Isolation of the myosin-V/kinesin heteromotor complex by sucrose gradient fractionation.
The molecular motors myosin-V and kinesin form a complex to transport vesicles within the neuron. Vesicles move long distances along microtubule tracks using kinesin motors and then switch to actin filaments for short distance movement using myosin-V motors. Affinity isolation of native kinesin with a glutathione S-transferase-tagged myosin-V globular tail fusion protein provided evidence that the two proteins can interact with one another to form a heteromotor complex. This complex provides a mechanism by which one motor can regulate the activity of the other, enabling vesicles to switch easily from one track to the other. Although the myosin-V/kinesin interaction has been shown, binding partners of the complex remain unknown. In this study, experiments were designed to isolate and identify proteins within the heteromotor complex from squid brain. Squid optic lobe extracts were loaded onto a 5%-30% linear sucrose gradient and spun at 200,000 g for 14 h. Fractions from the gradient were analyzed by SDS-PAGE and probed for the presence of myosin-V and kinesin via immunoblot. The peak fractions containing both motors of the complex were analyzed further by two-dimensional gel electrophoresis. The 2-D gels revealed approximately 50 protein spots between 30 and 66 kDa and isoelectric points in a pH range of 4-10. Approximately 10 spots in the peak fraction were enriched relative to the extract, and these proteins will be identified by N-terminal sequencing. No high molecular weight bands were visible on the blots in this pH range. Our previous studies have shown that enolase, [alpha]- and [beta]-tubulin, and elongation factor 1[beta] are myosin-V binding partners by affinity isolation. The sucrose density gradient fractionation provides an additional method to isolate binding partners of myosin-V.
Supported by NSF Grant IBN-0131470, Dartmouth College Richter Grant, and MBL-Shifman Award to JPF.
John Paul Flores, Y. L. Lee, and George M. Langford
Dartmouth College, Hanover, New Hampshire
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|Author:||Flores, John Paul; Lee, Y.L.; Langford, George M.|
|Publication:||The Biological Bulletin|
|Date:||Oct 1, 2004|
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