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

Abstract

Cells of eukaryotic organisms exhibit microtubules with various functions during the different developmental stages. The identification of multiple forms of alpha- and beta-tubulins had raised the question of their possible physiological roles. In the myxomycete Physarum polycephalum a complex polymorphism for alpha- and beta-tubulins has been correlated with a specific developmental expression pattern. Here, we have investigated the potential heterogeneity of gamma-tubulin in this organism. A single gene, with 3 introns and 4 exons, and a single mRNA coding for gamma-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 gamma-tubulins. However, immunological studies identified two gamma-tubulin polypeptides, both present in the two developmental stages of the organism, uninucleate amoebae and multinucleate plasmodia. The two gamma-tubulins, called gamma s- and gamma f-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 gamma-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 gamma-tubulins exhibited different sedimentation properties as shown by ultracentrifugation and sedimentation in sucrose gradients. Moreover, gamma s-tubulin was tightly bound to microtubule organizing centers (MTOCs) while gamma f-tubulin was loosely associated with these structures. This first demonstration of the presence of two gamma-tubulins with distinct properties in the same MTOC suggests a more complex physiological role than previously assumed.