- Research article
- Open Access
C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex
- Stephen MT Hoke†1,
- Gaoyang Liang†1, 2,
- A Irina Mutiu1,
- Julie Genereaux1 and
- Christopher J Brandl1Email author
© Hoke et al 2007
- Received: 18 January 2007
- Accepted: 08 August 2007
- Published: 08 August 2007
Spt7 is an integral component of the multi-subunit SAGA complex that is required for the expression of ~10% of yeast genes. Two forms of Spt7 have been identified, the second of which is truncated at its C-terminus and found in the SAGA-like (SLIK) complex.
We have found that C-terminal processing of Spt7 to its SLIK form (Spt7SLIK) and to a distinct third form (Spt7Form3) occurs in the absence of the SAGA complex components Gcn5, Spt8, Ada1 and Spt20, the latter two of which are required for the integrity of the complex. In addition, N-terminally truncated derivatives of Spt7, including a derivative lacking the histone fold, are processed, indicating that the C-terminus of Spt7 is sufficient for processing and that processing does not require functional Spt7. Using galactose inducible Spt7 expression, we show that the three forms of Spt7 appear and disappear at approximately the same rate with full-length Spt7 not being chased into Spt7SLIK or Spt7Form3. Interestingly, reduced levels of Spt7SLIK and Spt7Form3 were observed in a strain lacking the SAGA component Ubp8, suggesting a regulatory role for Ubp8 in the truncation of Spt7.
We conclude that truncation of Spt7 occurs early in the biosynthesis of distinct Spt7 containing complexes rather than being a dynamic process linked to the action of the SAGA complex in transcriptional regulation.
- Apparent Molecular Mass
- Tandem Affinity Purification
- Saga Complex
- Histone Fold
- Centromeric Plasmid
The multisubunit SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex is a prototype for transcriptional coactivators that interface between DNA binding transcriptional regulators, the chromosomal template and the basal transcriptional machinery . SAGA is a dynamic entity, with components existing in subcomplexes having related but distinct functions [2–6].
The Ada components of SAGA (ADA2, NGG1/ADA3, GCN5/ADA4) were first identified based on their involvement in regulated transcription in Saccharomyces cerevisiae [7–11]. SAGA possesses histone acetyltransferase (HAT) activity mediated by Gcn5 [2, 12], which stimulates or represses transcription in a promoter specific fashion [13–17]. The structural core of SAGA is composed of a subset of the TBP-associated factors (TAFs) that are also found in TFIID [18, 19]. Spt7, Ada1 and Spt20, members of the TBP-subclass of Spt (Suppressors of Ty insertions) [2, 20–22] proteins, are located near the core of the complex and are required for its integrity, forming an interface between TAFs and other SAGA-specific components . The additional Spt proteins, Spt3 and Spt8 are involved in direct interactions with TBP [3, 22–25]. Tra1, a 437 kDa, phosphatidylinositol-3-kinase related protein [26, 27] is found at the periphery of the complex and interacts with DNA-binding transcriptional regulators thus conferring promoter specificity to SAGA [28–32]. Through mass spectrometry analysis of purified SAGA complex, other components have been identified [33–36]. One of these proteins, Sus1, is associated with the nuclear pore mRNA export machinery  suggesting that SAGA may have a broader role in nuclear processes than previously envisioned.
Two forms of Spt7 have been described. The full-length protein of 1332 amino acid residues is found within the SAGA complex . The second, a C-terminally truncated form likely arising from processing between residues 1125 and 1150 is found in the SLIK (SAGA-like) complex [3–6]. As suggested by the alternate name for this complex, SALSA (SAGA altered, Spt8 absent), SLIK also differs from SAGA in that it lacks Spt8 [3, 4]. The absence of Spt8 can be attributed to the fact that it interacts with Spt7 in the region deleted in the truncated form [4, 6]. The truncation of Spt7 and the subsequent loss of Spt8 may be a mechanism to create a complex that acts without effects mediated through TBP; for example, the derepression of the HIS3 promoter under starvation conditions [3, 4]. A further difference between SAGA and SLIK is that Rtg2, which is required for the retrograde response pathway in yeast , is found specifically in SLIK . Two other Rtg proteins, Rtg1 and Rtg3 are transcription factors required for the expression of retrograde target genes such as CIT2 . Pray-Grant et al.  propose that SLIK facilitates transcriptional activation by Rtg1 and Rtg3 and perhaps also at a distinct set of promoters. However, the exact role of SLIK is unclear as the interpretation of transcriptional effects upon disruption of RTG2 is complicated since Rtg2 is required for nuclear import of Rtg1 and Rtg3 .
