<p>Supplemental Figure Legends</p><p>SF1: Fbw7 dimers and monomers interact indistinguishably with cyclin ET62A. A. </p><p>35S pulse-chase analyses of cyclin ET62A half-lives when co-transfected with Fbw7 dimers (wt-Fbw7) or monomers (Fbw7 ∆ D ), or with vector control in 293A cells. </p><p>Quantified signals were plotted on the right. B. In vitro ubiquitylation of cyclin E or cyclin ET62A with recombinant Fbw7 dimers (D) and monomers (M).</p><p>SF2: Alternative Fbw7 dimer-deficient mutants confirm the observations made with Fbw7∆ D. A. Illustration of truncation mutants. M240 lacks the initial essential amino acids of the dimerization domain, but contains all residues missing in </p><p>Fbw7∆ D. B. M240 is a monomer. Cells were co-transfected with differentially tagged Fbw7 as indicated and lysates immunoprecipitated and western blotted. </p><p>PCNA is a loading control. C. Weak degron positions reveal dimer-dependence with M240 monomer mutant. Cells were transfected and blotted as indicated. </p><p>PCNA is a loading control. Assay as in Fig. 2A. D and E. Lack of dimerization and cyclin E/T380S turnover by a double-point mutant Fbw7 (Fbw7LI). Cells were assayed as above.</p><p>SF3. Analyses of the cyclin E-Fbw7 interaction. A. Degron analysis of the P-2 position substituting the cyclin E T380 degron with residues naturally found in other substrates in this position (glycine: Jun and PGC1, threonine: SREBP1). </p><p>Cells were transfected and blotted as indicated. PCNA is a loading control. B. </p><p>Co-immunoprecipitation of central T/S exchanges in both cyclin E degrons with Fbw7 dimers (Fbw7∆ F) and monomers (Fbw7 ∆ FD). 293A cells were co- transfected as indicated, immunoprecipitated with FLAG antibody to Fbw7 and western blotted. C and D. A P+2 proline introduction increases the cyclin ET62 </p><p>CPD competence. Co-immunoprecipitation analysis (C) of mixed lysates expressing the indicated input (D). E and F. Engineered Fbw7 propeller mutants interact with cyclin E in a dimer-dependent fashion. Assay as in (C and D).</p><p>SF4. Generation of dimer-deficient cell lines of endogenous Fbw7. A. Depiction of the exon/intron border of the targeted region in the Fbw7 gene. Yellow: deletion used in FLAG-Fbw7∆ D, red: deletion chosen for gene targeting </p><p>(EWLKMF). B. Confirmation the EWLKMF prevents Fbw7 dimerization. 293A cells were co-transfected with differentially tagged Fbw7 and lysates immunoprecipitated and blotted as indicated. Asterisk denotes heavy chain. C. </p><p>Illustration of gene targeting strategy used for both AAV vectors (see methods). </p><p>D. Depiction of Southern screening strategy to identify correct homozygous gene targeting and excision of Neo cassette, as well as a representative Southern blot.</p><p>SF5. Fbw7 monomers and dimers can be distinguished in non-denaturing gels. </p><p>Two amounts of recombinant Fbw7 monomers (M) and dimers (D) purified from </p><p>SF9 cells were separated on denaturing and non-denaturing gels and stained with Coomassie blue. SF6. Dimer-dependent SREBP-Fbw7 interaction. A. To prevent SREBP turnover, cells were transfected separately with the indicated Fbw7 constructs or with </p><p>SREBP1 or 2. Lysates were mixed, immunoprecipitated and blotted as indicated. </p><p>Asterisk marks heavy chain. B. SREBP1 binding to Fbw7 requires SREBP1 dimerization in vitro. GST pull-down of in vitro phosphorylated SREBP1 dimers </p><p>(WT) and SREBP1 monomers (∆ D) with Fbw7 dimers. C. lysate input for (B).</p><p>SF7. Pin1 overexpression does not disrupt Fbw7 dimers. Cells were co- transfected as indicated and blotted on denaturing (upper panels) and non- denaturing gels (lower panel). Pin1 overexpression does not convert Fbw7 dimers to monomers.</p><p>SF8. Dimerization regulates Fbw7 auto-ubiquitylation. A. Ectopic Fbw7 half-life is proteasome-dependent. 35S pulse-chase analysis of transfected Fbw7 in the absence or presence of the proteasome inhibitor velcade. Chase times in hrs. B. </p><p>Ectopic Fbw7 instability is F-box-dependent. Transfected 293A cells were analyzed by pulse chase, comparing wild-type Fbw7 with an F-box deletion mutant. C. Trans-ubiquitylation of Fbw7 in vitro. 