Figure 5. LMX1B interacts with ATG8s in... | Rockefeller University Press

Figure 5.

$LMX1B interacts with ATG8s in a compartment and context-dependent manner. (A) Schematic of human LMX1A and LMX1B functional domains showing predicted NLS and a possible LIR motif in LMX1B (left; the same schematic is shown in Fig. S4 C depicting the location of a possible LIR in LMX1A). Alignment of the proposed LMX1B LIR in different species (right). (B) Sequence alignments of LIR motifs in various human (Hs) proteins designated as tryptophan-, phenylalanine- and tyrosine-type LIRs (refers to the residue at P0 of the LIR). * C-terminal ATG4B LIR; # N-terminal ATG4B LIR. (C) GFP-TRAP co-precipitation of LMX1B-FLAG with GFP-ATG8 family members in HEK293T cells. Immunoblotting for p62 is included as a positive control for binding. 5% protein lysate from equivalent GFP-expressing cells is shown as “input.” (D) Anti-FLAG co-precipitation of GFP-LC3B from lysates of HEK293T cells stably expressing LMX1B-FLAG. A non-specific IgG control is included. Arrow indicates position of GFP-LC3B; * indicates the position of the antibody light chain. (E) CoIP of endogenous LMX1B and LC3B using antibodies against native LMX1B in HEK293T cells. Arrow indicates position of LMX1B and LC3B in the anti-LMX1B lane; * indicates position of antibody heavy chain. (F) GFP-TRAP co-precipitation of LMX1B-FLAG with wild-type or LIR docking mutant (F52A/L53A) GFP-LC3B in HEK293T cells. Immunoblotting for p62 is included as a positive control for binding. 5% of protein lysate was used as control for protein expression (input). (G) In vitro pull-down of LMX1B-FLAG from lysates of HEK293T stably expressing LMX1B-FLAG using sepharose beads covalently attached to recombinant His-ATG8 family members. Immunoblotting for p62 is included as a positive control for binding. (H) GFP-TRAP IP of nuclear and cytosolic fractions from lysates of HEK293T cells co-expressing GFP-LC3B and LMX1B-FLAG. The LMX1B–LC3B interaction occurs exclusively in the nucleus under basal conditions. Fractionation is demonstrated to the left using Lamin B and GAPDH as markers for nuclei and cytosol, respectively. (I) GFP-TRAP IP of nuclear and cytosolic fractions from lysates of HEK293T cells co-expressing GFP-LC3B and LMX1B-FLAG. Comparisons of pull-downs in full nutrients or following 2 h starvation. An LMX1B–LC3B interaction emerges in the cytosol during starvation. 5% of protein lysate from equivalent GFP-expressing cells is shown as a representative of “input.” Immunoblotting for p62 is included as a positive control for binding. (J) GFP-TRAP IP of LMX1B-FLAG in HEK293T cells co-expressing wild-type, acetylation-deficient (K49R/K51R), and acetylation mimic (K49Q/K51Q) GFP-LC3B. Immunoblotting for p62 and ATG3 is included as positive controls for binding. (C–J) Molecular weight markers are shown in kD. PSSM, Position Specific Scoring Matrix.$

LMX1B interacts with ATG8s in a compartment and context-dependent manner. (A) Schematic of human LMX1A and LMX1B functional domains showing predicted NLS and a possible LIR motif in LMX1B (left; the same schematic is shown in Fig. S4 C depicting the location of a possible LIR in LMX1A). Alignment of the proposed LMX1B LIR in different species (right). (B) Sequence alignments of LIR motifs in various human (Hs) proteins designated as tryptophan-, phenylalanine- and tyrosine-type LIRs (refers to the residue at P₀ of the LIR). * C-terminal ATG4B LIR; # N-terminal ATG4B LIR. (C) GFP-TRAP co-precipitation of LMX1B-FLAG with GFP-ATG8 family members in HEK293T cells. Immunoblotting for p62 is included as a positive control for binding. 5% protein lysate from equivalent GFP-expressing cells is shown as “input.” (D) Anti-FLAG co-precipitation of GFP-LC3B from lysates of HEK293T cells stably expressing LMX1B-FLAG. A non-specific IgG control is included. Arrow indicates position of GFP-LC3B; * indicates the position of the antibody light chain. (E) CoIP of endogenous LMX1B and LC3B using antibodies against native LMX1B in HEK293T cells. Arrow indicates position of LMX1B and LC3B in the anti-LMX1B lane; * indicates position of antibody heavy chain. (F) GFP-TRAP co-precipitation of LMX1B-FLAG with wild-type or LIR docking mutant (F52A/L53A) GFP-LC3B in HEK293T cells. Immunoblotting for p62 is included as a positive control for binding. 5% of protein lysate was used as control for protein expression (input). (G) In vitro pull-down of LMX1B-FLAG from lysates of HEK293T stably expressing LMX1B-FLAG using sepharose beads covalently attached to recombinant His-ATG8 family members. Immunoblotting for p62 is included as a positive control for binding. (H) GFP-TRAP IP of nuclear and cytosolic fractions from lysates of HEK293T cells co-expressing GFP-LC3B and LMX1B-FLAG. The LMX1B–LC3B interaction occurs exclusively in the nucleus under basal conditions. Fractionation is demonstrated to the left using Lamin B and GAPDH as markers for nuclei and cytosol, respectively. (I) GFP-TRAP IP of nuclear and cytosolic fractions from lysates of HEK293T cells co-expressing GFP-LC3B and LMX1B-FLAG. Comparisons of pull-downs in full nutrients or following 2 h starvation. An LMX1B–LC3B interaction emerges in the cytosol during starvation. 5% of protein lysate from equivalent GFP-expressing cells is shown as a representative of “input.” Immunoblotting for p62 is included as a positive control for binding. (J) GFP-TRAP IP of LMX1B-FLAG in HEK293T cells co-expressing wild-type, acetylation-deficient (K49R/K51R), and acetylation mimic (K49Q/K51Q) GFP-LC3B. Immunoblotting for p62 and ATG3 is included as positive controls for binding. (C–J) Molecular weight markers are shown in kD. PSSM, Position Specific Scoring Matrix.