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Supporting Information Supporting Information Zhou et al. 10.1073/pnas.0801313105 SI Text Falcon tubes for 15 min to clear the bulk of the cellular debris, ESI and MALDI MS Analysis of the Human eIF3 Subunits. After LC-MS and the supernatant was decanted and incubated with gentle separation, the effluent from monolithic column was passed mixing for 1 hour at 6°C with IgG-Fast Flow Sepharose resin (GE through a nanospray ionization interface into a QSTAR Elite Healthcare), using Ϸ400 ␮l of 50% resin suspension (prewashed mass spectrometer (Applied Biosystems). The eluent was split so with the same IPP150 buffer) per 40 ml of lysate supernatant. that 1 ␮l/min was directed to a 50-␮m ID PicoTip emitter (New The resin was collected in a Polyprep column (Bio-Rad) using Objective) maintained at 2,500 V. The first and second declus- manually applied pressure from a disposable syringe to maintain tering potentials were 30 and 15 V, respectively. high flow rates. The resin was washed three times with 20 ml of To identify the proteins, LC separation was repeated and the TEV cleavage buffer [10 mM Tris-HCl (pH 8), 150 mM NaCl, effluent from the monolithic column connected to a Probot 1.0 mM DTT) containing no detergent, resuspended in 3 ml of sample fraction system (Dionex) for spotting onto a MALDI the same buffer, and incubated with 600 units of AcTEV plate for digestion and subsequent sequence analysis. Auto- protease (Invitrogen) at room temperature for 1 h. After TEV mated spotting was carried out at 10 s intervals onto a MALDI cleavage, the solution was collected and the resin washed with plate prespotted with 0.5 ␮l of trypsin (0.16 ␮g/␮l) (Promega). another 2 ml of the same buffer. Analysis of the eluted proteins The dry sample spots were then overlaid with 0.5 ␮lof25mM by SDS/PAGE and proteomics experiments revealed the pres- ammonium bicarbonate, digested for 10 min in a humidifier, ence of all five of the ‘‘core’’ yeast eIF3 subunits (a, b, c, g, and vacuum-dried and overlaid with ␣-cyano-4-hydroxycinnamic i) plus eIF5 (Fig. S5). The yeast equivalent of eIF3j (Hcr1) was acid matrix before analysis by automated MALDI-TOF/TOF- not observed. Other weaker bands were often present but varied MS/MS (Applied Biosystems 4700). Data-dependent peak se- in intensity between different preparations. The combined elu- lection of the three most abundant MS ions was used for CID. ate and resin washings were concentrated to Ϸ200 ␮l and then Ar was used as the collision gas. Resulting data were analyzed by purified/buffer-exchanged by size-exclusion chromatography on GPS Explorer (Applied Biosystems), which involved a MAS- a Sephadex 200 column (GE Healthcare) in 200 mM ammonium COT (Matrix Science) database search using all entries in the acetate (pH 7). The complex eluted close to the void volume of National Center for Biotechnology Information (NCBI) data- the column (Ϸ600 MDa), indicating a larger apparent mass than base to determine candidate peptides. Mass tolerance for the the expected for a globular protein of 411 kDa, the combined precursor ion was set to 100 ppm; mass tolerance for the mass of the core yeast eIF3 subunits plus eIF5 (data not shown). fragment ions was 0.3 Da. Manual validation was performed for each MS/MS spectrum. Expression and Analysis of eIF3 Subcomplexes Formed in Insect Cells. Baculoviruses expressing N-terminally Flag-tagged eIF3 sub- Preparation of the Yeast eIF3. Yeast cultures were grown in YPD units e, f, h, k, and l and untagged subunits e and k were medium [10 g/liter yeast extract, 20 g/liter Bacto-Peptone (both generated from the appropriate pcDNA5 vectors (3) essentially from BD Bioscience); 20 g/liter glucose (Sigma)] in batches of as described (4). HA-tagged eIF3b was made by excising the 500 ml of culture per 2-liter flask, inoculated with 2.5 ml of HA-3b coding region from pAMV-pA-HA (3) and inserting it overnight culture, and incubated at 30°C and 220 rpm for 22 h. into pFASTBAC1 (Invitrogen). The eIF3m sequence (GenBank (N.B. At this point, the yeast culture was determined by OD600 accession no. BC019103.2) was amplified by PCR and inserted to have passed the diauxic shift—the point where the glucose in into the NdeI-XhoI sites of pET28c to generate His-6-tagged the medium is exhausted