Figure 4.

Acetylation of FAAP20 inhibits its degradation. A, left panel: representative images of localization of GFP-FAAP20 variants. U2OS cells were transfected with indicated GFP–FAAP20 WT or mutants, and their subcellular localization was analyzed by GFP epifluorescence. C147A/C150A, UBZ zinc finger mutant; S113A/S117A, CPD mutant; K83Q/K152Q, acetylation mimetic mutant. Scale bar: 10 μm. Right panel: expression of GFP-FAAP20 variants in U2OS cells. B, subcellular fractionation of U2OS cells transfected with Flag–FAAP20 WT or KQ (K83Q/K152Q) mutants. Tubulin and ORC2 immunoblots serve as controls for S and P fractions, respectively. C, a ribbon diagram of the FAAP20 UBZ–ubiquitin complex. The position of FAAP20 lysine 152 within the FAAP20 UBZ domain is shown in red (PDB 2MUR). D, interaction of the FAAP20 UBZ domain with ubiquitin in vitro. 293T cell lysates expressing GFP–FAAP20 WT or KQ mutants were subjected to GST pulldown using recombinant GST or GST-ubiquitin, followed by anti-GFP Western blot (top) or Ponceau S staining (bottom). E, degradation kinetics of Flag–FAAP20 variants. U2OS cells expressing Flag–FAAP20 WT, KQ (K83Q/K152Q), or KR (K83R/K152R) were incubated with 100 μg/ml of CHX for the indicated times, and cell lysates were analyzed by Western blot. The short-lived MCL-1 protein serves as a control for CHX treatment. F, quantification of Flag–FAAP20 levels in E. Error bar indicates mean ± S.D., n = 3 from three independent experiments, *, p < 0.05, KQ or KR compared with WT, Student's t test.