Two models for the relationship between the Spt7-containing complexes can be envisioned. In the first, processing at the C-terminus would occur as part of the biosynthesis of distinct SAGA and SLIK complexes. Alternatively, processing might occur during the normal functioning of SAGA, perhaps to irreversibly signal that a transcriptional event had occurred and/or enabling subsequent activity of the SLIK complex. In this study we performed experiments to differentiate between these models. We show that the conversion of Spt7 from its full-length SAGA-form (Spt7SAGA) to its truncated SLIK-form (Spt7SLIK) and a third C-terminally processed form (Spt7Form3) occurs in the absence of components of the complex including Ada1 and Spt20, which are required for the integrity of the complex. Processing to these forms occurs rapidly after its synthesis and in the absence of full-length Spt7. Taken together our results suggest that processing of Spt7 occurs in the biosynthesis of distinct Spt7-containing complexes.
Truncated forms of Spt7 occur in the absence of fully functional SAGA complex
C-terminal processing of Spt7 occurs in the absence of its N-terminal amino acid residues
The finding that Spt7SLIK and Spt7Form3 were formed in the absence of components of SAGA, including Spt20 and Ada1, suggested that processing is not linked directly to the activity of the complex. If processing were part of the functional dynamics of SAGA, that is, occurring as a result of its activities in the regulation of transcription, then one would predict that it would require Spt7 in a functional form. We thus examined whether N-terminal deletion derivatives of Spt7, expressed in BY3218 (spt7Δ0) would be processed (Figure 3). Each molecule was N-terminally TAP and Flag tagged to allow purification and identification of Spt7 forms. The two longest derivatives (residues 580–1332 and 873–1332) contain the elements of the protein required for partial function . By comparison, the shortest derivative (residues 1038–1332) lacks the histone fold, which is required for interaction with other components of the complex . A protein reduced by approximately 20 kDa, an estimated mass consistent with a truncation at the Spt7SLIK processing site, was seen for each of the derivatives (denoted as SL). A further truncation, approximately 20 kDa smaller than Spt7SLIK and potentially corresponding to processing at the Spt7Form3 site (F3), was evident for Spt7581–1332 and Spt7873–1332 (though at a reduced level). Note that the third-form equivalent was not seen for Spt71037–1332, because its putative cleavage site is removed from this construct. This result suggests that the elements required for processing are intrinsic to the C-terminal end of Spt7 and that the N-terminus of the protein is not required. Furthermore, the appearance of a cleavage product for Spt71037–1332 suggests that processing can take place independently from SAGA and is thus not linked to the action of the complex. For Spt7581–1332, a fourth form was apparent just below Form 3 (marked by *). We have not characterized this form further and can not exclude that it is a degradation product.
Further characterization of the 160 kDa form of Spt7
To examine if Spt7Form3 is found within complexes, TAP-Flag-Spt7 was expressed in BY4741 and a crude protein extract applied to a Superose 6 HR10/30 gel filtration column. The elution profile of the different forms of Spt7 was examined by Western blotting with anti-Flag antibody and the relative amounts of the three forms of Spt7 determined by densitometry (Figure 5c). All three forms of Spt7 eluted in fractions corresponding to a high molecular mass, the largest being Spt7SAGA, followed by Spt7SLIK. Spt7Form3 eluted from the column with an average apparent molecular size slightly less than Spt7SLIK. To determine if Spt7Form3 was associated with components of the SAGA complex, extracts were prepared from strains containing N-terminally Flag-tagged Spt7 and TAP-tagged versions of Gcn5, Ada2, Ubp8, Taf5, Spt3 and Spt8. Complexes containing the TAP-tagged molecules were partially purified and the presence of the different forms of Spt7 analyzed by Western blotting with anti-Flag antibody (Figure 5d, lanes 3–9). Identification of the forms was established by immunoprecipitation of Flag-Spt7 from crude extracts with anti-Flag antibody (compare lanes 1 and 2). As expected from the interaction of Spt8 with the C-terminal end of Spt7 [4, 6], Spt8 interacted only with full-length Spt7 (lane 8). The other SAGA components interacted with both Spt7SAGA and Spt7SLIK. Spt7Form3 did not co-purify with the SAGA components to the extent of either Spt7SAGA or Spt7SLIK.
The function of Spt7Form3 is unknown. The fact that Spt7Form3 appears as stable as the longer forms is consistent with it having a function(s), likely distinct from components of the SAGA complex. It is interesting to note that Spt7 has been found to associate with Ccl1, Rtf1 and Prp43, and that these interactions are not reciprocated by C-terminally TAP-tagged Spt7 or generally by other components of the SAGA complex (Saccharomyces Genome Database). In addition, we previously observed a possible SAGA-independent form of Spt7 by fractionation of crude extracts on a Mono Q column . We also must consider an alternative model, whereby Spt7Form3 is an intermediate along a pathway of degradation. In this regard, we did observe an apparent increase in Spt7Form3 during TAP-purification.
