Supporting Online Material, Matsumoto et al. Material and Methods Library. Poly(A) + mrna was purified from RAW264.7 cells stimulated with murine IFN-γ (100 units/ml) and bacterial LPS (100 ng/ml) for 0, 2, 4, 6, 8, 12, 18 and 24 hrs. cdnas were prepared using oligo(dt) and random primers, ligated to the EcoRI adaptor (5' AATTCGCGGCCGCGTCGAC 3') and cloned into the EcoRI site of pgad10 (Clontech) prey plasmids. The average insert size was 1.7 kbp and the range of insert size was 0.4 ~ 3.5 kbp. The resultant library was amplified once in E. coli to obtain the plasmid cdna library used for screening. PCR analysis with specific primers verified that the library contained apoptosis-related cdnas including caspase-3, -8, -9, Apaf-1, Bcl-2 and apoptosis inducing factor (AIF). Bait plasmids. An Nco I and Xho I digest of caspase-3 (S1) was integrated into pas2-1 (Clontech). The entire open reading frame of AIF was derived from I.M.A.G.E. clones (ATCC) 1520292 and 25192, and sub-cloned into pas2-1 (between Nde I and EcoR I sites). DNA sequencing confirmed construct identities, and the expression of bait proteins (fused to binding domains, BD) was verified by immunobloting with monoclonal antibodies to the BD or bait-specific proteins or both. Modified two-hybrid screens. The YHB1 gene was deleted from yeast strain CG-1945 and the absence of NO consumption was verified (S2). Yeast two-hybrid screening was then performed in the CG-1945 yhb1 host. Cells were sequentially transformed with bait [selection in tryptophan (Trp)-deficient medium] and library [selection in Trp-leucine (Leu)-deficient medium] plasmids (S3). Cells containing pairs of interacting proteins were selected by their growth on histidine (His)-deficient medium and by expression of β-galactosidase (β-gal) activity. Specifically, interacting proteins reconstitute the active transcription factor Gal4, which drives transcription of β-gal and HIS3. Method 1: Auxotrophic selection was carried out on agar plates (15 cm diameter) made with Complete Supplement Mixture deficient in His-Trp-Leu (Q-Bio gene) (50 mm phosphate buffer, ph 7.2). Yeast were cultured for 4 days at 30 ºC in the presence or
absence of DETA-NO (40 µl of a 0.3M solution; Cayman Chemical). Clones that showed at least 3-fold greater growth in DETA-NO were selected and further analyzed for β-gal activity with O-nitrophenyl β-d-galactopyranoside (ONPG) as substrate. Prey plasmids were isolated from positive yeast clones and then re-introduced into the bait strain to confirm bait-prey interactions. Method 2: Cells seeded on 1.5% agar were covered with 3% low-melting-point agar, which in turn was layered with culture medium. NO donors (e.g. DETA-NO, 300 µm final concentration) were added to the liquid layer every 24 hours. Colonies were grown for 4 days as described in Method 1. Method 3: Auxotrophic selection was carried out in His-Trp-Leu-deficient buffered medium (see Method 1). Transformation with the cdna library was followed by overnight growth in medium deficient in Trp and Leu. Transformants were then grown for 3 days in His- deficient medium supplemented with DETA-NO (typically 200 µm final concentration). The plasmid DNA was harvested and transformed into E. coli. Individual clones were isolated, retransformed into bait strains, and reassessed for NOdependent growth and β-gal activity. Immunoprecipitation. Thirty million cells were lysed by homogenization in 1 ml IP buffer [10 mm NaPi, 100 mm NaCl, 1 mm EDTA, ph 7.9, with protease inhibitor cocktail (Roche)]. The supernatant obtained by centrifugation at 20,000x g for 10 min was used for immunoprecipitation. Caspase-3 immunoprecipitates (2.5 µg anti-caspase-3 monoclonal antibody, Transduction Laboratories) were washed, separated on 10% SDS- PAGE, and blotted with anti-asm antibody (Santa Cruz Biotechnology, and kindly provided by K. Sandhoff); 5% of the immunoprecipitate was blotted for caspase-3. Caspase-3 activity. Caspase-3 activity was measured with the EnzChek Caspase-3 kit (Molecular Probes) with Z-DEVD-AMC as substrate, and evaluated at 340/450 nm (excitation/emission). Acid sphingomyelinase activity. ASM activity was measured essentially as described (S4), using BODIPY FL C 5 -sphingomyelin (Molecular Probes) as substrate (1.5 nmol) in
