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. 2016 May 13;291(27):14373–14384. doi: 10.1074/jbc.M116.729343

FIGURE 9.

FIGURE 9.

Defective SUMOylation of VCP causes altered co-factor binding, impaired ERAD, and increased vulnerability to stress insults. A, defective SUMOylation of VCP leads to altered co-factor binding. His-tagged Ubc9 fusion-directed SUMOylation (UFDS) constructs of VCP were transfected in HEK293 cells and the binding to UFD1, NPL4, p47, or E4B were determined. B and C, the pathogenic or SUMO-deficient mutant VCP showed increased binding to co-factor NPL4 and p47. HEK293 cells transfected with CFP-SUMO1 together with various VCP constructs were analyzed as in A. Results represent mean ± S.E. n = 3 experiments. D and E, pathogenic or SUMO-deficient VCP mutants fails to degrade ERAD substrates. HeLa cells stably expressing the ERAD substrate TCRα-GFP were first transfected with siRNA targeting 5′ UTR to knockdown endogenous VCP, and then transiently transfected with the WT or mutant VCP as indicated. The degradation of ERAD substrate TCRα-GFP was determined by antibody against GFP. Results represent mean ± S.E. n = 3 experiments. F, confirmation of decreased SUMOylation of Ter94KK. Drosophila Ter94 and TER94KK were subcloned in mammalian expression vector and transiently expressed in HEK293 cells. The SUMOylation capacity of Ter94 and TER94KK were assessed by immunoprecipitation (IP) using an anti-V5 antibody followed by immunoblotting. Direct analysis of Ter94 SUMOylation in flies was attempted but not successful as the SUMO signals were beyond the detection limit. G, survival curve of male Elav[C155]/+ (black), Elav[C155]>Ter94 (green), and Elav[C155]>Ter94KK (red) after 1 mm arsenite treatment. Ter94KK is K57R,K59R double mutant, homologous to the human VCP K60–3 mutant. There are only two Lys residues in Ter-94 at this region. Each genotype contained at least 4 vials with 20 flies each, and 2 other replicate experiments showed similar results. Data are represented as mean ± S.E.; n ≥ 4 vials, Elav[C155]>Ter94 (green) were compared with Elav[C155]>Ter94KK for statistical analysis. All transgenic flies have normal lifespan without exposure to stress.