Figure S1 A B - images.nature.com · of PIP-4 were incubated with the PH domain of FAPP, which was shown previously to mediate its binding to PIP-4. After the incubation, liposomes

s u p p l e m e n ta ry i n f o r m at i o n

www.nature.com/naturecellbiology 1

Figure S1 Interaction of different proteins with lipids. (A) BARS bind to a subset of lipids. Purified lipids are spotted onto filter and then incubated with purified BARS followed by immunoblotting with antibody against BARS. (B) Binding of FAPP to defined liposomes that contain increasing

level of PIP-4. Liposomes that mimic Golgi composition and increasing level of PIP-4 were incubated with the PH domain of FAPP, which was shown previously to mediate its binding to PIP-4. After the incubation, liposomes were pelleted and then immunoblotted for FAPP.

Figure S1

A B

PAPCPEPS

CHOL

PI

PIP-3

PIP-4

PIP2-3,4

PIP2-3,5

PIP2-4,5

PIP3-3,4,5

0.5 1.0 2.0 5.0 ( g)

Blot: BARS

SM0 5 15 30In

put PIP-4 (%)

FAPP

© 2008 Macmillan Publishers Limited. All rights reserved.


2 www.nature.com/naturecellbiology

Figure S2 DAG neither interacts with BARS nor supports its ability to tubulate liposomes. (A) Pure lipids as indicated were spotted onto filter and then incubated with BARS followed by immunoblotting with antibody against BARS. (B) Liposomes that mimic Golgi composition and increasing level of specific lipid, as indicated, were incubated with purified BARS. Liposomes

were then pelleted by centrifugation and then immunoblotted for BARS. (C) Liposomes with different specific additional lipid, as indicated, were incubated with BARS and then examined by EM (images on left); bar, 200 nm. The fraction that exhibited tubulation was quantified, with mean and standard error from three experiments shown (graph on right).

Figure S2

DAGPAA B

0.5 1.0 2.0 5.0 ( g)

Inpu

t

0 5 15 30

Liposome ( %specified lipid )

Inpu

t

0 5 15 30

Liposome ( %specified lipid )PA

DAG

Blot: BARS

Blot: BARS

C

0 15 300

20

40

60

80

100DAG PA

Liposome(%)

Tubu

latio

n (%

)

PADAG




Figure S3 Characterization of BARS that lacks an associated acyltransferase activity. (A) Recombinant BARS expressed from bacteria that contained (XL1 blue) or lacked (JC201) acyltransferase activity were assayed for acyltransferase activity, as reflected by the generation of PA. (B) Purified lipids were spotted onto filter and then incubated with BARS followed by immunoblotting with antibody against BARS. (C) Liposomes that mimic

Golgi composition and increasing level of a specific lipid as indicated were incubated with purified BARS. Liposomes were then pelleted by centrifugation and then immunoblotted for BARS. (D) Liposomes bound by BARS were examined by EM (images on left); bar, 200 nm. The fraction that exhibited tubulation was quantified, with mean and standard error from three experiments shown (graph on right).

A BFigure S3

PAPCPEPS

CHOL

PIPIP-3

PIP-4

PIP2-3,4

PIP2-3,5

PIP2-4,5

PIP3-3,4,5

0.5 1.0 2.0 5.0

SM

( g)

JC20

1

X11B

lue

PalmitatePA

p-CoA C

Inpu

t

0 5 15 30

Liposome ( % specified lipid )

PA

PI

Blot: BARSPIP-4

Blot: BARS

DLiposome (15% specified lipid)

0 15 30 0 15 300

20

40

60

80

100

PIP-4 PI PA

Tub

ulat

ion

(% li

poso

me

obse

rved

)

Liposome (%specifed lipid)

BARS WT (BL21) WT (JC201)

PA PI PIP-4

-BARS

+BARS(JC201)




Figure S4 Inhibition of DGK activity does not have a significant effect on COPI vesicle formation. (A) DGK inhibitors, R59022 (#1) and R59949 (#2), were added to final concentrations of 10 µM. COPI vesicle formation was assessed by the fraction of β−COP released from Golgi membrane at the second stage of the vesicle reconstitution system. The mean from

three experiments with standard error is shown. (B) Myc-tagged DGK-α was expressed in CHO cells, and then isolated using an anti-myc antibody. Purified DGK was then assayed for enzymatic activity in presence of vehicle (DMSO) or 10 µM of the indicated inhibitor. Data are shown as percentage of control. The mean from three experiments with standard error is shown.

