A single gamma-tubulin gene and mRNA, but two gamma-tubulin polypeptides differing by their binding to the spindle pole organizing centres

Cells of eukaryotic organisms exhibit microtubules with various functions during the different developmental stages. The identification of multiple forms of α- and βtubulins had raised the question of their possible physiological roles. In the myxomycete Physarum polycephalum a complex polymorphism for α- and β-tubulins has been correlated with a specific developmental expression pattern. Here, we have investigated the potential heterogeneity of γtubulin in this organism. A single gene, with 3 introns and 4 exons, and a single mRNA coding for γ-tubulin were detected. They coded for a polypeptide of 454 amino acids, with a predicted molecular mass of 50,674, which presented 64-76% identity with other γ-tubulins. However, immunological studies identified two γ-tubulin polypeptides, both present in the two developmental stages of the organism, uninucleate amoebae and multinucleate plasmodia. The two γ-tubulins, called γs- and γf-tubulin for slow and fast electrophoretic mobility, exhibited apparent molecular masses of 52,000 and 50,000, respectively. They were recognized by two antibodies (R70 and JH46) raised against two distinct conserved sequences of γ-tubulins. They were present both in the preparations of amoebal centrosomes possessing two centrioles and in the preparations of plasmodial nuclear metaphases devoid of structurally distinct polar structures. These two γ-tubulins exhibited different sedimentation properties as shown by ultracentrifugation and sedimentation in sucrose gradients. Moreover, γstubulin was tightly bound to microtubule organizing centers (MTOCs) while γf-tubulin was loosely associated with these structures. This first demonstration of the presence of two γ-tubulins with distinct properties in the same MTOC suggests a more complex physiological role than previously assumed. SUMMARY