Figure 6.

Role of Cdc48/Npl4/Vms1 Complex in UBB⁺¹-Triggered Cytotoxicity

(A–C) UBB⁺¹ was expressed in strains with elevated levels of Cdc48 or Cdc48-S565G (A) and strains deleted for NPL4 (B) and VMS1 (C). Clonogenicity was determined 2 days after inducing expression before (B) and after acetate stress (A and C). The CFUs obtained using cells carrying vector controls were set to 100% in every experiment.

(D) Clonogenicity of UBB⁺¹-expressing cultures in strains with endogenous (vector control) and elevated levels of Vms1 (Vms1), respectively. Clonogenicity was determined 2 days after inducing expression followed by acetate treatment. The CFUs obtained using cells with endogenous and elevated levels of Vms1, respectively, but lacking UBB⁺¹, were set to 100% in every experiment (not shown).

(E and F) Cell death and oxidative stress was measured 1, 2, and 3 days after inducing expression of UBB⁺¹ and/or Vms1.

(G–I) UBB⁺¹ was expressed in wild-type strain with endogenous (vector ctrl) and increased levels of Vms1 (Vms1). Cellular oxygen consumption (G), mitochondrial membrane potential (H), and cellular ATP levels (I) were determined 2 and 3 days after inducing expression. The oxygen consumption (G), mitochondrial membrane potential (H), and ATP levels (I) measured using cells with endogenous Vms1 were set to 100% in every experiment.

(J) Protein alterations in crude mitochondria upon Vms1 expression. Mitochondria were isolated from cultures expressing UBB⁺¹ in cells with increased or endogenous levels of Vms1, respectively. Protein alterations were quantified by SILAC in two independent experiments. Changes were shown that were significant in both experiments. Blue-labeled proteins are inversely regulated as compared to Figure 5A.

(K) Cellular levels of basic amino acids upon Vms1 expression. Basic amino acids were isolated from cultures expressing UBB⁺¹ in cells with endogenous (vector control) or increased levels of Vms1 (Vms1), respectively. The mean values of amino acids from cells with endogenous Vms1 levels were set to 1.0 for every amino acid.

Data: percentage change values (A–D, G–I) and mean values (E, F, K), respectively. Error bars: SE. p values: ^xp < 0.1, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. See Tables S1, S3, and S4 and Figure S6.