Figure 3.

Effects of mutations in the C terminus on the association of MID1 with microtubules. (a) COS-7 cells were transfected with a cDNA encoding a GFP-MID1 fusion protein lacking the MID1 C terminus. Microtubules were stained with anti-tubulin antibodies and a Cy3-labeled secondary antibody. (b) COS-7 cells were transfected with a GFP-MID1 fusion protein with a 4-bp deletion in the MID1 C terminus. This protein mutant was previously identified in family “W.” Microtubules were stained with anti-tubulin antibodies and a Cy3-labeled secondary antibody. (c) Reduced microtubule association capacity of GFP-MID1 lacking Met-438 of MID1, the MID1 mutant identified in family “OS5.” Microtubules were stained with anti-tubulin antibodies and a Cy3-labeled secondary antibody. (d) Western blot of sedimented HeLa microtubules reassembled in the presence of cytosol of wild-type GFP-MID1 (lanes 1–4) or mutated GFP-MID1 (lanes 5–8), respectively. Each lane was loaded with 15 μg of protein of the microtubule-containing pellets (lanes 3 and 7), the same amounts of the respective cytosols (lanes 2 and 6), the acetone-precipitated supernatant left after microtubule reassembly (lanes 4 and 8); in lanes 1 and 5, 100 μg of the cell homogenates were loaded. A GFP-specific antibody detected protein of the expected size (100 kDa) only in the microtubule-containing pellet and the cytosol derived from the wild-type expressing cells (lanes 2 and 3), but not in the same fractions derived from the mutant-expressing cells (lanes 6 and7). A small amount also was detected in the acetone-precipitated supernatant from the experiments with the wild-type cells (lane 4). The homogenates (lanes 1 and 5) show that expression levels of GFP-MID1 was comparable in both experiments.