Supplemental Information

The 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 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 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. The crystal structures of IFT25 (Homo sapiens, pdb code 1TVG) and IFT25/IFT27 (Chlamydomonas reinhardtii, pdb code 2YC2) were superimposed using Pymol (Ver. 1.7) and the root mean square deviation (r.m.s.d.) was computed to be 0.669 Å. Hence IFT25 assumes a similar conformation by itself or when incorporated into the IFT25/IFT27 complex.

A DNA acTub ARL6 GFP B acTub GFP ARL6 DNA acTub IFT27

acTub IFT27 WT LAP -/-

DNA acTub BBS5 GFP Arl6 + IFT27[K68A]

-/- 100 acTub GFP BBS5 Ift27 50 IFT27

positive cilia (%) 0 WT Arl6-/-

-/- -/- C D NG3-BBS1 in IMCD3 cells WT Ift27 Arl6 IFT27

Arl6 * Actin MEFs

Liew et al., Figure S4 Figure S4, related to Figure 4: IFT27 regulates ciliary localization of ARL6 and the BBSome but ARL6 does not regulate IFT27 localization. (A) Similar to IFT27LAP and IFT27[T19N]LAP (Figure 4D and 4E), IFT27[K68A]LAP transfection into Ift27-/- MEFs rescues the ciliary accumulation of ARL6 and BBSome. (B) Ciliary localization of IFT27 is unaffected by loss of ARL6. WT and Arl6-/- MEFs were immunostained for IFT27, and the ciliary signal for IFT27 was found to be similar in both cell lines. (C) IFT27 does not regulate protein stability of ARL6, and vice versa. Whole cell lysates from WT, Ift27-/- and Arl6-/- MEFs were normalized for concentration, resolved by SDS-PAGE and immunoblotted for IFT27, ARL6 and Actin. ARL6 levels are similar in WT and Ift27-/- MEFs, while IFT27 levels are similar in WT and Arl6-/- MEFs. (D) NG3-BBS1 localizes to cilia in the stable IMCD3 cell line. Scale bar: 5 μm.

A B RFP-IFT88 NG3-BBS1 0s siControl siIFT27 BBS1 IFT88

60s BBS1 IFT88

1 1

0.5 0.5

Intensity 0 1 2 3 4 5 0 1 2 3 4 5 Cilia length (µm)

B T B T

Liew et al., Figure S5 Figure S5, related to Figure 6: IFT27 knockdown disrupts the co- localization between IFT88 and BBSome. (A) Additional kymographs of tagRFP.T-IFT88 and NG3-BBS1 co-movement (see Figure 6G). The fluorescent foci tracks for IFT88 (red) and NG3-BBS1 (green) are indicated in the bottom panels. Scale bar: 2 μm. (B) Additional images for structured illumination microscopy of IFT88 and NG3-BBS1 in Control (siControl) and IFT27-depleted (siIFT27) cilia as in Figure 6H. Colocalization between IFT88 (red) and NG3-BBS1 (green) is displayed on a line profile (bottom).

SUPPLEMENTAL MOVIE LEGENDS Movie S1, related to Figure 5 Representative examples of Fluorescence Loss After Photobleaching (FLAP) assay for siControl and siIFT27 cilia. Cytoplasmic NG3-BBS1 was photobleached before time point 2 and the fluorescent signal decay in cilia was recorded. Scale bar: 5 μm.

Movie S2, related to Figure 6 Representative examples of Fluorescence Recovery After Photobleaching (FRAP) assay for siControl and siIFT27 cilia. Ciliary NG3-BBS1 was photobleached and the fluorescent recovery rate in cilia was recorded at 5 s intervals. Scale bar: 5 μm.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Recombinant Protein Expression and Purification PLCδ was expressed as a derivative of pGEX2TK. OFD1c was expressed as a derivative of pGEX4T1. GST, TULP3 and SEC8 were expressed as derivatives of pGEX6P. All proteins were purified on Glutathione Sepharose 4B resin (GE Healthcare) and eluted with reduced glutathione, pH 8.0.

LAP purifications and mass spectrometry IMCD3 cells were lysed with LAP300 buffer (50 mM HEPES, pH 7.4, 300 mM

KCl, 1 mM EGTA, 1 mM MgCl2, 10% glycerol, 0.1 mM GDP/GTPγS/no nucleotide, 0.5 mM DTT, and protease inhibitors) containing 0.3% NP-40 before initial centrifugation at 16,000 × g for 15 min. [KCl] in supernatant was lowered to 200 mM before further centrifugation at 100,000 × g for 1 h to yield the S100 supernatant for capture on anti-GFP beads. Complexes were eluted by HRV3C (IFT27LAP) or TEV (LAPIFT88) mediated cleavage. For mass spectrometry, eluates were precipitated, resuspended in 100 mM ammonium bicarbonate (10% acetonitrile) and digested overnight with trypsin. Peptides were acidified with 0.1% formic acid, desalted using home-made stage-tips as previously described (Rappsilber et al., 2003), dried, then resuspended in 0.1% formic acid and analyzed by LC-MS/MS. Peptides were separated across a 90-min gradient on a C18 reverse-phase micro-capillary column connected to an LTQ-Orbitrap Velos Pro mass spectrometer. For each cycle, one full MS scan was acquired at a resolution of 60000 followed by the acquisition of 20 MS/MS spectra on the linear ion trap from the 20 most abundant MS ions. MS/MS spectra were searched against the IPI mouse database (Ver. 3.6) using the Sequest algorithm and peptide matches were filtered to <1% false-positives using a target-decoy database strategy. Spectra were also searched against the IPI human database (Ver. 3.6) to identify spectral counts for the human IFT27/RABL4 bait. Interacting proteins for each sample were assessed by spectral counting.

