Fig. 1.

Fractionation and identification of organelle division proteins. (A) All fractions were separated with 10% SDS/PAGE and stained with high-sensitivity CBB. Each set of three lanes corresponds to the fractions from nonarrested (DMSO-treated), S/G2-arrested (camptothecin-treated), and M-arrested (oryzalin-treated) cells, from left to right. Each lane contained the total indicated fraction from the starting cell lysate of ≈8 mg of protein except for the (S) fraction, of which only 1/20th was loaded. The cell lysate was centrifuged on 40% iodixanol bedding to give the top layer of the soluble fraction (S), the bottom pellet (40P) and the middle layer at the interface, which was subsequently centrifuged on 35% iodixanol bedding to give the bottom pellet (35P), and on 20% iodixanol bedding to give the bottom pellet (20P) and upper layers (20U). The pellets 35P and 20P were subsequently further extracted with 1 M NaCl (NaCl) and 2% heptylthioglucoside (HTG), to give an insoluble pellet (P) in which the organelle division proteins were concentrated. The filled arrowheads indicate Dnm2 and open arrowheads indicate Dnm1. Arrows indicate bands subjected to mass spectrometry. The molecular weight marker (m) is indicated on the left. (B) Predicted domain architecture of CMR185C Mda1. From the bands indicated by arrows in A, a protein was identified by mass spectrometry as being encoded by CMR185C in the C. merolae genome; this protein was later named Mda1. (C) Characterization of the affinity-purified antibody to CMR185C. C. merolae total protein was separated and immunoblotted by using antibody raised against the recombinant N-terminal region (1–297) of CMR185C. (D) Protein expression throughout the cell cycle and in arrest. Equal culture volumes of cells were harvested at indicated times (hours) after the onset of synchronization and immunoblotted for Dnm1, α-tubulin (αTub), and Mda1. Cells were also arrested with camptothecin (Cpt) at S/G₂ phase or with oryzalin (Orz) in M phase.