detergent-solubilizedchloroplast + pSSU

translocation intermediate + ATP

0 0.1 0.5 1 5 0 0.5 5 (mM ATP)

880

kD

440

66

1 2 3 4 5 6 7 8

Supplemental Figure 1. The 1-MDa Translocation Complex Is Not A Stromal Chaperonin Complex. (Lanes 1-5) After pea chloroplasts not carrying radiolabeled preproteins were solubilized with 1% digitonin, the supernatant was mixed with [35S]pSSU and indicated concentrations of ATP. After incubation for 10 min on ice, the samples were subjected to 4-14% BN-PAGE and autoradiography.(Lanes 6-8) After pea chloroplasts carrying [35S]pSSU were solubilized with 1% digitonin, the supernatant was mixed with indicated concentrations of ATP. After incubation for 10 min on ice, the samples were subjected to 4-14% BN-PAGE and autoradiography.

translocation complex

Supplemental Data Kikuchi et al., (2009) A 1-Megadalton Translocation Complex Containing Tic20 and Tic21 Mediates Chloroplast Protein Import at the Inner Envelope Membrane in Arabidopsis and pea.

TP

pSSU pFNR

Bind BindTP

pSSU

pFNR

BN

-PA

GE

SD

S-P

AG

E880

kD

440

66

translocation complex

pSSU

pFNR

free preproteins

Supplemental Figure 2. The 1-MDa translocation complex was generated using ferredoxin:NADP+ oxidoreductase as a preprotein. Pea chloroplasts were incubated with [35S]pSSU or [35S]precursor form of the ferredoxin:NADP+ oxidoreductase (pFNR) to generate translocation intermediates as described in Methods. Washed chloroplasts were solubilized with 1% digitonin and subjected to BN-PAGE (top) and SDS-PAGE (bottom). TP, 10% of the translation products added to each reaction.

TP 1 2 3 4

Autoradiogram

Immunoblotα-PsToc75

5 6 7 8

PIC

Thl- -- -+

+ ++

- -- -+

+ ++

*

Supplemental Figure 3. Partial degradation of the Toc complex, which had occurred during the isolation of chloroplasts, did not affect the formation of the 1-MDa translocation complex.Pea chloroplasts were isolated in the absence (-) or presence (+) of protease inhibitor cocktail (PIC). After the translocation intermediate was generated using [35S]pSSU under low ATP concentrations as described in Methods, chloroplasts were washed twice with HS buffer not containing PIC to enable surface proteolysis, and then incubated with (+) or without (-) 100 μg/ml thermolysin (Thl) for 20 min on ice. After inactivation of thermolysin, reisolated chloroplasts were solubilized in BN-PAGE sample buffer containing 1% digitonin for 10 min on ice. After ultracentrifugation, the supernatant was divided into three aliquots, one was subjected to BN-PAGE and autoradiography (top, lanes 1-4), the second was subjected to BN-PAGE and immunoblotting with anti-PsToc75 antibody (top, lanes 5-8), the third was mixed with 10% SDS and 2-mercaptoethanol to final concentrations of 3.3% and 5%, respectively, denatured by heating at 95� C for 2 min and subjected to 15% SDS-PAGE and autoradiography (bottom, lanes 1-4). Brackets indicate the positions of the 1-MDa translocation complex, intact Toc complex, and degraded Toc subcomplexes. TP, 10% of the [35S]pSSU translation product added to each reaction. SSU-DP, degradation product of SSU. An asterisk denotes a nonspecific cross-reacting band to an unidentified protein complex which migrates in large quantities at this position

kD

440

880

Rbs

66

132

kD

440

880

Rbs

66

132

BN

-PA

GE

SD

S-P

AG

E pSSU

SSU-DP

translocationcomplex intact Toc

complex

Toc subcomplex(350-500 kD)

Toc subcomplex(250-350 kD)

bottom top

ME 1 2 3 4 5 6 7 8 9 10 11 12

Thl -

62

83

kD

Thl +

Tic110

Toc75

pSSU(19 kD)

pSSU(19 kD)

SSU-DP(8 kD)

Supplemental Figure 4. Sucrose density gradient separation of outer- and inner-envelope membrane vesicles. The translocation intermediate was generated as described in Methods. Pea chloroplasts carrying [35S]pSSU were treated with or without thermolysin for 20 min on ice. Thermolysin was inactivated as described in Materials and methods. Chloroplasts carrying preproteins were lysed by freeze/thaw cycles under hypertonic conditions to separate the outer- and inner-envelope membranes. After low-speed centrifugation to remove thylakoids, the supernatant containing envelope membrane vesicles was separated by 0.4 M-1.2 M sucrose density gradient centrifugation. All 12 separated fractions were analyzed by SDS-PAGE and autoradiography or immunoblotting with anti-PsToc75 or -PsTic110 antibodies. ME, mixed-envelope membrane fraction prior to the sucrose gradient separation; Thl, thermolysin; SSU-DP, degradation product of SSU.

