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

摘要

Evidence indicates that elongation factor-1 alpha (EF-1 alpha), a ubiquitous and abundant protein factor involved in the first step of peptide elongation, is also associated with the cytoskeleton in a variety of organisms. Although the effects of these associations on EF-1 alpha's translational function have not been examined, the associations do appear to result in non-passive effects on the cytoskeleton. A carrot homolog of EF-1 alpha, pp 50, has been reported to interact with microtubules in vitro, inducing the formation of microtubule bundles that can be dissociated by Ca2+/calmodulin. The characterization of anti-pp 50 antibodies is reported here. Immunocytochemistry, using anti-pp 50 and anti-tubulin antibodies, was used to investigate the co-localization of pp 50 and microtubules in situ. In carrot protoplasts fixed after detergent lysis, at least a fraction of pp 50 appears to be associated with microtubules. Treatment of such protoplasts with amiprophos-methyl (APM) reduced both the presence of microtubules and the co-localizing pp 50-associated fluorescence. In taxol-treated protoplasts, increases in both microtubules and the colocalizing pp 50-associated fluorescence were observed. When carrot protoplasts were fixed prior to detergent extraction, confocal laser scanning microscopy likewise revealed co-localization. Furthermore, what is likely to be a fluorescence resonance energy transfer (FRET) between fluorochromes associated with anti-pp 50 and anti-tubulin reporters was observed, indicating that some pp 50 is intimately associated with microtubules. The in situ cytoarchitectural evidence is consistent with a function previously proposed for pp 50 based on in vitro experiments - that pp 50 is a plant microtubule-associated protein (MAP) whose function can be modulated by a Ca2+/calmodulin signal transduction mechanism in plant cells. [References: 51]