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. 2023 Jan 23;21(1):e3001950. doi: 10.1371/journal.pbio.3001950

Fig 3. Dfm1 reduces misfolded membrane protein toxicity through a chaperone-like activity.

Fig 3

(A) Western blot of aggregated (pelleted) versus non-aggregated (soluble) membrane proteins at the ER. Lysates from dfm1Δ cells containing HMG2-GFP, with either add-back of WT DFM1-HA, EV, DFM1-5Ashp-HA, DFM1-AR-HA, and DFM1-AxxxG-HA were blotted using anti-GFP to detect Hmg2. Top: Total fraction. Middle: ER pelleted fraction. Bottom: ER DDM solublilized fraction. (B) Western blot of aggregated versus non-aggregated membrane proteins at the ER as in (A) but with add-back of either WT DFM1-HA, EV, DFM1-F58S-HA, DFM1-L64V-HA, DFM1-K67E-HA, DFM1-Q101R-HA, and DFM1-F107S-HA. (C) DDM solubilized Hmg2-GFP binding to Dfm1-HA was analyzed by Co-IP, using anti-GFP to detect Hmg2 and anti-HA to detect Dfm1-HA. As negative control, cells not expressing Hmg2-GFP were used. Sec61 was analyzed as another negative control for nonspecific binding using anti-Sec61 (3 biological replicates, N = 3). (D) Graphic depicting integrated model of Dfm1’s function in misfolded membrane protein stress. Top: Misfolded membrane proteins in the absence of Dfm1 forming aggregates within the ER membrane. Bottom: Cells with WT Dfm1 or 5Ashp-Dfm1 promoting non-aggregated misfolded membrane proteins and preventing cellular toxicity. Data Information: All detergent solubility assays were performed with 3 biological replicates (N = 3). DDM, dodecyl maltoside; ER, endoplasmic reticulum; EV, empty vector.