In this report we show that processing of the C-terminus of Spt7 to the form found in the SLIK complex and to a novel third form does not require a functional SAGA complex. C-terminal truncation occurs in the absence of key components of SAGA and SLIK complexes. Spt20 and Ada1 are necessary for the integrity of a complete SAGA complex with their deletion resulting in severe transcriptional effects [6, 22], yet processing occurs in their absence. In addition, since the truncated Spt7 molecules were present in a gcn5Δ0 strain, histone acetylation is not required for processing. Also suggesting that processing of Spt7 is independent of the function of SAGA, we found that a truncated derivative of Spt7 that lacks the histone fold is capable of being processed in a strain lacking full-length Spt7. Finally a time course revealing the appearance and disappearance of the Spt7 forms showed that Spt7SAGA is not chased into the shorter forms. We thus suggest that processing of Spt7 is an independent event in the biosynthesis of distinct Spt7 containing complexes rather than being linked to transcriptional regulation by the SAGA complex.
Processing of Spt7 showed partial dependence on the ubiquitin protease Ubp8. The role of Ubp8 is unclear but based on a partial effect upon deletion of Sgf11, it may require the association of Ubp8 with SAGA. We were intrigued by the possibility that the effect of Ubp8 may be related to our previous observation of the ubiquitylation of Spt7 dependent on the E3-ubiquitin ligase Tom1 ; however, because the relative amounts of the Spt7 forms appear unchanged in a tom1Δ0 strain (not shown) we have concluded that these observations are unrelated.
Our study has identified a third form of Spt7 with an apparent molecular mass of ~160 kDa. The identification of this form with three different tagged derivatives suggests that it is not an artifact of tagging; however, we can not fully exclude the possibility that N-terminal tagging results in its appearance. Gel-filtration experiments suggest that Spt7Form3 is found within a complex; albeit, distinct from SAGA or SLIK. As Spt7 was N-terminally tagged, the mobility of Spt7Form3 on SDS-PAGE suggests a molecule containing amino acid residues 1 to ~950. Determining the size of the released C-terminal fragment would facilitate mapping of the cleavage site; however, we have been unable to identify this molecule using C-terminally TAP-tagged Spt7.
The wild-type yeast strains BY4741 (MATa hisΔ0 leu2Δ0 met1Δ0 ura3Δ0) and BY4742 (MATα hisΔ0 leu2Δ0 lys2Δ0 ura3Δ0) and the consortium constructed isogenic knockout strains  BY3218 (spt7Δ0), BY10809 (ubp8Δ0), BY17285 (gcn5Δ0), BY14619 (rtg2Δ0), BY12666 (spt8Δ0), BY14433 (sgf73Δ0), BY13418 (sgf29Δ0), BY2781 (sgf11Δ0), BY1038 (ada1Δ0) and BY17390 (spt20Δ0), and strains containing C-terminally TAP-tagged SAGA components were purchased from Open Biosystems or Research Genetics. CY1811 is a derivative of BY4741 that contains a YIplac211-GAL-Flag3-SPT7 integrated at the endogenous locus.
Oligonucleotides used in this study
3' fragment of SPT7
5'fragment of SPT7
5' truncations of SPT7
N-terminal Flag epitope
TAP-tagged derivatives of Spt7 with 752, 460 and 295 C-terminal amino acids were constructed by PCR using oligonucleotides 4009-1, 4536-1 and 4536-2 as 5' primer, and 4009-2 as 3' primer. Each was cloned into YCpDed-TAP-Flag as a Not I-Eco RI fragment to generate YCpDed-TAP-Flag-SPT7580–1332, YCpDed-TAP-Flag-SPT7872–1332 and YCpDed-TAP-Flag-SPT71038–1332.
The parent construct for plasmids expressing Flag3-Spt7 is pCB1450. This molecule contains a N-terminal Flag epitope (engineered with oligonucleotides 2821-1 and 2821-2) flanked upstream by the Pst I site of the pUC polylinker and downstream by a Not I site. Genes to be expressed can be inserted as Not I-(Sac I)Eco RI fragments. The polylinker Hin dIII site was deleted allowing insertion of promoter fragments as Pst I-Hin dIII fragments. The GAL10 promoter was synthesized by PCR using oligonucleotides 2764-1 and 2764-2 and inserted into pCB1450. SPT7 from YCpDed-TAP-Flag-SPT7 was inserted as a Not I-Eco RI fragment and two additional Flag epitopes were inserted at the Not I site using oligonucleotides 4213-1 and 4213-2. For integration into yeast the GAL-Flag3-SPT7 module was inserted into YIplac211 . Integration into the endogenous locus was possible after digestion with Sal I.
