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. 2019 Apr 18;74(2):347–362.e6. doi: 10.1016/j.molcel.2019.02.010

Figure 3.

Figure 3

Mutational Dissection of NDP52-FIP200 Interaction

(A) Diagram of NDP52 mutation and deletions. ΔSKICH is a deletion from position 1–127. ΔSKICH ΔCLIR is a deletion from 1–148. CLIR null is a V136S mutation in the LC3C binding domain. LIR-like null pertains to AAAA substitution of DYWE at position 203–206 within the non-canonical LIR domain. C425A is a mutation in ubiquitin-binding ZF2 motif. Diagram of FIP200 mutations and deletions. Q4A refers to 4 alanine substitutions within the ATG13-binding domain. LZA refers to 4 alanine mutations in the leucine zipper domain.

(B) Western analysis of Flag-NDP52 WT and mutants (see Figure 3H for NDP52 mutation map), and HA-FIP200 were transiently transfected in HEK293T cells followed by HA immunoprecipitation.

(C) Western analysis of HA-FIP200 immunoprecipitation performed on HEK293T cells overexpressing HA-FIP200 and GFP-NDP52 or GFP-NDP52 CLIR/LIR-like null. Arrow indicates NDP52 band. Asterisks indicate background bands.

(D) Western blot analysis of HEK293T cells transfected with full-length, N1 (1–800), and C1 (800–1,591) HA-FIP200 co-transfected with Flag-NDP52, then subjected to HA immunoprecipitation.

(E) Western blot analysis after HA immunoprecipitation performed in HEK293T cells transfected with HA-FIP200 C1 (800–1,591), C2 (1,300–1,591), C3 (1,400–1,591) and Flag-NDP52.

(F) Western blot analysis after HA immunoprecipitation performed on HEK293T cells transfected with HA-LZ (1,286–1,413 of FIP200), HA-LZA (1,286–1,413 of FIP200; AAAA substitution at positions 1,371, 1,378, 1,385, and 1,392) and Flag-NDP52.

(G) Western blot analysis after HA immunoprecipitation performed on HEK293T cells transfected with full-length HA-FIP200, HA-FIP200 Q4A (full-length FIP200 with AAAA substitutions at positions 582–585), HA-FIP200 LZA (full-length FIP200 with AAAA substitution at positions 1,371, 1,378, 1,385, 1,392), and Flag-NDP52.

(H) Confocal imaging of WT HeLa cells stably expressing mCherry-Parkin and GFP-NDP52 and transiently transfected with WT FIP200 leucine zipper (HA-LZ) or alanine mutant (HA-LZA), then treated with OA for 3 h. Cells were then fixed and stained for HA. Scale bars: 10 μm.

See also Figures S1 and S2.