Deshaies-2005-01-00987B
NIH Support- This work was supported in part by an NIH Research Project Grant
(GM065997) to R.J.D.
Supplementary Discussion for Table S2
Based on our findings with Gcn4, we propose a class of regulatory factors whose activity
is required in a reaction, but whose subsequent turnover or removal promotes completion
of the reaction or subsequent reaction cycles. Such ‘activation by destruction’ appears to
play a role in numerous cellular processes, especially those involving DNA or RNAmediated reactions. For example, VIP1 and VirE2 enable uptake of the agrobacterium
genome into the host nucleus. Subsequently, VirF, an F-box protein, promotes the
degradation of VirE2 and VIP1, exposing the genome so that it can integrate 1. Given the
diversity of other examples listed in Table S2, it is plausible that ‘activation by
destruction’ may represent a regulatory mechanism for a large, underappreciated class of
factors and may be an important determinant of infection and disease. The challenge
remains for most of these factors to understand the mechanism by which turnover
contributes to activator function.
Supplementary Discussion for Figure S2
Inspection of the microarray data from the Velcade analysis indicates that proteasome
inhibition significantly (>2x) depressed the transcription of ~80 Gcn4 targets 2. Dozens
of additional Gcn4 targets were down-regulated 1-2-fold.
Supplementary Discussion for Figure S2
ChIP analysis of Gal4 was performed with a strain in which the chromosomally-encoded
Gal4 was fused to a tandem affinity purification (TAP) tag 3. To facilitate isolation, this
tag contains the Z -domain from Protein A that recognizes immunoglobulin G (IgG). To
confirm that Gal4-TAP was functional and regulated normally we repeated the
proteasome inhibition analysis with MG132 and the pdr5, GAL4-TAP strain. As shown
in figure S2, GAL1 transcripts were produced normally in this strain and their induction
was inhibited by MG132 treatment.
Using the Gal4-TAP strain, we demonstrated that, unlike Gcn4, Gal4 occupancy at the
GAL1 promoter did not change upon proteasome inhibition (Fig. 2c) This is presumably
because the relative concentration of Gal4 allows it to constitutively occupy all of its
several dozen target sites 4. By contrast Gcn4 does not fully occupy all of its sites, which
may number in the thousands, in the growth conditions used 5.
Supplementary Discussion for Figure S3
One hypothesis to explain the effects of proteasome inhibition on Gcn4 and Gal4mediated transcription proposes that MG132 treatment disrupts cellular signaling
Deshaies-2005-01-00987B
upstream of the activators 6. This explanation appears unlikely to apply to Gcn4, as the
end-product of signaling in the Gcn4 pathway is the accumulation of the activator on
cognate promoters 7, and proteasome inhibition promoted such accumulation (Fig. 2b).
In contrast, Gal4 levels at cognate promoters is not regulated by signaling, but promoter
association of the transcriptional repressor, Gal80, is diminished upon galactose induction
8
. Therefore, we tested a strain in which the chromosomal GAL80 locus was TAP-tagged
to monitor the behavior of Gal80 upon galactose induction in the presence of MG132.
Proteasome inhibition had no effect on the promoter-association dynamics of Gal80TAP, as judged by ChIP (Fig. S3). These findings imply that the impact of proteolysis on
Gcn4 or Gal4-dependent transcription is not due to the role of the proteasome in cellular
signaling.
