FIGURE 3:

Microtubule sliding velocities are decreased in both pacrg and fap20 mutants and can be restored to WT levels upon addition of their respective protein. (A) Compared with WT, the mutants pacrg (pf12), fap20 (RL11), and pacrg; fap20 have a significantly slower microtubule sliding velocity (Tables 1 and 2) in sliding buffer. This decrease in velocity is rescued upon the addition of 2.5 µg of purified expressed His-PACRG, His-FAP20, or both proteins to 25 µg of mutant axonemes. Axonemes from cells rescued with their WT gene (pf12R and fap20R) have sliding velocities similar to mutant axonemes with their WT expressed protein added (Tables 1 and 2). Axonemes from pf18, a central pair mutant, were used as a control. No increase in sliding velocity was observed with addition of either expressed protein. n.s., not significantly different; asterisk, significantly different than the same strain’s value in sliding buffer. (B) Pelleting assay control experiment using purified PACRG protein and buffer. Anti-PACRG Western blot of purified His-PACRG shows minimal His-PACRG pelleting (P) in the absence of axonemes. Most of the protein is found in the supernatant (S). (C) Pelleting assay using purified PACRG protein and isolated axonemes. Anti-PACRG Western blots of axonemes incubated with His-PACRG (25 μg of axonemes with 2.5 μg of His-PACRG) and pelleted. Most of the His-PACRG is found in the pellet (P) fraction, indicating that His-PACRG binds pacrg, fap20, and pacrg:fap20 axonemes, all of which have no or minute amounts of PACRG endogenous protein. A small amount of His-PACRG pellets with pf18 axonemes, which assemble PACRG protein, was used as a control. For complete results of the pelleting assay, see Supplemental Figure 2.