Characterization of Reticulomyxa filosa Tubulin-Isotypes in 2D-PAGE, Immunfluorescence, and MALDI-TOF Mass-Spectrometry: Unusual Tubulins and the Tyr-Glu Paradoxon

Ralf N. Breuker and Manfred Hauser, Lehrstuhl für Zellmorphologie, Ruhr-Universität Bochum, 44780 Bochum, FRG; Kerstin Strupat, Institut für Medizinische Physik und Biophysik, Westfälische Wilhelms-Universität, 48149 Münster

Tubulins are a superfamily of actually at least five members: a- and b-tubulin, the main components of microtubules, g-tubulin [1], that is part of the MTOCs [2], and d- and e-tubulin [3] with yet unknown function. Each tubulin may exist in several isotypes [4]. Most isotypes may be posttranslationally modified. The most common modification is the detyrosination of a-tubulin by carboxypeptidase and tyrosination by tubulin-tyrosine-ligase [5]. Detyrosination of a-tubulin is commonly a marker for stable microtubules [6].
Recently, Linder et al. [7] identified and sequenced the genes for tubulin expression in Reticulomyxa. They found two isoforms of a-tubulin with a calculated molecular weight of 49,7 kD and two forms of b-tubulin with 52,5 and 49,6 kD. We localized the a- and one of the b-tubulins on 2D-PAGES of Reticulomyxa extracts and confirmed the calculated weights by means of IR-MALDI-TOF Mass-Spectrometry of blotted proteins. We also found a new a-tubulin isoform with a molecular weight of only approximately 45kD. Monoclonal antibodies in western blot do not cross-react between the different a-tubulin-isoforms, so we conclude, that they share only few common epitopes.
When evaluating the tyrosination/detyrosination state of the different a-tubulins, we found, that the 49,7 kD a-tubulins described by Linder do not react with any of the tyr- or glu-tubulin specific antibodies in western blot, but bind to the 20C6 anti-tyr-tubulin mAb in immunfluorescence. The 45 kD a-tubulin reacts with both, 27C2 anti-tyr-tubulin and ID5 anti-glu-tubulin mAb in a competitive manner. Therefore we conclude, that the 45 kD a-tubulin of Reticulomyxa is one of the most aberrant tubulins yet characterized, and further on, that "there is not necessarily everything tyr-tubulin, that reacts with an anti-tyr-tubulin antibody".
References: 1. Oakley et al. Nature 338, 662-664 (1989). 2. Joshi et al. .Nature 356, 80-83 (1992). 3. Burns Cell Motil. Cytoskel. 31, 255-258 (1995). 4. Luduena Mol. Biol. Cell 4, 445-458 (1993). 5. Wehland et al. Cell Sci. 88, 185-203 (1987). 6. Webster et al. Proc. Natl. Acad. Sci. USA 84, 9040-9044 (1987). 7. Linder et al. Eur. J. Cell Biol. 69-Suppl. 42, abstract 148 (1996).

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