Figure 2.
The Consequences of UBQLN Deficiency in Cells
(A) Wild-type (WT) or UBQLN triple knockout (TKO) cells expressing ATP5G1-HA were pulse-labeled with 35S-methionine for 30 min and chased for 1 hr with unlabeled methionine. Immunoprecipitated ATP5G1-HA was detected using autoradiography. Where indicated, a mixture of CCCP and valinomycin (C/V) was included during the pulse-chase to inhibit mitochondrial import. The right image shows an anti-HA immunoblot of untreated total cell lysate to detect steady-state levels of ATP5G1-HA. The positions of precursor (pre) and mature (mat) forms of ATP5G1 are indicated.
(B) WT, TKO, and Myc-UBQLN1 rescue cells (resc.) were transfected with ATP5G1-HA and analyzed by pulse-chase labeling in the presence of C/V. Pulse time was 5 min, and chase times were from 0 to 40 min. Where indicated, the proteasome was inhibited with MG132.
(C) Detergent-free cytosolic lysates from cells pulse-labeled for 30 min with 35S-methionine were subjected to the separation protocol shown on the left. The indicated fractions were analyzed by SDS-PAGE and autoradiography. The positions of Myc-UBQLN1 (only expressed in the rescue cells) and the precursor and mature forms of ATP5G1-HA (expressed in both cells) are indicated.
(D) WT, TKO, and rescue cells transfected with ATP5G1-HA were pulse-labeled for 30 min with 35S-methionine, separated into detergent soluble (S) and insoluble (P) fractions, and either analyzed directly by autoradiography (top), immunoblotting with anti-HA (bottom), or immunoprecipitation with anti-HA and autoradiography (middle).
(E) WT, TKO, and rescue cells were transfected with plasmids expressing GFP, GFP-Omp25, GFP-Omp25ΔTM, or ATP5G1-HA, separated into detergent-soluble and insoluble fractions, and equivalent amounts of each fraction immunoblotted for the respective antigens. Endogenous Bag6 and the Ponceau-stained blot from one of the experiments are shown to illustrate uniformity of fractionation. The fractions for any given sample were analyzed on the same gel, and the image was assembled from the same exposure in each case.
See also Figures S3 and S4.