Deshaies-2005-01-00987B
Supplementary Table 1
STRAIN
Relevant genotype
Reference
RJD 796
204, ura3, leu2, trp1, cdc34-2
9
RJD 1114
S288c, MATa, ura3, leu2, his3, pre1-1 pre4-1
10
RJD 1115
S288c, MATa, ura3, leu2, his3
10
RJD 1174
S288c, Mat a, his3 200, leu2-3,112, ura3-52
9
RJD 1721 (BY4741) MATa, his3-1, leu2-0, met15-0, ura3-0
Research Genetics
RJD 2125
RJD 1174, GCN4 (Myc9)::HIS3
9
RJD 2141
RJD 2125, cdc34-2
9
RJD 2152
RJD 2125, cdc4-1
9
RJD 2159
RJD 1174, gcn4 (Myc9)::URA3
RJD 2185
RJD 1174, gcn4-3T2S (Myc9)::HIS3
RJD 2262
RJD 1174, HIS4-LacZ-LEU2
This study
RJD 2264
RJD 2262, srb10::hisG
This study
RJD 2266
RJD 2262, pho85::URA3
This study
RJD 2268
RJD 2262, srb10::hisG, pho85::URA3
This study
RJD 2291
RJD 2185, HIS3P-LacZ::LEU2
This study
RJD 2292
RJD 2125, HIS3P-LacZ::LEU2
This study
RJD 2296
gcn4 (HA3), HIS3P-LacZ::LEU2
This study
RJD 2308
RJD 2296, cdc34-2
This study
RJD 2313
RJD 2291, cdc34-2
This study
RJD 2314
RJD 2292, cdc34-2
This study
RJD 2505
W303, can1-100, leu2-3,-112, his3-11,-15, trp1-1,
ura3-1, ade2-1, pep4::LEU2, pdr5::KANR
This study
9
Deshaies-2005-01-00987B
RJD 3035 (RG 2409) RJD 1721, pdr5::KANR
Research Genetics
RJD 3047 (SUB313) MATa, lys2-801, leu2-3,112, ura3-52, his3-A200,
trpl-1, ubil::TRP1, ubi2-2:ura3, ubi3-ub2,
ubi4-2::LEU2, [pGALP-UBI][pCUP1P-UBI]
11
RJD 3048 (SUB314) as RJD 3047, except [pCUP1P-ubi]
11
RJD 3049 (SUB315) as RJD 3047, except [pCUP1p-ubi-K48R]
11
RJD 3037
RJD 3035, GCN4 (Myc9)::HIS3
This study
RJD 3039
RJD 3035, gcn4-3T2S (Myc9)::HIS3
This study
RJD 3042
RJD 3035, GAL4 (TAP):: KANR
This study
RJD 3045
RJD 3035, GAL80 (TAP):: KANR
This study
RJD 3041
BY4741, GAL4 (TAP):: KANR
Research Genetics
RJD 3044
BY4741, GAL80 (TAP):: KANR
Research Genetics
RJD 3137 (RH1796) W303, ade2-101, leu2-3, 112, suc2-9, trp1-901,
GCRE-LacZ::URA3, gcn4::LEU2
12
RJD 3138
RJD 3137 + pME1092 (GCN4P-GCN4, TRP1)
12
RJD 3139
RJD 3138, pdr5::KANR (from cross w/ RJD 2505)
This study
RJD 3140
RJD 3138, CDC34 (from cross w/ RJD 796)
This study
RJD 3141
RJD 3138, cdc34-2 (from cross w/ RJD 796)
This study
RJD 3142
RJD 2262, cdc34-2
This study
RJD 3143
RJD 2264, cdc34-2
This study
RJD 3144
RJD 2266, cdc34-2
This study
RJD 3145
RJD 2268, cdc34-2
This study
Deshaies-2005-01-00987B
SUPPLEMENTARY TABLE 2
Target
Turnover
factor
Biological Process
ref
Gcn4
SCF(Cdc4)
Turnover of Gcn4 stimulates transcription of target genes.
VIP1, VirE2
VirF
See accompanying supplementary text.
1
Laforin
Malin
Malin targets laforin, a protein phosphatase, for turnover.
Recessive mutations in either give rise to accumulation of
glycogen particles, resulting in Lafora disease.
13
p21, p27
SCF
p21 or p27 promotes cyclin-CDK nuclear accumulation,
but also inhibits CDK. SCF-dependent turnover of p21
and p27 activates CDK to promote cell cycle progression.
14
Securin
APC
Securin promotes proper localization of separase. APC
targets securin for destruction to activate separase and
chromosome segregation.
15
MuA
ClpX(P?)
ClpX removes a protomer of MuA transposase from the
complex intermediate to complete phage Mu transposition.
ClpP role is unknown.
16
Deshaies-2005-01-00987B
Supplementary Methods
Media
Many of our analyses were performed with minimal drop-in medium, which here consists
of yeast nitrogen base (YNB- Difco) and glucose plus only those amino acids and bases
(typically leucine, histidine, and uracil) that the cell cannot synthesize due to auxotrophy.
