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Protoplasma

, Volume 190, Issue 3–4, pp 141–150 | Cite as

In situ immunocytochemical evidence that a homolog of protein translation elongation factor EF-1α is associated with microtubules in carrot cells

  • N. A. Durso
  • J. D. Leslie
  • R. J. Cyr
Article

Summary

Evidence indicates that elongation factor-1α (EF-1α), 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-1α'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-1α, 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 microtubuleassociated protein (MAP) whose function can be modulated by a Ca2+/calmodulin signal transduction mechanism in plant cells.

Keywords

Calcium Calmodulin Carrot cells Elongation factor-1-alpha Immunofluorescence microscopy Microtubule-associated protein 

Abbreviations

APM

amiprophos-methyl

BSA

bovine serum albumin

EF-1α

elongation factor-1-alpha

FRET

fluorescence resonance energy transfer

MAP

microtubule-associated protein

PBS

phosphate buffered saline

SDS-PAGE

sodium dodecylsulfate polyacrylamide gel electrophoresis

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Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • N. A. Durso
    • 1
  • J. D. Leslie
    • 1
  • R. J. Cyr
    • 1
  1. 1.Department of BiologyThe Pennsylvania State UniversityUniversity Park

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