Figure 4.

Pivotal Mitochondrial Impairment upon UBB⁺¹ Expression

(A) Oxidative stress levels were measured by flow cytometry 2 days after inducing expression.

(B) Mitochondrial fragmentation. UBB⁺¹ and RFP fused with a mitochondrial targeting sequence were expressed. 2 days after induction, cultures were shifted to fresh media repressing expression, and after 3 hr the proportion of cells with fragmented mitochondria was quantified.

(C–E) Cellular oxygen consumption (C), mitochondrial membrane potential (D), and cellular ATP levels (E) were determined 2 and 3 days after inducing UBB⁺¹ expression. The oxygen consumption (C), mitochondrial membrane potential (D), and ATP levels (E) measured using cells carrying vector controls were set to 100% in every experiment.

(F and G) Protein alterations in crude mitochondria. UBB⁺¹ was expressed for 24 hr and crude mitochondria were isolated by differential centrifugation. (F) Immunoblot demonstrating the steady-state levels of Rip1, cytochrome c (Cyt. c), and the mitochondrial outer membrane protein Por1 as loading control. (G) Quantification of (F). The immunoreactive signals obtained using cells carrying vector controls were set to 100% in every strain and experiment.

(H and I) UBB⁺¹-triggered cytotoxicity in strains deleted from genes encoding mitochondrial cell death (H), and ER-associated proteins (I), respectively. Clonogenicity was determined 2 days after inducing expression followed by acetate treatment. The CFUs obtained using cells carrying vector controls were set to 100% in every experiment.

Data: mean values (A and B), and percentage change values (C–E, G–I), respectively. Error bars: SE. p values: ^∗p ≤ 0.05, ^∗∗p < 0.01. See Table S1 and Figure S4.