Supplemental Material

This article contains the following supporting material:

• Figure 1 - (A) Wild type, MYO2-AAA and MYO2-EEE strains were fixed and Myo2p was visualized by immunofluorescence microscopy. The bottom panel shows the three strains after transformation to express GFP-Sec4p, fixed for 15 min. and GFP-Sec4p visualized by microscopy. The percentage of cells showing polarized Myo2p and Sec4p distribution is indicated. (B) The three strains were arrested using α -factor for 2h, then fixed for 2h for visualization of Myo2p and actin by indirect immunofluorescence microscopy. Cells expressing GFP-Kar9p or GFP-Tub1p were fixed for 15 min for visualization of GFP-Kar9p and GFP-Tub1p. Percentage of cells with polarized localization of Myo2p, Act1p and Kar9p is indicated (n=100). In the bottom panel GFP-Tub1p, the percentage of cells with microtubule orientated towards the projection tip is indicated, n=100. (C) The indicated cells were labeled with FM4-64 for 30 min. Excess dye was washed out and the cells were diluted with fresh medium (10 fold) and chased for 6 hrs. The vacuoles were then visualized by microscopy. Vacuole inheritance is calculated as percentage of cells with inherited vacuole in the bud (n=100).Mitochondrial distribution was visualized after incubating cells with Mitotracker (Molecular Probes, Inc) for 30 min, washing and incubating in medium for 30 mins. The percentage of small and medium size cells with mitochondria in the bud is indicated (n=100). Polarization of the TGN was determined in cells expressing GFP-Ypt31p and found to be indistinguishable between the three strains (data not shown).

• Figure 2 - Myo2p fractionates similarly in wild type, MYO2-AAA and MYO2-EEE strains. Exponentially growing cells were harvested and total proteins were prepared as indicated Equal amounts of proteins were centrifuged at 100,000 rpm. Equal volume of total proteins, supernatant (S100) and pellet (P100) were loaded on 5% SDS-PAGE and Myo2p was visualized by immunoblotting.

• Table 1