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K. Melcher: Mutational analysis of GAL80 (Supplemental Data)
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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
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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)
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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)
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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
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K. Melcher: Mutational analysis of GAL80 (Supplemental Data)
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oligonucleotide] to 10 nM. Reactions were separated by native PAGE, and subjected to
autoradiography,
Gal80 dimerization assay. Reactions containing
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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 1Interaction 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 2Interaction of Gal80 mutant proteins with Gal80-VP16. To analyze Gal80
dimerization, in vitro translated
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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:
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K. Melcher: Mutational analysis of GAL80 (Supplemental Data)
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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 3Interaction 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.
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