Processivity of chimeric class V myosins

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Unconventional myosin V is crucial for intracellular cargo transport, taking long steps along actin filaments.
• Processive run length is key for myosin V's long-distance cargo movement.
• Understanding structural features influencing run length is vital for cell biology.

Purpose of the Study:

• To investigate the structural determinants of myosin V processive run length.
• To analyze the properties of chimeric myosins combining mouse and yeast (Myo4p) class V myosins.
• To elucidate the role of specific myosin V regions, particularly loop 2, in actin binding and processivity.

Main Methods:

• Construction and analysis of chimeric myosin V constructs, including Y-MD (yeast motor domain on mouse myosin V neck and rod).
• Measurement of myosin V run lengths at varying salt concentrations.
• Biochemical assays, including steady-state ATPase assays, to determine kinetic parameters like K(m) for actin.

Main Results:

• A Y-MD chimera exhibited longer run lengths than wild-type mouse myosin V at low salt.
• Mouse myosin V run length showed minimal salt dependence, while Y-MD's decreased steeply with ionic strength.
• Yeast loop 2, when incorporated into mouse myosin V, also showed steep salt dependence and a higher K(m) for actin.
• Yeast loop 2 has no net charge, contrasting with the +6 charge of wild-type loop 2.

Conclusions:

• A positively charged loop 2 is important for maintaining myosin V processivity at physiological ionic strength.
• High affinity for actin, influenced by loop 2 charge, is critical for sustained processive movement.
• Structural variations in loop 2 significantly impact myosin V's ability to maintain long run lengths.