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SUPPLEMENTAL INFORMATION
Structural basis for the Golgi-association by the pleckstrin homology domain of the ceramide trafficking protein CERT
Toshihiko Sugiki1,2,3, Koh Takeuchi3, Toshiyuki Yamaji4, Toshiaki Takano2, Yuji Tokunaga1,2,3, Keigo Kumagai4, Kentaro Hanada4, Hideo Takahashi3,5*, and Ichio Shimada1,3*


  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

  2. Japan Biological Informatics Consortium (JBiC), Aomi, Koto-ku, Tokyo 135-8073, Japan

  3. Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Aomi, Koto-ku, Tokyo 135-0064, Japan

  4. Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

  5. Graduate School of Nanobioscience, Yokohama City University, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan

Figure S1




Figure S2


Figure S3

Figure S4



Figure S5



Figure S6




Supplemental Figure legends
Figure S1. Chemical shift perturbation (CSP) experiment of the CERT PH domain, using Ins(1,4)P2. (A) Superimposed 2D 1H-15N HSQC spectra of 0.1 mM uniformly 15N-labeled CERT PH domain with increasing concentration of Ins(1,4)P2 as shown in the inset: 0 mM (black), 0.125 mM (blue), 0.250 mM (light blue), 0.500 mM (green), 1.000 mM (orange), and 2.000 mM (red). (B) Normalized chemical shift changes (Δδ) of individual residues in the CERT PH domain induced by the addition of 2.000 mM Ins(1,4)P2. Red and orange dashed lines indicate 0.2 ppm and 0.1 ppm normalized chemical shift changes, respectively. Numbers on the horizontal axis indicate the residue number of the backbone amide signals in the CERT PH domain (24-117). For side chain, the names of the resonances are indicated in the figure. (C) The mapping of the residues that were significantly perturbed by the titration of Ins(1,4)P2 on the surface representation of the CERT PH domain structure. Red and orange surfaces indicate the side chains of the residues with >0.2 ppm and 0.1-0.2 ppm CSP, respectively. Residues colored dark gray (Tyr36, Ile37, Gly39, Asn50, Pro78, Pro102, and Asp103) indicate those without information.
Figure S2. Interaction analyses between the PtdIns(4)P-embedded liposome and the CERT PH domain wild-type by SPR. SPR sensorgrams of the CERT PH domain wild-type bind to PtdIns(4)P-embedded liposome. The resonance units were plotted against concentration of the CERT PH domains (left) and dissociation constant (KD) values of the wild-type was calculated assuming 1:1 binding (right).

Figure S3. TCS experiments using PtdIns(4)P-free liposome. (A) Signal intensity reduction ratios in the TCS experiment. Ratios of the signal intensities with and without saturation were plotted against the residue numbers. Red and orange dashed lines indicate >0.2 and 0.1-0.2 signal intensity reduction ratios, respectively. The error-bars were calculated from the root sum square of the S/N ratios in the spectra with and without irradiation. The bottom axis is the same as in Figure S1. (B) The mapping of the residues affected by the transferred cross-saturation on the surface of the CERT PH domain. Red and orange surfaces indicate the side chains of the residues with >0.2 and 0.1-0.2 signal intensity reduction ratios, respectively. Residues colored dark gray indicate those without information, as described in the legend of Figure S1.


Figure S4. Intracellular localization of CERT mutants. The HeLa transfectants were stained with rat anti-HA and mouse anti-GM130 (Golgi apparatus marker) antibodies. Details of the experimental procedure can be found in the Supplemental Experimental Procedures section.
Figure S5. Structural and functional differences between the CERT PH domain and the FAPP1 PH domain.

(A) Comparison of the tertiary structures and the amino acid sequences of the β1/β2 loop regions between the CERT PH domain and the FAPP1 PH domain (PDB code 2KCJ). (B) Superimposed 2D 1H-15N HSQC spectra of the CERT PH domain wild-type (black) and its S31Y/D42P mutant (red). The {15N-1hhhhhHHH} correlation signals of Ile37 and Gly39, which could not be observed in the spectrum of the wild-type due to severe signal broadening, appeared in the spectrum of the S31Y/D42P mutant (denoted by black arrows). (C) The 15N R2 relaxation dispersion experiments of the CERT PH domain wild-type and its S31Y/D42P mutant. The Ser31 and Asp42 (blue) and Thr34 (red sphere) are located on the root moiety and the head region of the β1/β2 loop, respectively (left panel). Comparison of the 15N-spin relaxation dispersion profiles of Thr34 of the CERT PH domain wild-type (black) and its S31Y/D42P mutant (red, right panel).


