Fig. 4. Bro1 is a soluble cytoplasmic protein that associates with endosomal membranes. (A) Cell lysates were centrifuged at 13,000 g and 100,000 g. The total protein content of the P13, P100 and S100 fractions was examined by western blotting. Alkaline phosphatase (ALP) is a vacuolar membrane protein, and 3-phosphoglycerate kinase (PGK) is a soluble cytoplasmic enzyme. (B) Fluorescence microscopic localization of GFP-Bro1 and FM 4-64. In the schematic diagram shown above the micrographs, the predicted domains of Bro1 are indicated as BOD (`Bro1 domain'), CC (coiled-coil) and PRD (proline-rich domain). The inset in the top row of panels shows cells subjected to a continuous incubation with FM 4-64 in order to demonstrate the colocalization of GFP-Bro1 with FM 4-64-positive endosomal structures (arrows). Arrows in the bottom set of panels indicate class E compartments. (C) The P13 fraction from vps4 cells was loaded at the bottom of a sucrose density gradient and centrifuged to equilibrium. Fractions of the floating material (F), the non-floating material (NF) and the pellet (P) were collected and analyzed by western blotting. (D) Subcellular fractionation and western blotting was performed as described in A using vps4 cells transformed with low-copy plasmids that encode the vps4^K179A (pMB24) or vps4^E233Q (pMB49) alleles (Babst et al., 1997).