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K. Melcher: Mutational analysis of GAL80 (Supplemental Data) 1 Supplemental Data Gal80p-Gal4p interaction. Interactions with the Gal4 activation domain were analyzed by incubating increasing amounts of wildtype or mutant Gal80 protein with a recombinant Gal4p derivative and a 32 P-labelled consensus Gal4p binding site oligonucleotide (Supplemental Data Fig. 1). The Gal4p derivative [Gal4p(1-147+34)] consists of the N-terminal DNA binding/dimerization domain directly fused to the C-terminal 34 amino acid core activation domain (MELCHER and XU 2001). The amount of Gal4p(1-147+34)-DNA complex supershifted by Gal80p was quantitated to estimate the relative affinities of Gal80p-Gal4p activation domain interactions. Gal80p variants with changes of amino acids in the very Cterminus and between amino acids 118-215 were mildly to moderately compromised in the Gal4p interaction. All other Gal80p variants were severely defective in interacting with Gal4p(1-147+34) (Supplemental Data Fig. 1). Gal80p-Gal80p interaction. Gal80p-Gal80p interactions were analyzed by complex formation with a Gal80p derivative with altered gel mobility. A fusion between Gal80p and the negatively charged activation domain of Herpes simplex VP16 (Gal80pVP16) migrates faster on native gels than Gal80p by itself. Hence, a mixture between both proteins gives rise to three dimer combinations with distinct mobilities: a slowly migrating Gal80p homodimer, a fast migrating Gal80pVP16 homodimer, and a Gal80p/Gal80pVP16 heterodimer with intermediate mobility (LEUTHER 1993; MELCHER and XU 2001) (Supplemental Data Fig. 2, left panel). To estimate the strength of dimerization, equal concentrations of radioactively labelled wildtype and mutant Gal80 proteins were incubated with increasing amounts of unlabelled Gal80pVP16 and formation of Gal80p/Gal80pVP16 heterodimers was determined. The Gal80p variants displayed a broad range of dimerization defects. With few exceptions (see below), the variants that were severely defective in the Gal80p-Ga80p interaction were K. Melcher: Mutational analysis of GAL80 (Supplemental Data) 2 the same as those that were particularly defective in the Gal80p-Gal4p interaction (Fig. 2 and Supplemental Data Fig. 1 and 2). Gal80 mutant proteins migrated aberrantly and/or as diffused bands, indicating that these variants were misfolded. As expected, aberrantly migrating proteins were generally stabilized by correctly folded, dimerization-competent Gal80pVP16 (Supplemental Data Fig. 2). Gal80p-Gal3p interaction. Finally, I also tested interaction of wildtype and mutant Gal80p with Gal3p. N-terminally His6GST-tagged Gal3p was expressed and purified from S. cerevisiae as described (MELCHER 2000). Then glutathione sepharose bound His6GST-Gal3p was incubated with labelled Gal80 proteins in the presence of galactose and ATP. As shown in Supplemental Data Fig. 3, wildtype Gal80p was retained quantitatively by 600 nM His6GST-Gal3p and to more than 80% by 60 nM His6GST-Gal3p. In contrast, binding of all Gal80 mutant proteins to His6GST-Gal3p was significantly compromised while background retention by His6GST alone was increased for several of the Gal80 variants (Supplemental Data Fig. 3). MATERIALS AND METHODS DNA-Gal4p-Gal80p gelshift assays. 1.5 nM (mutants L52S to S224P) 32 P-labelled consensus Gal4p binding site oligonucleotide (MELCHER and JOHNSTON 1995) was incubated with 2 nM purified Gal4p(1-147+34) and increasing amounts of in vitro translated Gal80p, basically as described (MELCHER and XU 2001). To increase sensitivity in reactions involving in vitro translated Gal80D260N to F394S, concentration of the Gal4-DNA complex was reduced by lowering [Gal4p(1-147+34)] to 0.6 nM and increasing [Gal4 binding site 2 K. Melcher: Mutational analysis of GAL80 (Supplemental Data) 3 oligonucleotide] to 10 nM. Reactions were separated by native PAGE, and subjected to autoradiography, Gal80 dimerization assay. Reactions containing 35 S-methionine labelled in vitro translated Gal80p and Gal80VP16 (upper left panel) were performed as previously described (MELCHER and XU 2001). For all other reactions, in vitro translated labelled wildtype and mutant Gal80 proteins were adjusted with unprogrammed lysate to the same relative concentrations and incubated without ("input") or with fourfold increasing amounts of in vitro translated unlabelled Gal80-VP16. Complexes were resolved by native PAGE and gels were fixed for 30 minutes in 40% methanol/10% acetic acid prior to drying and autoradiography. Gal80-Gal3 interaction assay. Pulldown reactions with in vitro translated Gal80 proteins and indicated amounts of immobilized His6GST and His6GST-Gal3p were performed basically as described (MELCHER 2000). FIGURES Figure 1Interaction of Gal80 mutant proteins with Gal4(1-147+34). Radioactively labelled Gal4p binding site oligonucleotides were incubated with a constant amount of Gal4(1147+34) and increasing amounts of in vitro translated Gal80 proteins. Reactions were separated by native PAGE and subjected to autoradiography. Each set of reactions included wildtype Gal80p for calibration of relative strength of binding. *DNA: labelled Gal4 binding site oligonucleotide; 4: Gal4p(1-147+34); 80: Gal80p. Figure 2Interaction of Gal80 mutant proteins with Gal80-VP16. To analyze Gal80 dimerization, in vitro translated 35 S-methionine labelled Gal80 wildtype and mutant proteins were incubated with Gal80p fused to the negatively charged VP16 activation domain (Gal80VP16). Reactions were separated by native PAGE and subjected to autoradiography. Top left: 3 K. Melcher: Mutational analysis of GAL80 (Supplemental Data) 4 dimerization of labelled wildtype Gal80 and labelled Gal80-VP16. Other panels: radioactively labelled Gal80 proteins were incubated without ("input") or with increasing amounts of in vitro translated unlabelled Gal80-VP16. *80VP16/80VP16: radioactively labelled/unlabelled Gal80-VP16; *802: homodimer of radioactively labelled Gal80p; *80+80VP16: heterodimer of labelled Gal80p and unlabelled Gal80-VP16; *80VP162: homodimer of labelled Gal80VP16. Figure 3Interaction of Gal80 mutant proteins with Gal3p. Radioactively labelled Gal80p (*80) was incubated in a pulldown reaction with 600 nM His6GST (GST) or increasing amounts (60 and 600 nM, respectively) of His6GST-Gal3p (GST-Gal3) immobilized on glutathione sepharose. Reactions were washed, separated by SDS PAGE and subjected to autoradiography. REFERENCES LEUTHER, K. K., 1993 PhD thesis, pp. University of Texas Southwestern Medical Center, Dallas, TX. MELCHER, K., 2000 A modular set of prokaryotic and eukaryotic expression vectors. Anal. Biochem. 277: 109-120. MELCHER, K., and S. A. JOHNSTON, 1995 GAL4 interacts with TATA binding protein and coactivators. Mol. Cell. Biol. 15: 2839-2848. MELCHER, K., and H. E. XU, 2001 Gal80-Gal80 interaction on adjacent Gal4 binding sites is required for complete GAL gene repression. EMBO J. 20: 841-851. 4