Tandem affinity purification (TAP) was carried out as described  with minor modifications. Yeast cells were grown in YPD to an A600 = 1.5 and lysed by grinding in liquid nitrogen . All further steps were performed at 4°C. Per liter of starting culture, cell extracts were suspended in ~5 ml of IPP150 (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% NP-40) with protease inhibitors (1 mM PMSF, 0.1 mM benzamidine hydrochloride, 2 μg/ml pepstatin A, 2 μg/ml leupeptin and 0.1 mg/ml trypsin inhibitor). The extract was cleared by centrifugation at 40,000 g for 1 h and incubated with 400 μl of IgG-Agarose (Sigma-Aldrich Canada Ltd) suspension for 2 h. After washing with 30 ml of IPP150 and 10 ml of TEV cleavage buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, 0.1% NP-40), IgG beads were suspended in 1 ml of TEV cleavage buffer and incubated with ~100 U TEV protease for 2 h. 3 μl of 1 M CaCl2 was added to the TEV-cleaved eluent which was then diluted with 3 ml of calmodulin binding buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM magnesium acetate, 1 mM imidazole, 2 mM CaCl2, 1 mM beta-mercaptoethanol, 0.1% NP-40). For the second step of purification the protein was applied to 400 μl of Calmodulin-Sepharose (Stratagene Inc) and incubated for 2 h. After the calmodulin beads were washed with 30 ml of calmodulin binding buffer, bound protein was eluted with calmodulin elution buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM magnesium acetate, 1 mM imidazole, 20 mM EGTA, 1 mM beta-mercaptoethanol, 0.1% NP-40), in 200 μl fractions. 20 μl of the peak fractions were separated by 5% SDS-PAGE and subjected to Western blotting.
Yeast extracts were prepared by glass bead disruption  or grinding in liquid nitrogen . Protein was separated by SDS polyacrylamide gel electrophoresis, transferred to PVDF membranes (Roche Applied Science) using a wet transfer system (Bio-Rad) in 48 mM Tris, 40 mM glycine, 0.0375% (6.5 mM) SDS and 20% methanol for 1 h at 100 V and detected using a 20% solution of Immobilon Western (Millipore Corp). Anti-Flag antibodies (M2, Sigma-Aldrich Canada Ltd) were used at a ratio of 1:4000; anti-myc antibody (Myc1-9E10 cell line) was used at a ratio of 1:2000. Densitometric scanning of films was performed using AlphaImager 3400 software (Alpha Innotech, Inc). Serial dilutions of samples were analyzed to provide standard curves for suitable film exposure.
Time course of the induction and decay of Spt7
A 10 ml culture of CY1811 was grown to an A600 ~3.0 in minimal media containing 0.6% casamino acids (Difco) and 2% raffinose. The culture was diluted with 10 ml of prewarmed media containing 2% galactose and grown for 30 minutes at 30°C. 80 ml of YP media containing 2% glucose was added and the cells grown for 90 minutes. This became time 0, and a 15 ml sample was taken. Cells were pelleted by centrifugation, washed in ice-cold water and frozen at -80°C. Additional samples were taken after 30, 60, 120 and 240 minutes. Yeast extracts were prepared by glass bead disruption in 20 mM Hepes-NaOH (pH 8.0), 240 mM NaCl containing protease inhibitors. Equal volumes were separated by SDS-PAGE (5%) and Western blotted with anti-Flag antibody.
Approximately 8.5 mg of whole cell extract from BY4741 containing YCpDed-TAP-Flag-SPT7 was prepared in 40 mM Tris-HCl (pH 7.7), 300 mM NaCl, 0.1% Nonidet P-40 and 10% glycerol, and subsequently applied to a Superose 6 HR10/30 column (Pharmacia Inc) at a flow rate of 0.3 ml/min. Protein from 250 μl aliquots of 500-μl selected fractions were precipitated with 10% trichloroacetic acid, acetone washed, resuspended in SDS loading buffer and separated on a 5% SDS-polyacrylamide gel.
We would like to thank Amy Tong and Drs. Brenda Andrews, Charlie Boone and Kathleen Gould for providing reagents as well as David Canton, David Litchfield, Meagan Davey, David Edgell, Kerri Kobryn and David Haniford for their discussions on this manuscript. This work was supported by a Canadian Institutes of Health Research operating grant to CB. SH is supported by a NSERC studentship, GL and AM by Western Graduate Research Scholarships.
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