Fig. 3

Missense and frameshift variants of KICS2 compromise the formation of the KICSTOR complex (A and B) Interaction analysis of key components of the KICSTOR complex using FLAG-tag-specific co-immunoprecipitation (coIP) of lysates from HEK293T cells transfected with HA-tagged wild-type (WT) KICS2, its p.Tyr393Cys and p.Asp296Glu missense variants, or the truncated frameshift variant p.Lys260Asnfs?18 (KICS2-fs), along with MYC-tagged KPTN and FLAG-tagged SZT2, or an empty control vector (mock). SZT2 was purified via its FLAG tag together with its binding partners KICS2 and KPTN. Western blot analysis of input samples and eluates, followed by SYPRO Ruby total protein staining, shows successful purification and precipitation of FLAG-SZT2 (A). Gray arrowheads mark Fab bands. KPTN and KICS2-WT were co-purified as interaction partners of SZT2, while all three KICS2 variants showed diminished binding (B). The previously reported SZT2 interaction partner LAMP2A was not co-precipitated. Overexpressed proteins were detected using tag-specific antibodies. GAPDH served as the loading control. (C) Relative quantification of co-precipitated MYC-KPTN and HA-KICS2, normalized to their expression levels, from n = 3 biological replicates. While KPTN binding was not influenced by the presence of the three KICS2 variants, all variants showed diminished binding to SZT2, with the KICS2-fs demonstrating the strongest reduction (???p < 0.001, ??p < 0.01, and ?p < 0.05, one-sample t test and two-way ANOVA followed by Tukey?s multiple comparison test). (D and E) The interaction analysis was validated using FLAG-tag-specific coIP of lysates from HEK293T cells transfected with HA-tagged WT KICS2, its p.Tyr393Cys and p.Asp296Glu missense variants, the truncated fs variant p.Lys260Asnfs?18 (KICS2-fs), or an empty control vector (mock), along with MYC-tagged KPTN and FLAG-tagged SZT2. HA-KICS2 was purified via its HA tag together with its binding partners SZT2 and KPTN. Western blot analysis of input samples and eluates, followed by SYPRO Ruby total protein staining, shows successful purification and co-precipitation of FLAG-SZT2 (D). SZT2 and KPTN were co-purified as interaction partners of WT KICS2, while interaction with all three KICS2 variants showed diminished binding or, in the case of SZT2 and the C-terminally truncated fs variant of KICS2, even an absence of interaction (E). Overexpressed proteins were detected using tag-specific antibodies. GAPDH served as the loading control. (F) Relative quantification of co-precipitated FLAG-SZT2 and MYC-KPTN, normalized to their expression levels, from n = 3 biological replicates. STZ2 binding to KICS2 variants was strongly reduced for the p.Tyr393Cys variant and practically absent for the C-terminally truncated fs variant. KPTN binding was also reduced, though it was not absent in the case of KICS2-fs (????p < 0.0001, ???p < 0.001, ??p < 0.01, and ?p < 0.05, one-sample t test and two-way ANOVA followed by Tukey?s multiple comparison test).