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20140826 Supplementary Material FINAL Supplemental Information The Intraflagellar Transport Protein IFT27 Promotes BBSome Exit from Cilia through the GTPase ARL6/BBS3 Gerald M. Liew, Fan Ye, Andrew R. Nager, J. Patrick Murphy, Jaclyn S. Lee, Mike Aguiar, David K. Breslow, Steven P. Gygi, and Maxence V. Nachury SUPPLEMENTAL MATERIALS INVENTORY Figure S1, related to Figure 1 Figure S2, related to Figure 2 Figure S3, related to Figure 3 Figure S4, related to Figure 4 Figure S5, related to Figure 6 SUPPLEMENTAL EXPERIMENTAL PROCEDURES SUPPLEMENTAL INFORMATION REFERENCES Movie S1 Movie S2 100 B LAP A G1 G2 G3 G4 G5 IFT27 N GDPAVGKT D^T DSAGK NKTD ETSVK C GxxxxGK(S/T) D(x)nT DxxGQ NKxD E(A/C/S/T)SA(K/L) 50 WT K68A T19N protein (%) [T19N] [K68A] GFP LAP Actin Relative levels of IFT27 0 WT K68A T19N C siRNA acTub IFT88 GFP acTub IFT88 GFP LAP LAP 100 Ctrl IFT27 GFP 50 mIFT27 IFT27[T19N] IFT27[T19N] positive cilia (%) 0 Actin ctrl mIFT27 Control siRNA siRNA IFT27[T19N]LAP D 100 50 protein (%) S Relative levels of IFT27 0 Parental T19N K68A WT IFT88 Eluate E IFT27LAP Control T19N K68A WT Spectral Spectral Spectral Spectral Count % Count % Count % Count % NCBI M.W. (Unique Sequence (Unique Sequence (Unique Sequence (Unique Sequence Protein Gene ID (kDa) Peptides) Coverage Peptides) Coverage Peptides) Coverage Peptides) Coverage IFT172 67661 197.5 - - - - 139 (97) 57.9 116 (83) 51.2 IFT88 21821 93.1 - - - - 48 (34) 38.6 38 (26) 36.5 IFT81 12589 79.3 - - - - 95 (65) 59.0 71 (51) 50.9 IFT80 68259 87.8 - - - - 57 (39) 54.4 48 (35) 52.1 IFT74 67694 69.3 - - - - 117 (81) 73.3 101 (67) 67.0 IFT70 72421 76.1 - - - - 32 (25) 38.4 29 (21) 32.5 IFT57 73916 48.8 - - - - 45 (32) 66.9 24 (18) 51.7 IFT56 264134 64.2 - - - - 55 (38) 56.5 45 (33) 48.0 IFT54 74019 71.0 - - - - 28 (20) 39.0 18 (14) 30.1 IFT52 245866 48.2 - - - - 28 (16) 31.9 26 (18) 33.8 Dyf-3 76779 48.0 - - - - 50 (33) 61.7 38 (26) 55.0 IFT46 76568 34.1 - - - - 18 (12) 52.8 18 (9) 38.9 IFT27S-tag 11020 26.1 - - 8 (3) 31.2 18 (5) 28.5 14 (4) 43.5 IFT25 72938 16.3 - - 13 (8) 36.4 21 (10) 55.2 17 (9) 55.2 IFT22 67286 20.8 - - - - 12 (10) 47.6 14 (10) 57.3 IFT20 90410 15.2 - - - - 11 (7) 47.0 9 (6) 36.4 ARL6 56297 21.0 - - 5 (3) 14.5 2 (2) 14.5 5 (4) 14.5 Specific Arl6 peptides identified: 1.R.MVVAKEELDTLLNHPDIK.H 2. R.RIPILFFANK.M Liew et al., Figure S1 Figure S1, related to Figure 1: Characterization of IMCD3-[IFT27LAP] cell lines (A) The G-domain of human IFT27 compared to the highly conserved residues in the G-domain of RAS superfamily GTPases (Colicelli, 2004; Vetter, 2001). Each of the five “G-boxes” within the ~20 kDa G-domain contribute residues that are critical for nucleotide binding and hydrolysis. By homology to mutations in other GTPases, the T19N mutation in the P-loop of IFT27 is predicted to abolish GTP binding to IFT27. Meanwhile, IFT27 does not possess a Glutamine or Histidine at the catalytic position 68 in the switch II region (equivalent to position 61 in Ras) and its ability to spontaneously hydrolyze GTP is therefore non-existent (Bhogaraju et al., 2011). However, in the example of Rap (which is also unable to spontaneously hydrolyze GTP) and several Rabs (which are competent for GTP hydrolysis), the amino acid at the equivalent position of Q61 in Ras is important for binding to the cognate GTPase Activating Protein (GAP). It is therefore conceivable that IFT27[K68A] is defective for GTP hydrolysis in vivo. (B) Expression levels of IFT27LAP, IFT27[K68A]LAP, and IFT27[T19N]LAP are comparable across cell lines. Cell lysate from each cell line was normalized for concentration, resolved by SDS-PAGE, and immunoblotted for GFP (IFT27LAP) and Actin. After normalization with the actin loading control, densitometry of the GFP immunoblot reveals that the levels of IFT27[K68A]LAP and IFT27[T19N]LAP proteins are 84.1% and 50% of the level of IFT27LAP, respectively. (C) Control siRNA treatment of IFT27[T19N]LAP IMCD3 cells does not permit IFT27[T19N]LAP to localize to cilia (left). Compare to Figure 1C where the depletion of endogenous IFT27 enables localization of IFT27[T19N]LAP to cilia in ~50% of the mouse IFT27 siRNA-treated cell population (right). To confirm knockdown of endogenous mouse IFT27, cell lysates from mouse IFT27 siRNA- treated cells or control siRNA-treated cells were normalized for concentration, resolved by SDS-PAGE, and immunoblotted for GFP (IFT27[T19N]LAP), IFT27 and Actin (center). (D) Densitometry of the S-tag immunoblot on LAP eluates in Figure 1D reveals that after LAP affinity purification and HRV3C cleavage elution, the amount of IFT27[K68A]S and IFT27[T19N]S proteins recovered are 83.7% and 21.1% of the level of IFT27S protein recovered, respectively. (E) Table displaying the spectral count, unique peptide count, and % sequence coverage for selected proteins identified in the mass spectrometry analyses of control, IFT27[T19N]LAP, IFT27[K68A]LAP, and IFT27LAP eluates. Specific peptides for ARL6 are listed as well. See Figure 1D for silver-stained gel and immunoblots. A LAPARL6 LAP WT [T31R] [Q73L] IFT27Myc T19N K68A WT T19NK68A WT T19NK68A WT T19NK68A WT Input WB: Myc IP: GFP WB: Myc IFT27 WT WT WT WT GDP/ GTP GDP/ GTP GDP/ GTP GDP/ GTP empty empty empty empty ARL6 none WT GDP/empty GTP ARL6 + EDTA + B GST-tagged protein M.W. (kDa) Arl6 GST PLCδ TULP3 OFD1 SEC8IFT25 GST PLCδ TULP3 OFD1 SEC8IFT25 IFT27 IFT27 97 66 45 • IFT27-GST 31 IFT25 21 • 14 • ARL6 Inputs Bound Liew et al., Figure S2 Figure S2, related to Figure 2: The interaction between IFT and ARL6 in the presence of EDTA is specific. (A) IFT27 preferentially interacts with the nucleotide-empty or GDP-bound form of ARL6. Co-transfections/co-immunoprecipitations were performed with all combinations of “GTP-locked” or “GDP-locked” variants of ARL6 and IFT27. IFT25 was co-transfected with IFT27Myc to ensure IFT27 stability. Note that the protein tags are switched around compared to Figure 