Thl Trp

0 10 100 0 10 100 (μg/ml)

880

kD

440

66

1 2 3 4 5 6

Supplemental Figure 5. The Arabidopsis 1-MDa translocation complex has identical protease accessibility properties to that of the pea complex. The translocation intermediate was generated as described in Methods, except that Arabidopsis chloroplasts were used. The chloroplast suspension was divided into six aliquots (lanes 1-6) and washed twice with HS buffer in the absence of protease inhibitor cocktail. Chloroplasts carrying [35S]pSSU were treated with the indicated concentrations of thermolysin (Thl) or trypsin (Trp) for 20 min on ice. After inactivation of the proteases, the chloroplast pellet was solubilized with digitonin and subjected to 4-14% BN-PAGE and autoradiography.

880

kD

440

66

880

kD

440

66

1 2 3 4 5 6 7 8 9 10 11

α-P

sToc

159

(1.8

μg)

α-P

orin

(4.

0 μ

g)

α-P

sToc

159

(7.1

μg)

α-P

sToc

34 (

0.9 μ

g)

α-P

sToc

34 (

3.4 μ

g)

α-P

sTic

110

α-A

tTic

40

α-P

sTic

22

α-S

ocpC

pn60α

α-S

ocpC

pn60β

α-S

ocpH

sp70

880

kD

440

66

Supplemental Figure 6. Antibody-shift BN-PAGE. The translocation intermediate was generated using pea chloroplasts as described in Methods. Chloroplasts carrying [35S]pSSU were solubilized in BN-PAGE sample buffer to a final concentration of 0.5 mg chlorophyll/ml for 10 min on ice. After ultracentrifugation at 100,000g for 10 min, 35 μl of the supernatant was mixed with purified anti-PsToc159 or -PsToc34 antibodies; or with anti-PsTic110, -AtTic40, -PsTic22, -spinach Cpn60α, spinach Cpn60β, or -spinach stromal Hsp70 antisera. After incubation for 30 min on ice, samples were mixed with Coomassie brilliant blue G-250 solution. After a clarifying spin, the supernatant was subjected to 4-14% BN-PAGE and autoradiography. As a control, purified antibody against porin was used (lane 1). The antiserum against AtTic40 was kindly provided by Dr. Hsou-min Li.

kD

440Rbs

66

132

Tic110 Tic40 Hsp93 Toc159

α-P

sTic

110

Pre

imm

une

α-A

tTic

40

Pre

imm

une

α-P

sHsp

93

Pre

imm

une

α-P

sToc

159

Pre

imm

une

*

Supplemental Figure 7. Tests for the binding ability of anti-Tic and -Toc antibodies to native proteins by immunodepletion. Pea chloroplasts were solubilized in BN-PAGE sample buffer to a final concentration of 0.5 mg chlorophyll/ml for 10 min on ice. Insoluble material was removed by ultracentrifugation at 100,000g for 10 min at 4 � C. Fifty to seventy microliters of the supernatant was incubated twice with 10 μl packed volume of rProtein A-Sepharose that had been coupled with IgG for 1 h in cold room (6� C) with rotational mixing. The unbound fraction (supernatant) was subjected to 4-14% BN-PAGE and immunoblotting. An asterisk denotes a position of non-specific cross-reaction band corresponding to the native Rubisco complex, which migrates in large quantities at this position. The antiserum against AtTic40 was kindly provided by Dr. Hsou-min Li.

Supplemental Figure 8. Immunoblot analyses of several proteins in albino mutants. Total proteins were extracted from 3-week-old wild-type Arabidopsis, homozygous tic21/pic1-1 (SALK_104852), tic20 (SALK_039676), and albino3 (CS16; Sundberg et al., 1997) mutants, which show albino phenotypes, grown on MS media containing 2% sucrose. Thirty micrograms of protein were subjected to SDS-PAGE and immunoblotting with indicated antisera. An antiserum against pea-seed ferritin detects several ferritin family proteins in Arabidopsis. An antiserum against Arabidopsis copper superoxide dismutase (CSD) 2 detects CSD1 isoprotein as well as CSD2. An antiserum against maize Alb3 detects an Arabidopsis homolog (Asakura et al., 2008). Antisera against cpHsp70, Tic20, and Tic21 were described in the text. The antiserum against pea-seed ferritin was kindly provided by Dr. Brigitte Touraine (Van Wuytswinkel et al., 1995; Gaymard et al., 1996). The antiserum against Arabidopsis CSD2 was kindly provided by Dr. Marinus Pilon (Kliebenstein et al., 1998; Abdel-Ghany et al., 2005).

