Representative images of cells stained with NBD-PC and FM4-64. This figure complements Fig. 3. (A) Three-dimensional models reconstructed from 200-nm thick-section electron tomograms of bro1Δ (GOY65) cells. This image depicts rare MVBs that contains an ILV. Normal-like endosomes are highlighted by yellow limiting membrane, while other colors depict flattened or tubular endosomes devoid of vesicles. The ILVs are highlighted in red. Scale bar = 100 nm. (B) Selection criteria for scoring positive NBD-PC cells. A usable cell is defined by having (1) readily identifiable FM4-64 labeling of vacuole membrane, (2) readily identifiable NBD-PC signal, and (3) defined vacuoles. An unusable cell is defined by having (1) a fragmented vacuole and (2) out of focus. A positive cell is defined by a diffuse NBD-PC signal within the lumen while lacking a distinct ring on the limiting membrane of the vacuole (defined by FM4-64), while a negative cell is defined by the colocalization of NBD-PC and FM4-64 on the limiting membrane of the vacuole. Scale bars = 5 µm. (C) Representative micrographs of bro1ΔBOD (bro1Δ::TEF1p-bro1ΔBOD; CTY2) and bro1ΔBOD vps4Δ (CTY5) cells stained with NBD-PC to assess lipid MVB sorting and FM4-64 to label vacuoles. Scale bars = 5 µm.
Figure S1.

Representative images of cells stained with NBD-PC and FM4-64. This figure complements Fig. 3. (A) Three-dimensional models reconstructed from 200-nm thick-section electron tomograms of bro1Δ (GOY65) cells. This image depicts rare MVBs that contains an ILV. Normal-like endosomes are highlighted by yellow limiting membrane, while other colors depict flattened or tubular endosomes devoid of vesicles. The ILVs are highlighted in red. Scale bar = 100 nm. (B) Selection criteria for scoring positive NBD-PC cells. A usable cell is defined by having (1) readily identifiable FM4-64 labeling of vacuole membrane, (2) readily identifiable NBD-PC signal, and (3) defined vacuoles. An unusable cell is defined by having (1) a fragmented vacuole and (2) out of focus. A positive cell is defined by a diffuse NBD-PC signal within the lumen while lacking a distinct ring on the limiting membrane of the vacuole (defined by FM4-64), while a negative cell is defined by the colocalization of NBD-PC and FM4-64 on the limiting membrane of the vacuole. Scale bars = 5 µm. (C) Representative micrographs of bro1ΔBOD (bro1Δ::TEF1p-bro1ΔBOD; CTY2) and bro1ΔBOD vps4Δ (CTY5) cells stained with NBD-PC to assess lipid MVB sorting and FM4-64 to label vacuoles. Scale bars = 5 µm.

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