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1 DOI:.38/ncb327 a b Sequence coverage (%) IP: -GFP isoform IP: GFP IP: -GFP IP: GFP Sequence coverage (%) IP: -GFP IP: GFP isoform IP: Control IP: Peptide Sequence Start End K.GVVEEWLSEFK.T 7 7 K.TLPETSLPNYATNLK.D 8 32 K.SSLVSSLYK.V 43 K.PSVYHEPSSIGSMALTESALSQHGLSK.V K.ALDDIIYR.A R.AQLELYPEPLLVANAIK.A R.CIQVEITPTSSR.I 3 3 K.RHEQPDNNNDATELGILVIPEISVTNVAGER.T R.TLGEIDAQHIQGVQETATDPR.T K.MMEDGINSPGR.V c WT KO d Human Macaque brain brain G W G W Human fibro. C P IB: Supplementary Figure /B are identified as interaction partners of /B, and is expressed as two splice forms, with the 58 splice form the major form in mammalian brain. (a) HeLa cells stably expressing -GFP, -GFP, or GFP only were subjected to GFP immunoprecipitation. The purified proteome was digested and analyzed by LC-MS/MS, and peptide and protein identities were determined using MaxQuant. Shown is the sequence coverage of (left) and (right) from two separate experiments: -GFP vs. GFP IP (black bars) and -GFP vs. GFP IP (white bars). (b) Top, Domain cartoon of the two splice forms of (isoform, 58 corresponding to NCBI NP_59.2; isoform 2, 47, corresponding to NCBI XP_ ), with regions common to both splice isoforms in cyan and regions unique to each splice isoform in yellow (isoform ) and magenta (isoform 2). Bottom, Confirmation of the identity of the 47 splice form. Immunoprecipitations using an anti- antibody raised against residues 2-38 of human or a negative control (rabbit IgG) were performed from lysates of primary human skin fibroblasts and analyzed by SDS-PAGE followed by staining. The band at ~47 in the anti- sample was excised and analyzed by mass spectrometry. Shown is the list of identified peptides; those colored in magenta are unique to isoform 2 (XP_ ). (c) Immunoblot analysis of from lysates from lung of wild-type (WT) and knockout (KO) mice. (d) Immunoblot analysis of in lysates from gray matter (G) and adjacent white matter (W) samples from human or macaque brain (parietal cortex) and control (C) or patient-derived, -deficient (P) primary human skin fibroblasts, demonstrating that isoform is the major form in brain, and isoform 2 is the major form in skin fibroblasts. Arrowheads denote background bands at ~43 in mouse lung (c) and ~57 in fibroblasts (d). Shown are representative immunoblots from three independent experiments.
2 a b GFP--N mcherry GFP--N mcherry GFP--N PM-mCherry GFP--N PM-mCherry -GFP mcherry -GFP mcherry -GFP PM-mCherry -GFP PM-mCherry + and Supplementary Figure 2 is a soluble, cytosolic protein that is recruited to the plasma membrane by and. COS-7 cells were transfected with the indicated combinations of the following plasmids: GFP--N, -GFP, soluble mcherry (mcherry), a plasma membrane-targeted mcherry containing the first residues of Lyn kinase (PM-mCherry) either alone (a) or in combination with -HA and -mtagbfp (b). The cells were imaged live by confocal microscopy; shown are single z-plane images. Scale bars, 2 µm. 2
3 a : KD WT : + + -N: + b c -N C57R L53P Supplementary Figure 3 Purification of PI4KIIIa complexes for in vitro kinase assay and representative electron density maps for the -N/ crystal structure. (a) 3xFLAG-tagged PI4KIIIa (wild-type or a kinase-dead point mutant) were expressed in Expi293 cells alone or co-expressed with or both and -N. The kinases (or kinase complexes) were purified by anti-flag affinity chromatography and analyzed by SDS- PAGE, staining with blue. Arrowhead indicates Hsp, which partially co-purified with all samples (and whose identity was verified by mass spectrometry). (b) Top, Experimental electron density map, contoured at.σ, into which the initial model was built. The map was calculated with phases from a SAD experiment after density modification and sharpened using B-factors (-3Å 2 ). The initial model before refinement is shown in green. Bottom, 2Fo-Fc map of the same region, contoured at.σ, with B-factor sharpening (-3Å 2 ). The refined model is shown in green. (c) Point mutations in that underlie HCC are indicated in the /-N structure. Most of the disease-causing mutations in result in premature termination; two known disease-causing missense mutations (L53P and C57R), indicated here, likely cause misfolding. 3
