Abstract
Unconventional myosin V takes many 36-nm steps along an actin filament before it dissociates, thus ensuring its ability to move cargo intracellularly over long distances. In the present study we assessed the structural features that affect processive run length by analyzing the properties of chimeras of mouse myosin V and a non-processive class V myosin from yeast (Myo4p) (Reck-Peterson, S. L., Tyska, M. J., Novick, P. J., and Mooseker, M. S. (2001) J. Cell Biol. 153, 1121-1126). Surprisingly a chimera containing the yeast motor domain on the neck and rod of mouse myosin V (Y-MD) showed longer run lengths than mouse wild type at low salt. Run lengths of mouse myosin V showed little salt dependence, whereas those of Y-MD decreased steeply with ionic strength, similar to a chimera containing yeast loop 2 in the mouse myosin V backbone. Loop 2 binds to acidic patches on actin in the weak binding states of the cycle (Volkmann, N., Liu, H., Hazelwood, L., Krementsova, E. B., Lowey, S., Trybus, K. M., and Hanein, D. (2005) Mol. Cell 19, 595-605). Constructs containing yeast loop 2, which has no net charge compared with +6 for wild type, showed a higher K(m) for actin in steady-state ATPase assays. The results imply that a positively charged loop 2 and a high affinity for actin are important to maintain processivity near physiologic ionic strength.
Highlights
We showed that the yeast Myo4p motor domain is capable of supporting processive movement
We found that loop 2 plays an important role in determining processive run lengths in agreement with structural data that show that loop 2 tethers myosin V to actin in the ATP and transition states [22]
Wild-type mouse myosin V remained highly processive over a range of salt concentrations, whereas the processive run lengths of chimeras containing yeast loop 2 decreased sharply as the salt concentration was increased
Summary
Myosin V Constructs—The constructs described below contained yellow fluorescent protein (YFP) followed by a FLAG epitope (DYKDDDDK) at the C terminus to facilitate purification. The characteristic TIRF run length for Y-Loop under these conditions was only 0.27 m (7.5 steps) This implies that close to half of the filaments that landed and attached to a single myosin would have detached before reaching the 0.2-m cutoff, reducing the number of events. This effect is most severe at low myosin densities when actin is most likely to be attached to a single myosin, leading to a steeper dependence of the landing rate on myosin density and a higher n value To our knowledge this is the first time a landing rate assay has been performed on a weakly processive motor. The rate of the reaction was measured from the decrease in absorbance at 340 nm caused by the oxidation of NADH by lactate dehydrogenase This assay minimizes the time-dependent ADP-induced inhibition of actin-activated ATPase activity. Data were fitted to the Michaelis-Menten equation to obtain the maximum ATPase rate (Vmax) and Km
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