INPP5K is recruited to the ER via the interaction with ARL6IP1. (A) Left: INPP5K domain structure and deletion constructs used for the experiments shown in B and C. Right: ARL6IP1 domain structure and predicted topology. TM, transmembrane regions. L1 to L3, three cytosolically exposed regions. Segments highlighted in blue were replaced with flexible linkers of equivalent length comprising myc tags and these constructs were used for experiments shown in F. (B–H) Representative confocal images and corresponding line-scan analysis of COS-7 cells coexpressing the EGFP-INPP5K and mCh-ARL6IP1 constructs depicted in A. (B) WT INPP5K and WT ARL6IP1 colocalize in the ER. (C) The C-terminal 17-aa segment of INPP5K is dispensable for colocalization with ARL6IP1. (D and E) Neither the 5-phosphatase domain only (INPP5K^1–361) nor the SKICH domain only (INPP5K^276–448) is sufficient to bind ER-bound ARL6IP1. (F) The INPP5K^I363T patient mutation strongly impairs the recruitment to the ER. (G and H) The recruitment of INPP5K to the ER is strongly reduced when the N-terminal region of ARL6IP1 is replaced by another sequence (G) but remains unchanged when the C-terminal region of ARL6IP1 is replaced (H). The replacement of the L1 segment, but not the L3 segment, of ARL6IP1 nearly abolishes the recruitment of WT INPP5K to the ER. Hot spots of ARL6IP1^L3-myc possibly reflect misfolded proteins. Note these hot spots are not enriched for INPP5K (see Results). Scale bars: 5 µm. (I) Plot of the ratio of EGFP-INPP5K fluorescence intensity on the ER relative to the adjacent cytosol based on line-scan analysis of individual tubules from multiple cells. Data for cells expressing INPP5K^WT and ARL6IP1^WT were the same as those shown in Fig. 1 F. (INPP5K^WT and ARL6IP1^WT: n = 39 ER tubules from nine cells; INPP5K^1–361 and ARL6IP1^WT: n = 31 ER tubules from four cells; INPP5K^276–448 and ARL6IP1^WT: n = 35 ER tubules from four cells; INPP5K^I363T and ARL6IP1^WT: n = 31 ER tubules from four cells; INPP5K^WT and ARL6IP1^L1-Myc: n = 32 ER tubules from six cells; INPP5K^WT and ARL6IP1^L3-Myc: n = 32 ER tubules from four cells). ****, p < 0.0001; n.s., not significant (two-tailed t test). (J) Extracts of HeLa cells transfected with HA-INPP5K and the indicated EGFP-tagged constructs were subjected to anti-GFP immunoprecipitation (IP) and then processed for SDS-PAGE and immunoblotting (IB) with anti-HA antibody. Left: Representative blot from three independent experiments. The bar graph on the right shows quantification of HA-INPP5K coprecipitated with EGFP-tagged proteins normalized to input (from densitometric scans of gel bands). Data are presented as mean ± SEM, n = 3; **, p < 0.01; *, p < 0.05; n.s., not significant (two-tailed t test).