Isolation of a 90-kD microtubule-associated protein from tobacco membranes

Jan Marc, Deirdre E. Sharkey, Neil A. Durso, Min Zhang, Richard J. Cyr

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Abstract

The organization and function of microtubules in plant cella are important in key developmental events, including the regulation of directional cellulose deposition. Bridges connecting microtubules to each other and to membranes and other organelles have been documented by electron microscopy; however, the biochemical end molecular nature of these linkages is not known. We have partitioned proteins from a suspension culture of tobacco into cytosolic and membrane fractions, solubilized the membrane fraction with a zwitterionic detergent, and then used affinity chromatography and salt elution to isolate tubulin binding proteins. Dark-field microscopy of in vitro-assembled microtubules showed that the eluted proteins from both fractions induce microtubule bundling and, in the presence of purified tubulin, promote microtubule elongation. Gel electrophoresis of the eluted proteins revealed two distinct sets of polypeptides. Those in the membrane eluate included unique bands with apparent molecular messes of 98, 90, and 75 kD in addition to bands present in both eluates. The cytosolic eluate, in contrast, typically included relatively smaller proteins. The eluted proteins also bound to taxol-stabilized microtubules. Initial immunological characterization using monoclonal antibodies raised against the 90-kD polypeptide showed that it is colocalized in situ with cortical microtubules in tobacco protoplast ghosts.

Original languageEnglish (US)
Pages (from-to)2127-2138
Number of pages12
JournalPlant Cell
Volume8
Issue number11
DOIs
StatePublished - 1996

All Science Journal Classification (ASJC) codes

  • Plant Science
  • Cell Biology

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    Marc, J., Sharkey, D. E., Durso, N. A., Zhang, M., & Cyr, R. J. (1996). Isolation of a 90-kD microtubule-associated protein from tobacco membranes. Plant Cell, 8(11), 2127-2138. https://doi.org/10.1105/tpc.8.11.2127