Bro1^ΔBOD supports ILV formation. (A) Three-dimensional models reconstructed from 200-nm thick-section electron tomograms of bro1Δ (GOY65) and bro1^ΔBOD (bro1Δ::TEF1p-bro1^ΔBOD; CTY2) cells. The bro1Δ cells have class E compartments, which are flattened stacks of endosomal membranes that generally lack internal vesicles; these stacks are shown in different colors to differentiate individual membranes. For bro1^ΔBOD, the limiting membrane of MVBs are labeled yellow, the ILVs are highlighted in red, and the vacuole limiting membrane is labeled as red mesh. Scale bar = 100 nm. (B) WT (SEY6210), bro1Δ (GOY65), and bro1^ΔBOD (CTY2) were analyzed by ET and quantified for number of ILV per MVB. Asterisk indicates statistically significant differences compared with WT and bro1Δ (P < 0.0001). (C) WT (SEY6210) and bro1^ΔBOD (CTY2) were analyzed by ET and quantified to assess ILV size (diameter), individual BP size (surface area), and the frequency of incomplete ILV budding events (BPs per MVB); 12 MVBs from WT cells (SEY6210) containing 337 ILVs and 11 budding intermediates (BPs), 64 MVBs from bro1^ΔBOD cells (CTY2) containing 831 ILVs and 17 budding intermediates, and 32 class E endosomal compartments from bro1Δ cells (GOY65) were quantified. Data are represented as mean ± SEM. Asterisks indicate statistically significant differences compared with WT (BP per MVB, P = 0.0016; BP size, P = 0.0048).