Figure S6. Comparison of the tertiary structures between the CERT PH domains and other PH domains from Grp1 and DAPP1. Top: ribbon representations of the PH domains of CERT, Grp1 and DAPP1. The specificity of the PH domains were also indicated. In the Grp1 and DAPP1 PH domain structure, the residues that are known to be important for PIP recognition were colored and labeled. In the of the CERT PH domain structure, two basic, K32 and R43, and Y54 were indicated. Below: Superposition of the CERT PH domain with the Grp1 PH domain (left) and the DAPP1 PH domain (right). The sidechains shown in the top panels were also shown while the mainchain were simplified by a tube representation. For superposition, the side chains of K32 and R43 of the CERT PH domain and their corresponding residues of the GRP1 PH domain used as reference points. Inositol ring moiety of PtdIns(3,4,5)P3 were represented by yellow.
Supplemental Experimental Procedures
15N R2 relaxation dispersion experiments

The 15N R2 relaxation dispersion data were obtained by Carr-Purcell-Meiboom-Gill (CPMG)-type experiments with 12 sets of various frequencies of CPMG 15N refocusing pulses, νCPMG, of 50, 100, 150, 200, 250, 300, 350, 400, 500, 650, 800, and 1,000 Hz. The intensities of the {15N-1H} correlation signals of each data set were quantified by using Sparky (Goddard and Kneller, SPARKY 3 – NMR Assignment and Integration Software. University of California, San Francisco, CA), and the effective R2 relaxation rate (R2eff) was estimated from the equation R2eff = -ln(I/I0)/T, as the exponential decay rate of the signal intensity with (I) and without (I0) the constant-time relaxation delay (T, 40 ms). The relaxation dispersion profiles were calculated by plotting the R2eff versus 1/τCPMG, where τCPMG is the interval period between the CPMG 15N refocusing pulse, and by curve fitting with the equation for a two-state, slow exchange model (Richard-Carver equation), using the program NESSY (Bieri and Gooley, 2011).



Retroviral infection and Immunofluorescence Microscopy

Hemagglutinin(HA)-tagged CERT mutant cDNAs, which encodes single alanine-substitution of Lys32, Trp33, Arg43, Arg66, and Arg98, were subcloned into the EcoR I and Xho I sites in pMXs-IB, a retroviral vector. Preparation of retroviruses and their infection of HeLa-mCAT#8 cells were performed using the Plat-E system, as described previously (Morita et. al., Yamaji et. al.). The concentration of blasticidin-S for selection was 5 µg/ml. The HeLa transfectants were stained with rat anti-HA antibodies and mouse anti-GM130 (Golgi apparatus marker), followed by Alexa Fluor 488-anti rat IgG (green) and Alexa Fluor 594 (red) anti-mouse IgG. Immunofluorescence microscopy was performed as described previously (Yamaji et. al.), and the specimens were visualized with a confocal laser-scanning microscope, LSM510 META (Carl Zeiss, Jena, Germany) equipped with a C-Apochromat 63x/1.2W Corr objective.

Table S1. Dissociation constants (KD) between the CERT PH domain mutants and PtdIns(4)P-embedded liposomes


Mutant

1KD (× 10-6 M)

Wild-type

3.26 ± 0.23

K32A

305 ± 32

W33A

269 ± 17

T34A

39.1 ± 7.2

N35A

39.6 ± 5.7

Y36A

142 ± 30

I37A

19.2 ± 0.76

H38A

16.7 ± 1.0

G39A

4.39 ± 0.92

W40A

(N.D.)2

Q41A

7.47 ± 1.8

R43A

612 ± 14

K56A

3.52 ± 0.23

R66A

42.9 ± 3.9

H79A

(N.D.)2

R85A

(N.D.)2

S93A

2.19 ± 0.1

Y96A

38.6 ± 2.4

R98A

6.88 ± 0.41


1KDs were measured by the SPR method, as described in the Experimental Procedures.

2N.D. = No data. (the recombinant proteins could not be prepared.)

Supplemental Reference





Bieri, M., and Gooley, P.R. (2011). Automated NMR relaxation dispersion data analysis using NESSY. BMC Bioinformatics 12, 421.


Morita S, Kojima T, and Kitamura T (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7: 1063-1066
Yamaji T, Nishikawa K, and Hanada K (2010) Transmembrane BAX inhibitor motif containing (TMBIM) family proteins perturbs a trans-Golgi network enzyme, Gb3 synthase, and reduces Gb3 biosynthesis. J Biol Chem 285: 35505-35518








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