In the Drosophila male germ line, multiple post-mitotic microtubule arrays, including cytoplasmic microtubules, meiotic spindles and axonemes, are used to execute many different functions, which include sperm head shaping, meiosis and sperm motility. Our previous studies showed that the amino acid sequences of the tubulins have a profound impact on many aspects of microtubule structure and function, including formation and stability of the heterodimer, microtubule protofilament number, and axoneme assembly and organization (Fackenthal et al., 1993; Fackenthal et al., 1995; Hoyle et al., 1995; Hoyle et al., 2001; Hutchens et al., 1997; Nielsen and Raff, 2002; Nielsen et al., 2001; Popodi et al., 2005; Popodi et al., 2008; Raff et al., 1997; Raff et al., 2000). In my dissertation research, I examined the role of the C-terminus of alpha84B in tubulin dimerization, microtubule polymerization, and in assembly and organization of microtubule based-structures in the Drosophila post-mitotic male germ cells. Among a series of alpha84B C-terminal truncations that I constructed and tested, alpha84BDeltaC7 (7 C-terminal amino acids removed) was the largest C-terminal truncated a84B-tubulin that produced a stable protein. None of the larger truncations produced protein accumulated to any detectable level. Because the larger truncations all extend into the resolved part of the molecule in 3D structure of the tubulin dimer (Nogales, et al., 1998), I conclude that the proximal region of the alpha-tubulin C-terminus is required for folding.;I carried out functional studies to compare alpha84BDeltaC7 with full-length alpha84B, when it was expressed in the germ line. I found that alpha84BDeltaC7 has the capacity to form stable alpha,beta-tubulin heterodimers with various beta-tubulin species, comparable to full-length alpha84B. Moreover, dimers with alpha84BDeltaC7 can readily polymerize into microtubules. I also found that when it is expressed as the major alpha-tubulin in the post-mitotic male germ cells, alpha84BDeltaC7 can support all microtubule functions including axonemes. However, I demonstrated that alpha84BDeltaC7 cannot fully rescue the sterility of the a84B-tubulin mutation alpha84Bnc33, as full-length alpha84B does. This key finding revealed that the truncated alpha84B-tubulin is not equivalent to full-length a84B-tubulin, even though the truncated form alpha84BDeltaC7 can provide considerable function in all of the microtubule structures in the post-mitotic germ cells in which alpha84B normally is utilized. My dissertation research demonstrates that the requirements for the C-termini in microtubule function are significantly different for alpha84B-tubulin than for beta2-tubulin. Nonetheless, the functional deficits reveal the evolutionary basis for the strong conservation of the acidic alpha-tubulin C-terminus.
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