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Identification of Novel Myosin-V Binding Partners by Immunoprecipitation and Column Chromatography

¹ Dartmouth College, Hanover, New Hampshire
² Boston Biomedical Research Institute, Watertown, Massachusetts
³ Pomona College, Claremont, California

Myosin-V, an actin-based motor, and kinesin, a microtubule-based motor, interact to form a "heteromotor" complex. The proteins that couple the heteromotor complex to neuronal vesicles have not been determined. The goal of this study is to identify novel binding partners of the myosin-V/kinesin heteromotor complex in the neuron.

To identify proteins involved in myosin-V/kinesin binding to neuronal cargo, we used a recombinant glutathione S-transferase-labeled globular tail domain of myosin-V (GST-GTD) in immunoprecipitation and column affinity chromatography experiments. Immunoprecipitations were performed using bacterially expressed GST-GTD incubated with clarified squid brain extract. Proteins bound to GST-GTD were separated by SDS-PAGE and individually sequenced. As a complementary method for isolating myosin-V binding partners, an affinity column was generated by attaching GST-GTD to a GSTrap column. Clarified squid brain was applied to the affinity column. After two washes, proteins bound to GST-GTD column were eluted, separated by 2-D gel electrophoresis, and sequenced.

The proteins identified by peptide sequence analysis as myosin-V binding partners in squid brain were elongation factor-ß (EF-1ß), enolase, and - and ß-tubulin. Although the functional significance of these interactions has yet to be determined, it is known that localization of elongation factor-1 to activated synapses is important for maintaining long-term potentiation. The translocation of EF-1ß to the synapse may be myosin-V dependent. In addition, enolase, a glycolytic enzyme, diffuses across the cell membrane at high rates when the neuron is damaged. Myosin-V may be involved in transporting this enzyme to the cell cortex. The association of tubulin dimers in microtubules with the tail of myosin-V may provide a mechanism for linking the actin and microtubule cytoskeletons. Characterizing these interactions will be important in understanding novel roles of myosin-V in memory and in understanding the diversity of cargo transported by this molecular motor in neurons.

Supported by NSF Grant IBN-0131470 and NIH NS-040493.

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by DeSelm, C. J. Articles by Langford, G. M. Search for Related Content PubMed Articles by DeSelm, C. J. Articles by Langford, G. M.