Monomeric myosin V uses two binding regions for the assembly of stable translocation complexes

*Institute of Structural Biology, GSF–National Research Center, 81377 Munich, Germany;
^†Department of Chemistry and Biochemistry, Gene Center of the Ludwig Maximilians University Munich, 81377 Munich, Germany;
^¶Munich Center for Integrated Protein Science, Feodor-Lynen-Strasse 25, 81377 Munich, Germany,
^‡Department of Chemistry, Technical University Munich, 85748 Garching, Germany; and
^§Caesar Research Center, 53175 Bonn, Germany

Edited by Edward D. Korn, National Institutes of Health, Bethesda, MD, and approved October 29, 2007 (received for review July 19, 2007)

Abstract

Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most plus-end directed myosins, which constitute the vast majority of all myosin motors, form stable dimers and interact constitutively with their cargo complexes. To date, little is known about regulatory mechanisms for cargo-complex assembly. In this study, we show that the type V myosin Myo4p binds to its cargo via two distinct binding regions, the C-terminal tail and a coiled-coil domain-containing fragment. Furthermore, we find that Myo4p is strictly monomeric at physiologic concentrations. Because type V myosins are thought to require dimerization for processive movement, a mechanism must be in place to ensure that oligomeric Myo4p is incorporated into cargo-translocation complexes. Indeed, we find that artificial dimerization of the Myo4p C-terminal tail promotes stabilization of myosin-cargo complexes, suggesting that full-length Myo4p dimerizes in the cocomplex as well. We also combined the Myo4p C-terminal tail with the coiled-coil region, lever arm, and motor domain from a different myosin to form constitutively dimeric motor proteins. This heterologous motor successfully translocates its cargo in vivo, suggesting that wild-type Myo4p may also function as a dimer during cargo-complex transport.

Footnotes

^‖To whom correspondence should be addressed at:
Gene Center LMU/GSF, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
E-mail: niessing{at}lmb.uni-muenchen.de
Author contributions: A.H., C.K., R.-P.J., and D.N. designed research; A.H., T.-G.D., S. Jellbauer, K.R., and C.K. performed research; A.H., C.K., S. Jaklin, M.M., J.B., and R.-P.J. contributed new reagents/analytic tools; A.H., T.-G.D., S. Jellbauer, K.R., C.K., R.-P.J., and D.N. analyzed data; and A.H., M.M., R.-P.J., and D.N. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0706780104/DC1.