Genetic analysis of growth inhibition by GAL4-L kappa B-alpha in Saccharomyces cerevisiae.
I kappa B proteins bind to and regulate Rel/NF- kappa B transcription factors. We showed previously that a fusion protein (GAL4-p40) containing the DNA-binding domain of GAL4 and sequences of chicken l kappa B-alpha (p40) inhibits growth in the yeast Saccharomyces cerevisiae. We now show that p40 must be bound to DNA to inhibit yeast growth, p40 proteins, bound to DNA either as GAL4 or LEXA fusion proteins, inhibit yeast growth. In contrast, p40 proteins that cannot bind to DNA, such as full-length p40, a GAL4-l kappa B fusion protein containing a mutant GAL4 DNA-binding domain, and a fusion protein (GAD-p40) containing the transcriptional activation domain of GAL4 fused to p40, each failed to inhibit cell growth. As with GAL4-VP16, GAL4-p40 needs a functional cellular ADA2 gene to exert its growth-inhibitory effect in S. cerevisiae. Using a high copy suppression strategy, we have isolated three S. cerevisiae genes that restore normal growth to yeast expressing GAL4-p40 or LEXA-p40. We have termed these rescuing genes collectively as SIK genes, for "Suppressors of 1 kappa B." Expression of the SIK genes specifically suppresses the growth-inhibitory activity of GAL4-p40 and LEXA-p40 because SIK gene expression cannot block GAL4-VP16-mediated growth inhibition in S. cerevisiae. SIK1 encodes a novel protein that contains a COOH-terminal repeat that has been found in many microtubule-binding proteins. SIK2 encodes NH2-terminal acetyltransferase, and SIK3 encodes the yeast ribosomal S4 protein. None of the SIK proteins binds directly to p40 sequences in vitro, suggesting that the SIK proteins are likely to act downstream of the direct point of growth inhibition by GAL4-p40. Our results may be useful for devising strategies for identifying vertebrate inhibitors of l kappa B proteins and of other proteins that inhibit growth in S. cerevisiae.
Morin
P J
PJ
Department of Biology, Boston University, Massachusetts 02215, USA.
Downs
J A
JA
Snodgrass
A M
AM
Gilmore
T D
TD
D000595:Amino Acid Sequence; D002455:Cell Division / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal / Q000502:physiology; D006131:Growth Inhibitors / Q000235:genetics; D020672:I-kappa B Proteins; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013489:Suppression, Genetic / Q000502:physiology; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D006131:Growth Inhibitors; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D051996:Transcription Factor RelA; D014157:Transcription Factors