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. 2020 Dec 15;9:e62163. doi: 10.7554/eLife.62163

Figure 4. Influence of lipid=-binding site mutations on Dnf1 transport activity and localization.

(A) NBD-lipid uptake catalyzed by Dnf1 variants expressed on a dnf1,2∆ strain background. (B) NBD-lipid uptake catalyzed by Lem3 variants expressed on a lem3∆ background, in which lipid transport is mediated primarily by Dnf2–Lem3. (C) NBD-lipid uptake catalyzed by Lem3 variants expressed on a lem3∆ dnf2∆ background to assay the influence of Lem3 mutations solely on Dnf1 activity. In (A–C), cells were incubated with NBD-lipids for 30 min and uptake was measured by flow cytometry. The value represents percentage uptake of NBD-lipid in comparison to WT Dnf1 (A) or WT Lem3 (B–C) for each substrate lipid. The variance was assessed with one-way ANOVAs, and comparison to WT was calculated with Tukey’s post hoc analysis. n ≥ 9,± SD, * indicates p<0.05, **p<0.01, ***p<0.001. (D) Localization of GFP-tagged Dnf1 variants expressed in dnf1,2∆ cells. (E) WT Dnf1-GFP co-expressed with Lem3 variants. Arrowheads indicate nuclear envelope/endoplasmic reticulum location. Scale bar = 2 μm (n = 40).

Figure 4.

Figure 4—figure supplement 1. Expression levels of Dnf1 variants.

Figure 4—figure supplement 1.

(A) Immunoblot of FLAG-tagged Dnf1, Dnf1 R264A, Dnf1 Q610A, Dnf1 S611A, Dnf1 W652S and Dnf1 N1226 expressed in the dnf1,2∆ strain. The arrow indicates the full-length FLAG-Dnf1 band and arrowhead indicates a background band present in S. cerevisiae cell lysates recognized by the anti-FLAG antibodies. (B) Quantification of mutant FLAG-Dnf1 expression in dnf1,2∆ relative to WT FLAG-Dnf1. Expression (C) and quantification (D) of FLAG-Dnf1 in a lem3∆ strain expressing mutant forms of Lem3. Empty vector indicates the strain lacks any Lem3 and untagged indicates expression of Dnf1 without the FLAG tag. For quantitation, the full-length FLAG-Dnf1 band was quantified using ImageJ and normalized to the amount of the background band indicated with the arrowheads for each lane, and each mutant Dnf1 variant was plotted as the % of the WT FLAG-Dnf1 band intensity (n = 3).
Figure 4—figure supplement 2. Ability of Dnf1 variants to support viability of flippase-deficient cells.

Figure 4—figure supplement 2.

HIS3-marked plasmids (pRS313) carrying the indicated Dnf1 variants were used to transform a dnf1,2,3∆drs2∆ pRS416-DRS2 strain. Strains were spotted onto minimal media plates (SD) selecting for both plasmids, and pRS416-DRS2 was counter-selected on medium containing 5-fluoroorotic acid (SD-FOA). Strains were incubated at 30°C for 2 d and imaged. The growth phenotype on SD-FOA is an indicator of how well the Dnf1 variant functions in vivo.