ACTIN-DEPENDENT MITOCHONDRIAL MOTILITY IN MITOTIC YEAST AND CELL-FREESYSTEMS - IDENTIFICATION OF A MOTOR-ACTIVITY ON THE MITOCHONDRIAL SURFACE

Using fluorescent membrane potential sensing dyes to stain budding yea st, mitochondria are resolved as tubular organelles aligned in radial arrays that converge at the bud neck. Time-lapse fluorescence microsco py reveals region-specific, directed mitochondrial movement during pol arized yeast cell growth and mitotic cell division. Mitochondria in th e central region of the mother cell move linearly towards the bud, tra verse the bud neck, and progress towards the bud tip at an average vel ocity of 49 +/- 21 nm/sec. In contrast, mitochondria in the peripheral region of the mother cell and at the bud tip display significantly le ss movement. Yeast strains containing temperature sensitive lethal mut ations in the actin gene show abnormal mitochondrial distribution. No mitochondrial movement is evident in these mutants after short-term sh ift to semipermissive temperatures. Thus, the actin cytoskeleton is im portant for normal mitochondrial movement during inheritance. To deter mine the possible role of known myosin genes in yeast mitochondrial mo tility, we investigated mitochondrial inheritance in myo1, myo2, myo3 and myo4 single mutants and in a myo2, myo4 double mutant. Mitochondri al spatial arrangement and motility are not significantly affected by these mutations. We used a microfilament sliding assay to examine moto r activity on isolated yeast mitochondria. Rhodamine-phalloidin labele d yeast actin filaments bind to immobilized yeast mitochondria, as wel l as unilamellar, right-side-out, sealed mitochondrial outer membrane vesicles. In the presence of low levels of ATP (0.1-100 mu M), we obse rve F-actin sliding on immobilized yeast mitochondria. In the presence of high levels of ATP (500 mu M-2 mM), bound filaments are released f rom mitochondria and mitochondrial outer membranes. The maximum veloci ty of mitochondria-driven microfilament sliding (23 +/- 11 nm/sec) is similar to that of mitochondrial movement in living cells. This motor activity requires hydrolysis of ATP, does not require cytosolic extrac ts, is sensitive to protease treatment, and displays an ATP concentrat ion dependence similar to that of members of the myosin family of acti n-based motors. This is the first demonstration of an actin-based moto r activity in a defined organelle population.