Nucleotide Exchange Assay Typically, 0.2 to 0.4 μM of ARL6 was loaded for 1 h at 30°C with 2 μM [3H]GDP or [35S]GTPγS (diluted to 1 μCi/100 μL reaction using unlabeled nucleotide) in 1X

Loading buffer (50 mM HEPES, pH 7.0, 150 mM KOAc, 1 mM Mg(OAc)2, 0.2 mg/mL BSA, 0.1% Triton X-100, and 1 mM DTT). Exchange reaction was initiated by addition of at least 200-fold molar excess unlabeled nucleotide and 0.8 to 40 μM of IFT25/IFT27 or control (buffer or GST). At different time-points, reaction aliquots were diluted in 4 mL of ice-cold STOP buffer (25 mM HEPES, pH 7.0, 100 mM NaCl, 5 mM MgCl2, and 0.002% Triton X-100) and rapidly filtered through mixed cellulose membranes (Millipore, #HAWP02500). Membranes were then rapidly washed 3 times with 3 mL of STOP buffer each time. Preliminary control experiments showed that IFT25/IFT27 did not significantly bind nucleotide, as previously reported (Bhogaraju et al., 2011). Data obtained were fit to a single phase exponential decay equation using KaleidaGraph (Ver. 4.1). The Sec12-catalyzed exchange of nucleotide on Sar1p was used as a positive control for the nucleotide exchange assay itself (Barlowe and Schekman, 1993). See Figure S3A for a summary of of all conditions tested.

Immunofluorescence and Fixed-Cell Microscopy Ciliated cells grown on acid-treated 12 mm diameter #1.5 coverslips were fixed with 4% paraformaldehyde in PBS for 5 min at room temperature and extracted with -20°C methanol for 5 min. coverslips were then blocked (5% normal donkey serum, 3% BSA, 0.1% Triton X-100 in PBS) for 1 h at room temperature or overnight at 4°C. Primary and secondary antibodies (Cy3 or Cy5-labeled) [Jackson ImmunoResearch Laboratories] were diluted in IF buffer (3% BSA, 0.1% Triton X-100 in PBS), and antibody incubations were sequentially carried out for 1 h each with 5 IF buffer washes after each incubation. Preparations were then counterstained with Hoescht DNA dye and mounted on slides (Thermo Scientific, #3050-002) with Fluoromount-G (Electron Microscopy Sciences). Slides were examined under a Carl Zeiss Axio Imager M1 microscope and photographed with the 63X objective (oil immersion). Contrast adjustments were standardized for immunofluorescence channels to be compared in each set of panels using Slidebook software (Ver. 5.0, Intelligent Imaging Innovations) or ImageJ (NIH).

Analysis of Photokinetic Data For Fluorescence Loss After Photobleaching (FLAP), the integrated fluorescent intensity over time was plotted for each and fit to an exponential (b + m*e- kt). To assess the magnitude of photobleaching during data acquisition, in every experiment we monitored the cytoplasmic fluorescence of an unbleached cell in the same field of view as the FLAP cell. For unbleached cells, the fluorescence decreased by <2% over 2000 s.

For Fluorescence Recovery After Photobleaching (FRAP), the integrated fluorescent intensities of cilia over time were normalized by the first time point, and corrected for photobleaching during data acquisition (y = yobserved - yt=0 + yphotobleached; where yt=0 is the first time point following FRAP, and yphotobleached is described below). The results were plotted either as individual cilia or pooled by treatment, and fit by either an exponential or the initial rate. Only cilia between 5 and 6.5 μm in length were included in the analysis. Photobleaching during acquisition was determined using a paraformaldehyde-fixed sample by observing the loss of integrated fluorescent intensities using identical imaging conditions, and fitting to an exponential. Under TIRF microscopy, the half-life for NeonGreen fluorescent signal was 876+/- 308 s. The fluorescence lost at any moment due to -t/876 photobleaching equals yabsolute*(1 - e ), where yabsolute is the absolute number of NeonGreen particles in cilia (yabsolute = yobserved + yphotobleached). Therefore -t/876 yphotobleached = yobserved / (1/(1 - e ) - 1). All analyses were performed with KaleidaGraph 3.52.

SUPPLEMENTAL INFORMATION REFERENCES

Barlowe, C., and Schekman, R. (1993). SEC12 encodes a guanine-nucleotide- exchange factor essential for transport vesicle budding from the ER. Nature 365, 347–349.

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Vetter, I.R. (2001). The guanine nucleotide-binding switch in three dimensions. Science 294, 1299–1304.