assay buffer: 250 mm sodium acetate, ph 5.0, 10 mm EDTA. To assay Zn-stimulated ASM activity, 0.1 mm ZnCl was used without EDTA. Sample (30 µl) was incubated in assay buffer (70 µl) at 37 ºC for 1-3 hours. Reactions were terminated by adding 1.0 ml heptane and 0.29 ml isopropyl alcohol. Phases were then separated by adding 0.23 ml H 2 O, and the heptane phase was washed with 0.23 ml H 2 O. The fluorescence of the organic phase (containing BODIPY FL-ceramide) was assayed at 505/514 nm excitation/emission wavelengths. Mitochondrial purification. Isolation of mitochondria and determination of purity was as described (S5) with minor modification. Briefly, rat liver (10 g) or cultured cells collected from 15 dishes (15 cm diameter) were gently homogenized in 10 mm Tris-HCl, 200 mm mannitol and 50 mm sucrose, ph 7.4, using 10 strokes of a glass pestle (Wheaton Dounce). Following removal of nuclei and unbroken cells (centrifugation at 1000xg), heavy (3000xg) and light mitochondrial (20,000xg) fractions were separated further by Opti-Prep [60% solution (w/v) of iodixanol; Sigma] gradient centrifugation. Cytosolic (supernatant) and microsomal (pellet) fractions were separated at 100,000xg. Following protein quantification, each fraction was subjected to marker enzyme assays to determine purity of organelles. Acid phosphatase activity (lysosomal marker) was assayed by the hydrolysis of p-nitrophenyl phosphate to p-nirtophenolate anion and evaluated at 410 nm absorbance; succinate dehydrogenase activity (mitochondrial marker) was measured using sodium succinate as substrate and p-iodonitrotetrazolium violet (INT) as the electron acceptor, and evaluated at 490 nm (ε490 = 19,200 M -1 cm -1 ); catalase activity (peroxisomal marker) was derived from rates of H 2 O 2 consumption (S6). Removal of NO donor. DETA-NO treatment at neutral ph was followed by brief acidification (ph 5.0 x 10-min) to decompose the NO donor. ASM/procaspase-3 coincubations were then performed at ph 7.2.
Supporting Figures Fig. S1 Growth of yeast (CG-1945 yhb1) with sustained delivery of NO in the twohybrid assay. DETA-NO at less than 300 µm generates NO without inhibiting yeast growth (monitored by absorbance at 600 nm). Steady-state NO concentrations are maintained at ~100 nm-1 µm for several days as measured with an NO electrode (not shown).
Fig. S2 NO-dependent interaction of AIF with MIP-1α. Yeast ( yhb1) were transformed with Gal4 BD-AIF (Bait) and Gal4 AD-MIP-1α (Prey) (or Bait alone). (A) Robust growth of the AIF/MIP-1α clone requires NO (single asterisk, p<0.001 versus AIF/MIP-1α without NO; n=6). In contrast, the clone expressing AIF alone shows little growth in either the presence or absence of NO. Cells were grown in His-Trp-Leudeficient medium at 30 ºC for 72 hrs with or without 200 µm DETA-NO. (B) β- galactosidase activities in samples shown in A (single asterisk, p<0.001 vs. without NO; n=4). Supporting References and Notes S1. J. B. Mannick et al., Science 284, 651 (1999). S2. L. Liu, M. Zeng, A. Hausladen, J. Heitman, J. S. Stamler, Proc Natl Acad Sci USA 97, 4672 (2000). S3. P. L. Bartel, S. Fields, The Yeast Two-Hybrid System. A. Jacobson, Ed., Advances in Molecular Biology (Oxford University Press, New York, 1997). S4. E. Romiti et al., Mol Cell Biochem 205, 75 (2000).
S5. A. Okado-Matsumoto, I. Fridovich, J Biol Chem 276, 38388 (2001). S6. S. Nag, K. Saha, M. A. Choudhuri, Plant Science 157, 157 (2000).