and cells switch to ethanol as a carbon eIF3m, which was subsequently moved into pFASTBAC1. For source—but not to have reached the stationary phase. In this Flag-eIF3m, the PCR product was directly inserted into FLAG- state, the mRNA levels of proteins involved in translational FASTBAC1 (4). processes are lower than before the diauxic shift (1), but the total Sf9 insect cells were coinfected with various combinations of recoverable eIF3 is still higher due to the increased total cell baculoviruses designed to express human eIF3 subunits and density.) Cell pellets from a total of 12 liters were combined, analyzed essentially as described by Fraser et al. (4). Cells were flash-frozen, and stored at Ϫ80°C until processed. Cells were incubated for 72 h, lysed, and subjected to immunoprecipitation lysed by mechanically from partially defrosted cell pellets using with antibody-bound beads (Sigma) against the Flag or HA a bead-beater in the IPP150 buffer described by Gould et al. (2) epitope. The beads were washed, and bound proteins were eluted with added protease inhibitors (Pierce). The total lysate, plus with SDS buffer and subjected to SDS/PAGE, followed by further washings of the glass beads, was spun at 2,000 ϫ g in immunoblotting or staining with Coomassie blue. 1. DeRisi JL, Iyer VR, Brown PO (1997) Exploring the metabolic and genetic control of gene 4. Fraser CS, et al. (2004) The j-subunit of human translation initiation factor eIF3 is expression on a genomic scale. Science 278:680–686. required for the stable binding of eIF3 and its subcomplexes to 40 S ribosomal subunits 2. Gould KL, Ren L, Feoktistova AS, Jennings JL, Link AJ (2004) Tandem affinity purifica- in vitro. J Biol Chem 279:8946–56. tion and identification of protein complex components. Methods 33:239–244. 3. Zhang L, Pan X, Hershey JW (2007) Individual overexpression of five subunits of human translation initiation factor eIF3 promotes malignant transformation of immortal fibroblast cells. J Biol Chem 282:5790–800. Zhou et al. www.pnas.org/cgi/content/short/0801313105 1of13 Identified Subcomplexes Connectivity Map Inferences 0 k-l-e-d c a-b-g-i h-f-m j 1 k-l-e-d c b-g-i h-f-m I. Fidelity of Subcomplexes II. Minimal Connectivity 2 e-d c b-g-i h-f-m 3 k-l-e-d c h-f-m Pairs Modules Building on Connecting 4 k-l-e-d c h-f 5 Modules Modules k-l-e-d c h a b 6 k-l-e-d c 7 c a-b-g-i b g i 8 e a-b-g-i a b i 9 g a b k-l-e-d c g-i i g g 10 l-e-d c h-f-m Module A c 11 l-e-d c h-f b i 12 l-e-d c h A + 30+31 13 l-e-d c 14 k-l-e c k l i 15 k-l-e-d i l e d a b a 16 l-e-d k l e g g b 17 e-d k e d Module B e A + 8 e 18 b-g-i l c 19 g-i d f 20 b-g h f m m 21 h-f-m f k l e d 22 f-h h f h B + 13+14 23 h---m m c 24 h m f-m Module C 25 k-l-e 26 k-l k l e d 27 l-e c f 28 b---i h 29 a-b B + C + 12 m 30 c b---i yeast 31 c b-g 32 g-i Fig. S1. Here, we illustrate the process of building a connectivity map for the eIF3 complex, using only the observed subcomplexes from Table 1 and the data from the yeast eIF3 complex (presented in a column-aligned format at the left). The connectivity map is inferred through application of two hypotheses: (i). The ‘‘Fidelity of Subcomplexes,’’ which states that the intersubunit connections present in the observed subcomplexes are interactions retained from the original full complex. (ii) ‘‘Minimal Connectivity,’’ which states that the full complex contains the smallest set of connections between subunits required to explain those present in multiple subcomplexes. The first hypothesis allows us to identify connected pairs of subunits (indicated by red bars), and we define a pairwise- connected module as a set of pairs with common subunits. Minimal connectivity allows us to build up and connect modules from the subcomplexes and modules denoted below each complex. We use a colored bar and circle to indicate an interaction between a subunit and one or more subunits enclosed within the circle. The resulting ‘‘minimal map’’ contains the same overall connectivity as the model in Fig. 5. Zhou et al. www.pnas.org/cgi/content/short/0801313105 2of13 Fig. S2. MS/MS spectra of in-solution-generated subcomplexes were used to confirm their compositions. (a) eIF3l:k dimer. (b) eIF3i:g dimer. (c) eIF3k:l:e. (d) eIF3b:g:i. (e) eIF3k:l:e:d. (f) eIF3k:l:e:d:c.
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