Under these conditions, the cell must synthesize most of its amino acids (except those for
which it is auxotrophic), and to do so it relies on amino acid biosynthetic enzymes, whose
production depends on Gcn4. Consequently, gcn4∆ cells are severely compromised for
growth in this medium 17 (doubling time ~6 hours) and for transcription of many target
genes (see Figs. 1e, 3e, and S4). The expression of target genes in GCN4 cells in this
minimal medium is approximately half the maximal induction that is seen when cells are
shifted from rich or replete medium to starvation medium (data not shown), allowing for
the assessment of positive or negative effects on Gcn4-dependent transcription. To
induce amino acid starvation cells were cultured in minimal medium to mid-log phase
then harvested and resuspended in a similar medium that lacked leucine 7. In the cases
where the cells were prototrophic for leucine, starvation was induced by the histidine
analog, 3-aminotriazole (3-AT), which was added to 100mM in medium lacking histidine
7
.
Strains used for GAL1 analysis were grown in YP-raffinose (3%) to mid-log (OD 0.5-1).
Galactose was then added to 3% for 0.5 hr. For INO1 analysis, strains were grown in
synthetic complete medium (SC) plus inositol to mid-log, then washed and cultured in SC
lacking inositol for 0.5 hrs.
In all experiments involving MG132, cultures were grown to mid-log, then treated with
50 uM MG132 (dissolved in DMSO at 1000X) or DMSO for 0.5- 1 hour. Inductions
were carried out after 0.5 hour incubation with MG132 or DMSO.
Ubiquitin Derivative Analysis
Strains 11 were used that lacked all chromosomal copies of ubiquitin and that harbored a
plasmid expressing ubiquitin from the GAL1 promoter. In addition the strains carried
plasmids that expressed from the CUP1 promoter either wild type ubiquitin (RJD 4001),
no coding sequence (i.e. empty vector; RJD 4002), or the K48R version of ubiquitin
(RJD 4003) . These strains were grown in minimal medium with galactose, then
switched to glucose containing medium for 6 hrs to deplete WT ubiquitin and exclusively
express the ubiquitin driven by the CUP1 promoter. Samples were prepared for RT-PCR
as described in the text.
SCF Mutant Analysis
Thermosensitive mutant versions of CDC34 and CDC4 were analyzed at semi-permissive
temperature, 30oC because Gcn4-dependent transcription is down-regulated at the
restrictive temperature, 37oC, and defects associated with cell-cycle arrest are avoided at
Deshaies-2005-01-00987B
30oC. Despite having little growth defect at 30oC, cdc34-2 mutants are clearly defective,
as evidenced by synthetic lethality between cdc34-2 and other mutations even at low
temperatures 18.
Deshaies-2005-01-00987B
Supplementary Figure Legends
Figure S1- Proteasome inhibition represses the transcription of many Gcn4 targets and of
INO1, a target of the transcriptional activators, Ino2 and Ino4. a, GCRE6-LacZ,
pdr5cells were cultured in minimal medium, MG132 or DMSO was added for 0.5 hrs,
and cells were processed for RT-PCR analysis of ADH5, ARG1, and ASN1 transcripts. b,
pdr5cells were cultured in SC+inositol to mid-log phase, MG132 or DMSO were
added for 0.5 hours, and then the cells were resuspended in SC lacking inositol for 0.5
hours. Cells were then processed for RT-PCR analysis of INO1 and ACT1 transcripts.
Figure S2- Gal4-TAP functions similarly to wild-type Gal4. pdr5GAL4-TAP strains
were treated as in Fig. 1b.
Figure S3- Proteasome inhibition does not affect the dynamic association of Gal80 with
a Gal4 target. A pdr5, GAL80-TAP strain was prepared for ChIP analysis as in Fig. 2c,
except half of the culture was maintained in raffinose medium and half had galactose
added. PCR was performed with primers to the promoter region of GAL1.
Figure S4- Gcn4 is required for the association of RNA polymerase II (polII) with HIS4.
ChIP analysis of polII was performed as in Fig. 2b with GCN4-Myc9 and gcn4 strains
grown in minimal medium.
Deshaies-2005-01-00987B
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