Figure S4A

BARS+ + + + +ARFGAP1- + + + +DGKI 1DGKI 2

- - + - +- - - + +

0

20

40

60

80

100

Coa

tom

er R

elea

sed

(%)

B

vehicleInhibitor 2

0

25

50

75

100

vehicleInhibitor 1

0

25

50

75

100

myc

-DG

Ka

activ

ity(%

of c

ontr

ol)

* *




Figure S5 Further characterization of Golgi membrane. (A) Level of PLD2 on Golgi membrane isolated from cells that were either treated with siRNA against PLD2 or not treated. (B) Level of BARS on Golgi membrane isolated from cells that were either treated with siRNA against

PLD2 or not treated. (C) The anti-PLD2 antibody used to block PLD2 function detected one predominant band on washed Golgi membrane used for the COPI vesicle reconstitution system, as assessed by immunoblotting.

Figure S5

A B C

PLD2

No siRNA

siRNA

(PLD

2)

181115

82644837

26

Mw (kd)

Blot: PLD2

- +si PLD2:

PLD2

GRASP 55 BARS




Figure S6 Effect of siRNA against PLD2 on organelle markers. HeLa cells, either treated with siRNA against PLD2 or mock treated, were examined by immunofluorescence microscopy labeling for different proteins as

indicated; bar, 10 µm. To examine VSVG-KDELR at the Golgi, HeLa cells were transfected with a construct encoding for this protein, followed by examination at the permissive temperature.

Figure S6No siRNA siRNA (PLD2)

PDI

KDEL-r

Giantin

VSVG-KDELr




Figure S7 The effect of BFA on the Golgi is not affected perceptibly upon siRNA against PLD2. (A) BFA-induced Golgi tubules. HeLa cells were treated with siRNA against PLD2 or not treated. BFA was then added to cells for 5 minutes at room temperature (which allows Golgi tubules to be better visualized). Cells were then fixed followed by examination of giantin using

immunofluorescence microscopy; bar, 5 µm. (B) Kinetics of BFA-induced Golgi redistribution to the ER. HeLa cells were treated with BFA at 37oC for times as indicated, and then assessed for the fraction that showed giantin in the ER. At each time point, the mean from three experiments with standard error is shown.

Figure S7

BA

BFA / 5 minNo BFA

0 10 20 300

25

50

75

100

No siRNA

siRNA (PLD2)

min

ER D

istr

ibut

ion

of G

iant

in (%

)

No

siR

NA

siR

NA

PLD

2




Figure S8 Activities of different truncation domains of BARS. Binding to specific purified lipids (A), to liposomes of varying composition (B), and

assessment of liposome tubulation (C) were done as described above (Figs 1, S2, and S3).

Figure S8PAPCPEPS

CHOL

PI

PIP-3

PIP-4

PIP2-3,4

PIP2-3,5

PIP2-4,5

PIP3-3,4,5

0.5 1.0 2.0 5.0

BARS (WT)

SM

BARS (CTD)

0.5 1.0 2.0 5.0 0.5 1.0 2.0 5.0

BARS (SBD)

0.5 1.0 2.0 5.0

BARS (CTP)

( g)A

0 5 15 30

B

Inpu

t Liposome ( % )

PA

PIP-4PI

PA

PIP-4PI

BAR

SW

CTD

PA

PIP-4PISB

D

PA

PIP-4PIC

TP

0 15 30 0 15 30 0 15 30 0 15 30 0 15 30 0 15 30 0 15 300

20

40

60

80

100

PIP-4 PI PA

Frac

tion

of T

ubul

atio

n(%

)

Liposome (%)BARS NTDWT NBD CTD SBD NTP CTP

C




Figure S9 Full scans of gels. Full scans for gels in the indicated figures are shown.

BARS

BARS

4837

4837

Full scan for Fig1A170110806047

17011080

47

Full scan for Fig2A

1701108047

1108047

170

PLD2

PLD2

PLD1

PLD1

Figure S9

Full scan for Fig2B

11080

47

170

PLD2170110806047

170110806047

-COP

Full scan for Fig2D

-COP1701108060

Full scan for Fig3A

-COP

Full scan for Fig3D47

1701108060

-COP

Full scan for Fig3C352619

Membri 352619

352619

KDEL-r

p26


3017731033

1517741443

517751650

017761753

PA/PI/PIP-4 (mol%)

CHOL (mol%)

SM (mol%)

DOPS (mol%)

DOPE (mol%)

DOPC (mol%)

Table S1: Composition of Liposomes

DOPC, dioleoylphosphatidylcholine; DOPE, dioleoylphosphatidylethanolamine;DOPS, dioleoylphosphatidylserine; SM, sphingomyelin; CHOL, cholesterol;PA, phosphatidic acid; PI, phosphatidylinositol; PIP-4, phospatidylinositol-4-phosphate. PA, PI, and PIP-4 are also dioleoyl species.


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Figure S1 A B - images.nature.com · of PIP-4 were incubated with the PH domain of FAPP, which was shown previously to mediate its binding to PIP-4. After the incubation, liposomes