Membrane accumulation at class E compartments. (A–D) Quantification of endosome morphologies in the temperature-shifted vps4^ts cells from Fig. 1 (frequency relative to vps4^ts cells maintained at 26°C, n = 50–300 cell profiles). (E) Mean membrane surface areas of MVBs (including ILVs) relative to individual class E compartment cisternae in electron tomograms of wild-type cells (0.096±0.013 µm², N = 11), vps4Δ cells (0.240±0.061 µm², N = 10, P<0.05) and snf7Δ cells (0.300±0.0561 µm², N = 15, P<0.01). (F–I) Quantification of class E compartment biogenesis using confocal fluorescence microscopy. Class E compartments were labeled with FM 4-64FX, a fixable FM 4-64 analog. (F,G) Fluorescence micrographs of Sna3–GFP and FM 4-64FX in temperature-shifted vps4^ts cells. The indicated times include a 30-minute fixation step. Closed arrowheads, class E compartments marked by both fluorophores; open arrowheads, vacuoles depleted of Sna3–GFP. Scale bar: 2 µm. Images are representative of two independent experiments. (F) Cells shifted to 38°C for the indicated times. (G) Cells shifted to 38°C for 70 minutes and returned to 26°C for the indicated times. (H,I) Quantification of class E compartments marked by both Sna3–GFP and FM 4-64FX in the experiment shown in F and G [mean frequency (± s.e.m.) relative to cells maintained at 26°C, n = 140–220 cells over two independent experiments].

Vps21 is required for biogenesis of class E compartments but not MVBs. (A–D,G–J) Electron micrographs of the indicated strains. Scale bars: 100 nm. Arrowhead in B, MVB. (E) Quantification of MVBs and class E compartment cisternae represented as frequency of structure per cell profile, n = 50. (F) Quantification of cell profiles containing MVBs, class E compartments, or aberrant vesicle clusters; n = 50–100.

Vps21 activity is not required for endosomal membrane trafficking. Confocal fluorescence micrographs of FM 4-64-stained cells expressing GFP–Cps1. Scale bar: 2 µm.

Vps21 is concentrated at class E compartments in its active GTP-bound state. (A–C) Confocal fluorescence micrographs of FM 4-64-stained cells expressing the indicated GFP fusion proteins. Scale bar: 2 µm. Arrowheads, colocalization with FM 4-64-stained class E compartments. (D) Subcellular fractionation of wild-type and vps4Δ cells. PGK1 and mitoporin (Por1) were used as fiducial markers for cytosol and membrane fractions. T, total lysate after 1000 g spin; P15, membrane-associated 15,000 g pellet fraction; S15, cytosolic 15,000 g soluble fraction. (E) Extraction of Rab GTPases Vps21 and Ypt7 by RabGDI. P15 membrane pellets prepared from wild-type or vps4Δ cells were resuspended in lysis buffer or buffer supplemented with 3 µM Gyp1^TBC, and incubated for 15 minutes on ice. Indicated samples received 9 µM recombinant GDI immediately prior to another 15,000 g spin in which all samples were again separated to membrane-bound (P) and soluble fractions (S). Alkaline phosphatase (ALP) served as a fiducial marker for sedimentation of membranes.

ESCRT disruption inhibits the completion of Rab5–Rab7 conversion at endosomes. (A–E,I) Confocal fluorescence micrographs of FM 4-64-stained cells expressing the indicated GFP fusion proteins. Scale bars: 2 µm. Closed arrowheads, colocalization with FM 4-64-stained class E compartments; open arrowheads, FM 4-64-stained class E compartment without GFP colocalization. In E, identical exposure settings were used for GFP in wild-type and vps4Δ. Exposure settings for I were identical to those for A, and strains used for these panels all contained chromosomal integrations of Vps8–GFP controlled by the endogenous VPS8 promoter. (F) Western blot using antibodies against Ccz1 or Mon1 to detect proteins from total lysates that were bound to IgG Sepharose. The PEP4 gene was deleted in each strain to inactivate vacuolar proteases prior to lysis. (G) Western blot analysis of total cell extracts from strains used for D and E. PGK1 was used as loading control. (H) Diagram of Vps21 and Ypt7 GEFs and effector complexes.