Neuronal ceroid lipofuscinosis protein CLN3 interacts with motor proteins
and modifies location of late endosomal compartments
Cellular and Molecular Life Sciences
Kristiina Uusi-Rauva, Aija Kyttälä, Rik van der Kant, Jouni Vesa,
Kimmo Tanhuanpää, Jacques Neefjes, Vesa M. Olkkonen, Anu Jalanko
Corresponding author: Anu Jalanko, National Institute for Health and Welfare, Helsinki, Finland.
Phone: + 358 20 6108392; Fax: +358 20 610 4744 8480; e-mail:
CLN3E295K and CLN3∆ex7-8 mutations disturb interactions between CLN3, Xpress-Rab7 and Xpress-RILP proteins. COS-1 cells were transfected with CLN3 or CLN3 mutant proteins in the absence or presence of Xpress-tagged Rab7 and/or RILP proteins. Except Xpress-tagged Rab7 and RILP, other CLN3-interacting proteins were endogenously expressed. Co-immunoprecipitation experiments involving CLN3∆ex7-8 were performed with anti-CLN3 1-33 antibody, and experiments involving CLN3E295K were performed either with anti-CLN3 1-33 (c, d) or anti-CLN3242-258 (a) antibodies. Quantities of indicated immunoprecipitated proteins were analysed by quantitative Western blotting using Scion Image software (Scion Corp.). Total pixel values of each protein band of interest with background corrections were normalised according to the amount of corresponding immunoprecipitated CLN3/CLN3 mutant. Relative binding affinities were analysed in wild type–mutant pairwise manner (a, binding affinity of wild type = 1), or if replicates were analysed on the same blot, averaged absolute values were compared (c). Each data point represents mean ± s.e.m. of 2-3 experiments. Student’s one-tailed t-test was used for statistical analysis. Quantified binding affinities of CLN3E295K and CLN3∆ex7-8 mutant proteins to dynactin subunit p150Glued, kinesin-2 (monitored with anti-KIF3A), dynein (monitored with anti-dynein intermediate chain, DIC), and Xpress-Rab7 [*P = 0.004, **P = 0.093, ***P = 120] (a) and representative Western blot results (b) show that CLN3∆ex7-8 but not CLN3E295K interacts less efficiently with Xpress-Rab7 than the wild type CLN3 (in the absence of Xpress-RILP). Quantified binding affinities of wild type CLN3 and CLN3E295K to Xpress-tagged Rab7 and RILP under different conditions [P < 0.03 (*) and P < 0.003 (**)] (c) and representative Western blot results (d) show that CLN3E295K mutant binds significantly less Xpress-RILP than the wild type CLN3, especially when Xpress-Rab7 is co-expressed with Xpress-RILP and CLN3. In addition, compared to wild type protein, CLN3E295K interacts less efficiently with Xpress-Rab7 in cells simultaneously expressing CLN3 and Xpress-tagged Rab7 and RILP (c, compare to a)