4 a log 2 (Patient/Control) log 2 (Oligodendrocyte/Neuron) 5 b -GFP: + Control Patient log 2 (KO/WT Brain) log 2 (KO/WT Optic Nerve) c 4 d 2 Relative expression α-pi4p (Plasma membrane) Control Patient 2 µm e Oligo Neu WT KO WT KO Oligo Neu CNPase NeuN log 2 (KO/WT) Supplementary Figure 4 Biochemical evidence for defects in PI4KIIIa function in HCC fibroblasts and KO mice. (a) Quantification of changes in protein levels shown in Fig. 5. Shown are the ratios of protein levels between the two indicated samples from immunoblots in Fig. 5, quantified using densitometry. Top left, HCC patient and control fibroblasts (Fig. 5a)., PI4KIIIa, p =.49;, p =.26;, p =.8;, p =.3;, p =.9;, p =.; n = 3 independent experiments (with 3 total technical replicates for all, except 4 for PI4KIIIa). Top right, Primary cultured oligodendrocytes and cortical neurons (Fig. 5d)., PI4KIIIa, p =.9;, p =.43;, p =.48;, p =.27; n = 2 independent experiments (with 2 total technical replicates for all, except 5 for PI4KIIIa and 4 for ). Bottom left, KO and WT mouse brain (Fig. 5e).,, p =.2;, p =.3;, p =.24; n = 3 independent experiments (with 3 total technical replicates for all, except 4 for PI4KIIIa and 5 for ). Bottom right, KO and WT mouse optic nerve (Fig. 5f).,, p =.8;, p =.;, p =.56; n = 2 independent experiments (with 2 total technical replicates for all, except 3 for,, and ). Significance was calculated using either an unpaired two-tailed Student s t-test with equal variance (top left) or a two-tailed, paired ratio t-test (all others). Error bars represent standard deviation. (b) Rescue of PI4KIIIa complex levels in HCC patient fibroblasts by expression of -GFP. Immunoblot (IB) analysis of PI4KIIIa complex components in lysates from control fibroblasts or HCC patient fibroblasts transduced with either a control lentivirus ( ) or lentivirus containing a C-terminally tagged construct corresponding to isoform 2, the major form in fibroblasts (see Supplementary Fig. d). Shown are representative immunoblots from three independent experiments. (c) mrna is more abundant than mrna in human fibroblasts. cdna from human fibroblasts was analyzed by qrt-pcr using primers specific to and. Shown are relative amounts of to, with value normalized to (two-tailed, Student s t-test, unequal variance, p =.4; n = 3 independent experiments). Error bars represent standard deviation. (d) Plasma membrane PI4P levels are reduced in HCC fibroblasts. Immunofluorescence analysis of the plasma membrane pool of PI4P, using an anti-pi4p antibody, in control and HCC patient fibroblasts. Shown are representative average intensity projection images of a confocal z-stack. Quantification and statistical information is provided in Fig. 5c. (e) KO results in a more severe impact on PI4KIIIa complex levels in oligodendrocytes compared to neuronal cells. Left, Immunoblot analysis of PI4KIIIa complex components in cells of the oligodendrocyte (Oligo) and neuronal (Neu) lineage that were immunoisolated from wildtype and KO mice at postnatal day 8. Right, Quantification of these immunoblots, showing the relative amount of each PI4KIIIa complex component in the corresponding cell type in KO compared to in wild-type. Black bars, oligodendrocytes; gray bars, neuronal cells. Two-tailed Student s t-test, unequal variance,, PI4KIIIa: p =.68. : p =.5. : p =.457. : p =.4. : p =.62. : p =.6; n = 3 biological replicates (3 total technical replicates for all, except 4 for PI4KIIIa). Error bars represent standard deviation. 4
5 Supplementary Figure 5 Morphological analysis of myelination in KO and control mice. Light and transmission electron micrographs of myelin in transverse sections of spinal cord (ventral funiculus cervical region), optic nerve, and sagittal sections of two regions of the central area of corpus callosum from WT and KO male mice at age P5. No substantial differences in extent of myelination between the two genotypes can be observed. (a) Representative light microscopy images of semithin ( µm) sections, stained with toluidine blue. (b) Representative transmission electron microscopy images of ultrathin sections (6 nm) contrasted with osmium tetroxide and uranyl acetate. Shown are representative images (n = 3 KO mice; n = 2 WT littermate control mice). Scale bars: 2 µm (a); 2 µm (b). 5
6 Fig. b (top) Fig. b (bottom) Fig. d 5 GFP () FLAG () MYC () HA () Fig. 3a 5 Fig. 3b Fig. 5d Fig. 5a Fig. 5e Fig. 5f MBP SYP MBP 5 5 Supplementary Figure 6 Original scans of immunoblots and -stained gels included in Figures, 3, and 5 and Supplementary Figures, 3, and
7 Supplementary Fig. b Supplementary Fig. 4b Supplementary Fig. c Supplementary Fig. 4e Supplementary Fig. d CNPase NeuN Supplementary Fig. 3 Supplementary Figure 6 continued 7
8 Supplementary Table Legends Supplementary Table Mass spectrometry data for -GFP and -GFP immunoprecipitation. Shown are the average peptide intensities (LFQ) for each protein detected in the different replicates (-GFP vs. GFP or -GFP vs. GFP), the number of total peptides, unique peptides, statistical p value (two-tailed Student s t-test, unequal variance), and the log 2 of the abundance ration. n = 3 independent experiments. Supplementary Table 2 Data collection and refinement statistics. Shown are the data collection and refinement statistics for the -N/ crystal structure. Numbers in parentheses represent values in the highest resolution shell. 8
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a FL (1-2266) NL (1-1190) CL (1191-2266) HA-ICE1: - HA-ICE1: - - - FLAG-ICE2: + + + + FLAG-ELL: + + + + + + IP: anti-ha FLAG-ICE2 HA-ICE1-FL HA-ICE1-NL HA-ICE1-CL FLAG-ICE2 b IP: anti-ha FL (1-2266) NL
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