293A cells were co-transfected with MYC-Fbw7F and either FLAG-Fbw7 or FLAG-Fbw7F. Lysates were subjected to sequential immunoprecipitation to isolate MYC- and FLAG-tagged hetero-dimers, followed by an in vitro ubiquitylation reaction and immunoblotted. </p><p>The MYC blot was stripped and re-probed with Fbw7-specific antibody to demonstrate the 1:1 ratio of MYC- vs. FLAG-Fbw7. Note that MYC-∆ F and FLAG-Fbw7 co-migrate due to the F-box deletion. D. In vitro auto-ubiquitylation of recombinant Fbw7 monomers (M) and dimers (D) (Rx- reaction mix only). E. </p><p>Fbw7F dominantly stabilizes wild-type Fbw7 in a dimer-dependent fashion. </p><p>Cells were co-transfected and immunoblotted as indicated. F. Dominant Fbw7 stabilization by co-transfection requires dimerization. MYC-Fbw7 was co- transfected with FLAG-truncation mutants progressively deleting the dimerization domain (+ indicates dimerization) (Welcker and Clurman 2007).</p><p>SF9. Fbw7 dimerization facilitates substrate target lysine utilization. In vitro ubiquitylation of cyclin E with recombinant Fbw7 dimers and monomers comparing wt and K48R ubiquitin.</p><p>Supplementary methods</p><p>Cell culture, transfections. 293A and HCT116 were grown in standard conditions (10% FCS) except when assayed for SREBP (FCS was substituted for lipoprotein-deficient serum, Sigma). Aphidicolin (5µg/ml), nocodazole (40ng/ml) and bortezomib (0.5 µM) were used overnight. Cycloheximide was used at 50 </p><p>µg/ml. Transfections were performed by calcium-phosphate precipitation.</p><p>Cell lysis, immunoprecipitations, immunoblotting, kinase assays. Protein extracts were made with NP-40 or Tween 20 lysis buffer, and SDS-PAGE, western blot, and immunoprecipitation utilized standard methods (Welcker et al. 2003). Quantitative western blotting was performed with an ImageQuant </p><p>Bioanalyzer and ImageQuant TL (Fig. 3D). Endogenous Fbw7 and SREBP were immunoprecipitated prior to detection by western blot.</p><p>SREBP1 pull-down. Bacterial recombinant 6xHis-tagged wild-type and dimer- deficient (mutation of three Leu residues in the zipper) SREBP1a (aa 1-490) were purified with Ni2+-NTA and phosphorylated with recombinant GSK3 (NEB) using 1mM ATP. Phosphorylated SREBP was monitored with pT456 (pT426 in </p><p>SREBP1b) antibody and incubated with GST-purified GST-Fbw7 expressed in </p><p>293 cells on beads.</p><p>Generation of dimer-deficient HCT116 cell-lines. Adeno-associated viral (AAV) vectors were used to target both endogenous Fbw7 alleles as described (Hirata and Russell 2000; Grim et al. 2008). An internal promoter-driven Neo vector was used to create Fbw7∆ D cells and a splice-acceptor-Neo vector used to develop additional lines with EWLKMF deletions (termed A7 and B6 in Fig. 3B). </p><p>Homozygous deletions were obtained by removing Neo from the first targeted allele with Ad-Cre, followed by retargeting and a second round of Cre-mediated excision. Homozygous mutants were identified by PCR, Southern blotting, and sequencing of genomic DNA and cDNA (SF4 and not shown).</p><p>Pulse-chase. Cells were starved for 30 min in methionine-free DMEM containing</p><p>5% dialyzed FCS and labeled in 6 cm dishes with 1 ml of trans-35S-label (300 µCi/ml) for 30 min. Dishes were washed, ‘chased’ with cold methionine, and frozen at -800C. Cell lysates were subject to immunoprecipitation and electrophoresis. Gels were analyzed with a phosphor-imager and quantified with </p><p>ImageQuant TL. </p><p>Ubiquitylation. Ubiquitylation reactions contained purified E1 enzyme (1 µg), </p><p>UbcH3 E2 enzyme (1 µg), neddylated Cul1/Rbx1 (100 ng), ubiquitin (5 µg), 2 mM</p><p>ATP, 5mM MgCl2, 2 mM DTT, 50 mM Tris pH 7.5. Cyclin E (Figs. 1F, SF1B, and </p><p>SF9) was added as transfected cell lysates (10 µl, spiked with bortezomib) whereas Fbw7 (25 ng) was added as purified recombinant monomer (aa 263-</p><p>707) or dimer (aa 228-707). Recombinant Fbw7 dimers were prepared as described (Pierce et al. 2013). For auto-ubiquitylation, purified Fbw7 was used as substrate, either as washed IPs (SF8C) or recombinant proteins (all other assays). The Fbw7 monomer was purified as described in Hao et al., 2007.</p><p>Supplemental references</p><p>Grim JE, Gustafson MP, Hirata RK, Hagar AC, Swanger J, Welcker M, Hwang HC, Ericsson J, Russell DW, Clurman BE. 2008. Isoform- and cell cycle-dependent substrate degradation by the Fbw7 ubiquitin ligase. J Cell Biol 181: 913-920.</p><p>Hirata RK, Russell DW. 2000. Design and packaging of adeno-associated virus gene targeting vectors. J Virol 74: 4612-4620.</p><p>Pierce NW, Lee JE, Liu X, Sweredoski MJ, Graham RL, Larimore EA, Rome M, Zheng N, Clurman BE, Hess S et al. 2013. Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins. Cell 153: 206-215. Welcker M, Singer J, Loeb KR, Grim J, Bloecher A, Gurien-West M, Clurman BE, Roberts JM. 2003. Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E degradation. Molecular cell 12: 381-392.</p>