which code for alpha-tubulins with unusual C-terminal amino acids, GLY and ALA.

ABSTRACT

The ciliated protozoan Paramecium harbours a particularly large diversity of microtubule networks, ranging from the elaborate and stable ciliary axonemes and basal bodies to very dynamic cytoplasmic, cortical or intranuclear arrays. Their organization and individual cycle of assembly/disassembly are well known and extensive immunocytochemical studies of the post-translational modifications in the various microtubule systems have been reported. However, in order to better understand the biogenesis of these multiple and diverse microtubule arrays, it seemed necessary to characterize the tubulin gene family. We show that P tetraurelia possesses four alpha- and three beta-genes and we report the cloning and sequencing of two intronless alpha-genes, alpha PT1 and alpha PT2, which code for very similar polypeptides, differing only by their unusual C-terminal amino acids, respectively GLY and ALA. Partial sequencing of the two other alpha-genes suggests an absence of any further isotype diversity. In an attempt to study the expression of alpha PT1 and alpha PT2, polyclonal antibodies were raised against the twelve C-terminal amino acids corresponding to the deduced polypeptide sequences. The reactivity of these anti-sequence antibodies was studied on blots of soluble tubulin and in situ and compared with that of other well characterized anti-alpha-tubulin antibodies. The molecular data show that in Paramecium, like in other ciliates, microtubule diversity does not arise from tubulin isotype diversity. The immunocytological data indicate that the native C-terminal sequences are predominantly detected in transient or nascent microtubule arrays and lead us to propose 1) that the C-terminal TYR, absent in Paramecium and in most cilate species, has no intrinsic functional role; and 2) that post-translational modifications do not seem directly instrumental in the geometry and functions of microtubule arrays.