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. 2021 May 28;220(8):e202012104. doi: 10.1083/jcb.202012104

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© 2021 Yang et al.

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Figure S1. — The ESCRT machinery is essential for the degradation of VM proteins, related to Fig. 4.(A) Western blots showing the degradation of Zrt3*-GFP in WT and vps4Δ cells. (B) Quantification (±SD, n = 3) of protein levels in A. (C) Time-lapse imaging of Zrt3*-GFP in WT and vps4Δ strains during a 6-h rapamycin treatment. Both strains were grown in the same chamber. Arrows highlight the intralumenal structures in vps4Δ cells. (D) Western blots showing the degradation of Fth1-GFP in WT and vps4Δ strains. Asterisk indicates a 35-kD cleavage product of Fth1-GFP when expressed from a TET-OFF plasmid. (E) Quantification (±SD, n = 3) of protein levels in D. (F) Time-lapse imaging of Fth1-GFP in WT and vps4Δ cells during a 9-h rapamycin treatment. Both strains were grown in the same chamber. Arrows highlight the intralumenal structures in vps4Δ cells. (G–I) During rapamycin treatment, Zrt3* (G), Fth1 (H), and Cot1 (I) were sorted into punctate structures that colocalized with Vps4-3HA-mCherry. (J) Percentage of yeast cells that contain colocalized punctae. White dashed lines indicate the periphery of yeast cells. Numbers on top of the columns were the counted cell number. Dox, doxycycline; FL, full-length protein fused with GFP; Rapa, rapamycin. Scale bars, 3 µm.