Figure 5. Interplay of LRRK2 with CMA components.

(a) Immunoblot (duplicates) of rat liver lysosomes incubated with wild-type (WT) or G2019S mutant (G/S) LRRK2 alone or with 1mM GTP and/or GAPDH. Bottom: Quantification of lysosome-bound LRRK2 expressed as times the binding without additions. (n=6). (b) Effect of kinase inhibitor on WT, G/S, or the kinase-dead mutant D1994A (KiD) LRRK2 incubated with rat liver lysosomes in presence or not of GAPDH (5 µg). Right: Quantification expressed as fold-times the binding without additions. (n=3–5). (c,d) HEK293 cells transiently transfected with the indicated myc-or GFP tagged LRRK2 constructs (see scheme). Full (full length); M (KFERQ motifs (M1–M8)) (c) Binding to GST-hsc70. Lanes 1–6 show 1/10 inputs. NT: non transfected cells. Immunoblot for hsc70 is shown as loading control. Top right: Quantification of LRRK2 bound to hsc70. Where indicated, samples were co-incubated with RNase A to compete the KFERQ-mediated binding of hsc70 (n=3–4). (d) Co-immunoprecipiation (IP) of hsc70 with anti-myc or GFP in the same cells. Inputs (Inp: ¼ of starting volume). (e) Binding or LRRK2 to rat liver lysosomes treated with the indicated concentrations of trypsin. LAMP-1 is shown to confirm the integrity of the lysosomes. (f) Co-immunoprecipiation (IP) of LAMP-2A with anti-LRRK2 in lysosomes incubated with WT or G/S LRRK2. Inp: ¼ of starting sample). IgG: immunoglobulin. Values: LAMP-2A recovered in the IP (IP L2A) corrected for the amount of LRRK2 pulled down and expressed as folds WT. (g, h) LAMP-2A immunoblot of rat liver lysosomes incubated alone (none) or with WT or G/S LRRK2 (g) or lysosomes from control (Ctr) and WT and G/S LRRK2 transgenic mice and subjected to blue native electrophoresis (BNE). Arrow: 700KDa multimeric complex. Duplicates are shown. All values are mean+s.e.m. (differences compared to none or untreated (*) or to full length protein (§) were significant for p<0.05). Full-length blots/gels are in Supplementary Figure 12.