Fig 1.
Simplified model of the ESCRT assembly pathway.
Graphic representation of ESCRT proteins assembling at the endosomal membrane during the formation of endosomal ILVs. ESCRT-0 complex (pink) binds and clusters ubiquitinylated membrane protein (gray) and recruits ESCRT-I (blue/green). ESCRT-I recruits ESCRT-II (purple), resulting in the recruitment of ESCRT-III subunits (orange/yellow). ESCRT-III induces membrane deformation through the creation of the Snf7-Vps2-Vps24 heteropolymer and allows subsequent recruitment of the accessory proteins (green) Bro1p and Doa4p to deubiquitylate target proteins. Lastly, the ATPase Vps4p is recruited to complete ILV formation and disassemble the complex. Diagram created in BioRender. Berardi, L. (2025) https://BioRender.com/9vzuw43.
Fig 2.
Wbm0152 expression inhibits ESCRT-dependent ubiquitylated protein turnover.
RapIDeg yeast strains harboring either a vector control or the β-estradiol inducible wBm0152 or EcPAL expression vectors were assayed for vacuolar turnover of Fth1-GFP-FKBPx2, as in Methods. A) Representative images showing intracellular localization of Fth1-GFP in response to 1 µg mL-1 rapamycin; bar = 5 µ. Vacuolar localization of Fth1-GFP counted as either ‘diffuse’ lumenal localization (white arrow) or ‘vacuolar’ membrane localization (asterisk). B) Representative α-GFP immunoblot of whole cell lysates prepared from cells in A).C) Results of One-Way ANOVA performed on percentage of cells displaying indicated phenotype in each condition compared to the empty vector control. (****) p ≤ 0.0001; (***) p ≤ 0.001; (ns) not significant; n > 300 cells for each condition.
Fig 3.
Wbm0152 expression phenocopies ESCRT mutant growth defects.
BY4742 yeast strains harboring the indicated ESCRT subunit deletion, an empty vector control, or the constitutively expressing pYESTDH3-wBm0152 (Methods) were grown overnight at 30° C in CSM-URA media. Cultures were diluted to a final OD600 = 1.0, serially diluted 10-fold four times into sterile water, and 10 µL of each dilution was spotted to CSM media lacking uracil either lacking or containing 15 µg mL-1 Congo red. Plates were incubated at 30° C and imaged after 72h.
Fig 4.
wBm0152 expression inhibits intralumenal vesicle formation in vivo.
Yeast strains harboring either the vector control (A-C) or wBm0152 expression vector (D-F) were visualized via cryo-fixation electron tomography (Methods, bar = 100 nm) and membrane structures were modeled as indicated: MVBs, yellow; degradative vacuole, red; endosomal ILVs, red spheres; ER, green; lipid droplets; white. Raw images labeled to show degradative Vacuole (V), MVBs (MVB) and lipid droplets (LD). G) Quantification of total MVBs (left) or ILVs per MVB (right) observed across 100 cell profiles for the indicated yeast strain. H) Calculated diameters of all ILVs observed in cell profiles from (G). I) The total surface area of the limiting membranes of observed MVBs or ILVs from the tomogram profiles of the indicated strains were quantified, as outlined in Methods.
Fig 5.
Wbm0152 colocalizes with core ESCRT subunits in vivo.
A) SEY6210 yeast strains harboring the indicated chromosomal ESCRT subunit-GFP or Mnn9-GFP and the pYES-wBm0152-mRuby or pYES-Snf7-mRUBY expression vector were grown to saturation at 30° C in selective media. Cells were diluted 1:10 into fresh selective media containing 1 µM β-estradiol, outgrown for 6h at 30° C, and imaged. Bar = 5 µ. B) Truncated violin plot showing calculated Pearson Correlation Coefficient of the positive (Bro1/Snf7 colocalization) and negative controls (Mnn9/Snf7) colocalization along with the scores for the indicated ESCRT:wBm0152 colocalization. One-Way ANOVA analysis with the negative control PCC scores as the reference group was run. (****) p ≤ 0.0001; (ns) not significant; n ≥ 100 cells for each condition.
Fig 6.
wBm0152 binds ESCRT-III subunit Vps2 in vivo.
SEY6210 strains harboring either the N-terminus (VN) or C-terminus (VC) of a Venus-YFP molecule on the C terminus of the indicated ESCRT subunit were transformed with the corresponding copper-inducible pYESCUP1-wBm0152-VN or pYESCUP1-wBm0152-VC plasmid. Strains were grown in CSM media lacking uracil for 18h at 30° C with shaking, diluted 1:10 into fresh selective media lacking or supplemented with 0.5 mM CuSO4, and outgrown for 6 hours before imaging. Bar = 5 µ; images are representative of three separate experiments.
Fig 7.
Wbm0152 activity requires ESCRT assembly.
BY4742 yeast strains harboring the indicated ESCRT subunit deletion, an empty vector control, or the constitutively expressing pYESTDH3-wBm0152 (Methods) were grown overnight at 30° C in CSM-URA media. Cultures were diluted to a final OD600 = 1.0, serially diluted 10-fold four times into sterile water, and 10 µL of each dilution was spotted to CSM media lacking uracil either lacking or containing 15 µg mL-1 Congo red. Plates were incubated at 30° C and imaged after 72h.
Fig 8.
Wbm0152 binds Brugia malayi Vps2 ortholog (Bm6583b) in yeast.
A) Sequence alignment of Vps2 protein from yeast (yVps2) and Brugia Vps2 homolog (BmVps2) in which highlighted residues (*) indicate identical amino acids, (:) indicating highly similar amino acids and (.) indicating similar residues. B) Yeast strain SEY6210 expressing Snf7-VC and harboring either the pRS414-pVPS2-VPS2-VN (top) or pRS414-pVPS2-BmVPS2-VN expression vector were grown in CSM media lacking uracil and tryptophan for 18h at 30° C with shaking, diluted 1:10 into fresh selective media, and outgrown for 6 hours before imaging. C) Yeast strain SEY6210 harboring yeast wild type SEY6210 (left) or vps2∆ (right) strains harboring the plasmids pRS414-pVPS2-Bm6583b-VN (Methods) and pYESCUP1-wBm0152-VC were grown as in B), except fresh selective media either contained or lacked 0.5 mM CuSO4 during outgrowth. Bar = 5 µ; images are representative of three separate experiments.