IN-SITU IMMUNOCYTOCHEMICAL EVIDENCE THAT A HOMOLOG OF PROTEIN TRANSLATION ELONGATION-FACTOR EF-1-ALPHA IS ASSOCIATED WITH MICROTUBULES IN CARROT CELLS

Evidence indicates that elongation factor-1 alpha (EF-1 alpha), a ubiq uitous and abundant protein factor involved in the first step of pepti de elongation, is also associated with the cytoskeleton in a variety o f organisms. Although the effects of these associations on EF-1 alpha' s translational function have not been examined, the associations do a ppear to result in non-passive effects on the cytoskeleton. A carrot h omolog of EF-1 alpha, pp 50, has been reported to interact with microt ubules in vitro, inducing the formation of microtubule bundles that ca n be dissociated by Ca2+/calmodulin. The characterization of anti-pp 5 0 antibodies is reported here. Immunocytochemistry, using anti-pp 50 a nd anti-tubulin antibodies, was used to investigate the co-localizatio n of pp 50 and microtubules in situ. In carrot protoplasts fixed after detergent lysis, at least a fraction of pp 50 appears to be associate d with microtubules. Treatment of such protoplasts with amiprophos-met hyl (APM) reduced both the presence of microtubules and the co-localiz ing pp 50-associated fluorescence. In taxol-treated protoplasts, incre ases in both microtubules and the colocalizing pp 50-associated fluore scence were observed. When carrot protoplasts were fixed prior to dete rgent extraction, confocal laser scanning microscopy likewise revealed co-localization. Furthermore, what is likely to be a fluorescence res onance energy transfer (FRET) between fluorochromes associated with an ti-pp 50 and anti-tubulin reporters was observed, indicating that some pp 50 is intimately associated with microtubules. The in situ cytoarc hitectural evidence is consistent with a function previously proposed for pp 50 based on in vitro experiments - that pp 50 is a plant microt ubule-associated protein (MAP) whose function can be modulated by a Ca 2+/calmodulin signal transduction mechanism in plant cells.