References: Abdel-Ghany, S. E., Müller-Moulé, P., Niyogi, K. K., Pilon, M., and Shikanai, T. (2005) Plant Cell 17, 1233-1251.Asakura, Y., Kikuchi, S., and Nakai, M. (2008) Plant J. 56, 1007-1017.Gaymard, F., Boucherez, J., and Briat, J. F. (1996). Biochem. J. 318, 67-73.Kliebenstein, D. J., Monde, R.-A., and Last, R. L. (1998) Plant Physiol. 118, 637-650.Sundberg, E., Slagter, J. G., Fridborg, I., Cleary, S. P., Robinson, C., and Coupland, G. (1997) Plant Cell 9, 717-730.Van Wuytswinkel, O., Savino, G., and Briat, J. F. (1995). Biochem. J. 305, 253-261.

WT

tic21/pic1-1

tic20

alb3

WT le

aves

alb3

leave

s

WT ro

ots

alb3

root

s

tic20

leav

es

tic20

root

s

tic21/pic1-1

leav

es

tic21/pic1-1

root

s

Ferritin

cpHsp70

Tic20

Tic21

Alb3

CSD2

CSD1

Precursor forms

Processed forms

Anti-GFP

1 2 3 4

In WT In alb3

Rbc

S-n

t:GF

P

E1α

-nt:G

FP

Rbc

S-n

t:GF

P

E1α

-nt:G

FP

Supplemental Figure 9. Protein import into plastids in a transient expression system using protoplasts from the albino3 mutant. Protoplasts isolated from Arabidopsis wild-type and the homozygous albino3 mutant were transformed with RbcS-nt:GFP or E1α-nt:GFP. After 8 h of incubation at 22� C, proteins were extracted from transformed protoplasts and subjected to SDS-PAGE followed by immunoblotting with anti-GFP antibody. RbcS-nt, the N-terminal transit peptide of the small subunit of Rubisco; E1α-nt, the N-terminal transit peptide of the pyruvate dehydrogenase E1α subunit.

kD

175

62

83

47.5

32.5

α-P

sHsp

93

α-P

sTic

110

Inpu

t

Pre

imm

une

1 2 3 4

High ATP, MBS

pSSU-DHFR

mSSU-DHFR

Supplemental Figure 10. Immunoprecipitation After Chemical Cross-Linking with MBS. Energy-depleted Arabidopsis chloroplasts were incubated with [35S]pSSU-DHFR under high ATP concentrations (4 mM) in HS buffer containing 5 mM MgCl2, 5 mM DTT, 3 mM methionine, 3 mM cysteine, 5 μl/ml protease inhibitor cocktail, and 20 μM methotrexate (MTX) for 10 min at 25� C in the dark. Reisolated chloroplasts carrying [35S]pSSU-DHFR, at 0.24 mg chlorophyll/ml in HS buffer in the presence of 20 μM MTX, were cross-linked with 0.2 mM MBS. Chloroplasts carrying cross-linked products were solubilized under denaturing conditions containing 1% LiDS. The resulting extract was diluted 30-fold with 0.5% Triton X-100 in TBS followed by immunoprecipitation with anti-PsTic110 (lane 3) or -PsHsp93 (lane 4) antibodies, or with preimmune serum (lane 2). The eluates were subjected to 7.5% SDS-PAGE and autoradiography. Input represents 30% of the material used for immunoprecipitation (lane 1).

880

kD

440

66

1 2 3 4 5 6 7 8 9 10

DSP DTSSP

0 0.25 0.5 1 2 0 0.25 0.5 1 2 (mM)

cross-linkedsupercomplex

free pSSU

1 MDa complex

Supplemental Figure 11. Chemical cross-linking of the translocation intermediate. The translocation intermediate was generated as described in Methods. Pea chloroplasts carrying [35S]pSSU were divided into ten aliquots (lanes 1-10). Resuspended chloroplasts, to a final concentration of 0.25 mg chlorophyll/ml in HS buffer, were treated with the indicated concentrations of dithiobis (succinimidyl propionate) (DSP) or its water-soluble analog, 3, 3'-dithiobis (sulfosuccinimidyl propionate) (DTSSP) for 15 min on ice. Chloroplasts were recovered by centrifugation, washed twice with HS buffer containing 5 μl/ml protease inhibitor cocktail, and then solubilized in BN-PAGE sample buffer to a final concentration of 0.5 mg chlorophyll/ml for 10 min on ice. After insoluble material was removed by ultracentrifugation at 100,000g for 10 min, the supernatant was subjected to 4-14% BN-PAGE and autoradiography. Brackets indicate the positions of cross-linked supercomplexes, the 1-MDa translocation complex, and free pSSU.