Figure 5.
TRIM1 binds an N-terminal LRRK2 regulatory loop region via its B-box domain. (a) Coimmunoprecipitation of full-length myc-LRRK2 with GFP-TRIM1 domain constructs in HEK-293T cells (∆BB, TRIM1 construct lacking both B-box domains; ∆CT, TRIM1 lacking C-terminal domain; ∆RF, TRIM1 lacking ring-finger domain; ∆CC, TRIM1 lacking coiled coil domain; ∆FN3, TRIM1 lacking fibronectin III domain; details of constructs in Short et al. (2002). (b) Coimmunoprecipitation of full-length myc-LRRK2 with GFP-TRIM1 B-box domain constructs in HEK-293T cells (ΔRF denotes TRIM170–715; linker denotes TRIM170–117; BB1 denotes TRIM170–164; BB1,2 denotes TRIM170–212). (c) Coimmunoprecipitation of full-length myc-TRIM1 with GFP-LRRK2 domain constructs in HEK-293T cells. LRRK2 constructs include indicated amino acids from full-length LRRK2 sequence and are illustrated in Fig. S4. LRRK2822–892 is sufficient for interaction with TRIM1. (d) Live-cell confocal microscopy of GFP-LRRK2822–982 and mCherry-TRIM1 transiently transfected into H1299 cells. Inset shows higher magnification of region identified by the yellow box. Scale bar = 10 μM. (e) Schematic of LRRK2-TRIM1 domain interaction mediated by the LRRK2 Regulatory Loop (RL, green) and TRIM1BBox1 (red). (f) Coimmunoprecipitation of full-length myc-TRIM1 with GFP-LRRK2 WT and RL alanine scanning mutants. Mutants are full-length LRRK2 constructs with the three amino acid residues indicated mutated to three alanines. (g) Live-cell confocal microscopy of GFP-LRRK2 RL alanine scanning mutants and mCherry-TRIM1 transiently transfected into H1299 cells. Scale bar = 10 μM. (h) Ubiquitination of immunoprecipitated GFP-LRRK2 WT versus RL alanine scanning mutants. All coimmunoprecipitation and microscopy experiments are a representative image of at least three independent experiments. Source data are available for this figure: SourceData F5.

TRIM1 binds an N-terminal LRRK2 regulatory loop region via its B-box domain. (a) Coimmunoprecipitation of full-length myc-LRRK2 with GFP-TRIM1 domain constructs in HEK-293T cells (∆BB, TRIM1 construct lacking both B-box domains; ∆CT, TRIM1 lacking C-terminal domain; ∆RF, TRIM1 lacking ring-finger domain; ∆CC, TRIM1 lacking coiled coil domain; ∆FN3, TRIM1 lacking fibronectin III domain; details of constructs in Short et al. (2002). (b) Coimmunoprecipitation of full-length myc-LRRK2 with GFP-TRIM1 B-box domain constructs in HEK-293T cells (ΔRF denotes TRIM170–715; linker denotes TRIM170–117; BB1 denotes TRIM170–164; BB1,2 denotes TRIM170–212). (c) Coimmunoprecipitation of full-length myc-TRIM1 with GFP-LRRK2 domain constructs in HEK-293T cells. LRRK2 constructs include indicated amino acids from full-length LRRK2 sequence and are illustrated in Fig. S4. LRRK2822–892 is sufficient for interaction with TRIM1. (d) Live-cell confocal microscopy of GFP-LRRK2822–982 and mCherry-TRIM1 transiently transfected into H1299 cells. Inset shows higher magnification of region identified by the yellow box. Scale bar = 10 μM. (e) Schematic of LRRK2-TRIM1 domain interaction mediated by the LRRK2 Regulatory Loop (RL, green) and TRIM1BBox1 (red). (f) Coimmunoprecipitation of full-length myc-TRIM1 with GFP-LRRK2 WT and RL alanine scanning mutants. Mutants are full-length LRRK2 constructs with the three amino acid residues indicated mutated to three alanines. (g) Live-cell confocal microscopy of GFP-LRRK2 RL alanine scanning mutants and mCherry-TRIM1 transiently transfected into H1299 cells. Scale bar = 10 μM. (h) Ubiquitination of immunoprecipitated GFP-LRRK2 WT versus RL alanine scanning mutants. All coimmunoprecipitation and microscopy experiments are a representative image of at least three independent experiments. Source data are available for this figure: SourceData F5.

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