2B, i.e. ARL6 has an N- terminal LAP tag while IFT27 has a C-terminal Myc tag. Immunoprecipitation was performed with anti-GFP beads to capture ARL6 and interacting proteins from lysate. (B) Nucleotide-empty ARL6 preferentially interacts with IFT25/IFT27-GST and not with other proteins. To control for non-specific binding of nucleotide-empty ARL6, we tested for capture by five other protein baits (GST, PLCδ, TULP3, OFD1, SEC8) and found no capture of ARL6 in the presence of EDTA. The control protein baits are: GST-PLCδ (PH domain), GST-TULP3 (23-68 a.a.), GST- OFD1c (664-1011 a.a.) (Giorgio et al., 2007),and GST-SEC8 (554-975 a.a.). A Summary of GEF assays Ratio of Condition rate constants +/- IFT27 B Summary of aggregation assays 20 nM ARL6 0.92 +/- 0.8 μM IFT25/IFT27 20 nM ARL6 1.11 +/- 2 μM IFT25/IFT27 20 μM ARL6 + 20 μM EDTA + Endpoint (A.U.) 20 nM ARL6 (insect cells) 0.88 +/- 2 μM IFT25/IFT27 20 M GST 3.0 40 nM ARL6 20 M IFT25 3.0 1.12 200 M IFT25 2.8 +/- 2 μM IFT25/IFT27 10 M IFT25/IFT27-GST 2.8 20 μM ARL6 0.94 20 M IFT25/IFT27-GST 0.8 +/- 2 μM IFT25/IFT27 40 M IFT25/IFT27-GST 0.27 20 nM ARL6 10 M IFT25/GST-IFT27 0.21 1.01 +/- 20 μM IFT25/GST-IFT27 20 M IFT25/GST-IFT27 0.18 40 M IFT25/GST-IFT27 0.15 20 nM ARL6 2.08 +/- 20 μM IFT25/IFT27-GST 20 nM ARL6 + 100 μM XDP 1.26 +/- 20 μM IFT25/IFT27[D126N] C IFT25 (Homo sapiens) IFT25/IFT27 (Chlamydomonas reinhardtii) Superimposed crystal structures (r.m.s.d. = 0.669 Å) Liew et al., Figure S3 Figure S3, related to Figure 3: IFT25/IFT27 stabilizes nucleotide-empty ARL6 but exhibits vey limited guanine nucleotide exchange activity on ARL6. (A) Conditions tested for ARL6 nucleotide exchange assay. We excluded the contribution of IFT25/IFT27 in calculations of nucleotide binding to ARL6 because no significant binding of guanine nucleotides to IFT25/IFT27 was detected in our exchange assays, consistent with a previous report (Bhogaraju et al., 2011). The D126N mutation is predicted to switch the nucleotide specificity of IFT27 from guanine to xanthine (Hoffenberg et al., 1995). ARL6 was produced as an N- terminal GST-fusion protein, and the GST-tag was removed by HRV3C cleavage before size-exclusion chromatography. Typical loading efficiencies of [3H]GDP on ARL6 were 30-40% mol GDP/mol ARL6 after 1 hr. [3H]GDP loading onto uncleaved GST-ARL6 was very limited with a maximum of 4% loading after 1 hr. C-terminally GST-tagged ARL6 was poorly expressed and unstable. (B) Data summary for ARL6 turbidity assay. Various IFT25/IFT27 fusion proteins were tested for their ability to rescue EDTA-induced ARL6 precipitation at 37°C. GST and up to 200 μM of IFT25 did not rescue ARL6 precipitation. N-terminal GST-tagged IFT27 was significantly more effective than C-terminal GST-tagged IFT27 at rescuing ARL6 precipitation. (C) Comparison of the crystal structure of IFT25 by itself with the crystal structure of IFT25 when it is incorporated into the IFT25/IFT27 complex.
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