Isolation of Yeast PhosphoCTD-Associating Proteins (PCAPs)
In an effort to understand the consequences of CTD phosphorylation by CTDK-I, we undertook to identify proteins that specifically interact with the CTDK-I-phosphorylated form of the CTD (PCTD). Our approach used a combination of ion-exchange chromatography, affinity chromatography, and far-western blotting to identify such PC
roteins (PCAPs). CTDK-I was used to exhaustively phosphorylate a GST-CTD fusion protein in vitro [as assayed by a mobility shift on SDS–PAGE (e.g., ref 41
)], and this hyperphosphorylated PCTD fusion protein was used to make the matrix for affinity purification as well as the probe for far-western analysis. The general purification scheme for PCAPs is outlined in .
Schematic overview of methods used to purify phosphoCTD-associating proteins (PCAPs) from yeast. Rectangles represent ion-exchange columns; triangles represent salt elution gradients; trapezoids represent affinity columns.
(A) Methods 1 and 2
An extant yeast extract (40
) was first fractionated on a cation-exchange HiTrap S column (method 1). Bound proteins were eluted with increasing salt, subjected to SDS–PAGE, and visualized by Coomassie staining (). To identify potential PCAPs, replicates of the stained gels were transferred to nitrocellulose and probed in a far-western assay using radiolabeled recombinant β
-gal-PCTD. As seen in , this approach reveals a variety of PCAPs eluting from the column. For example, fractions 23, 25, and 27 have several bands that are detected by the PCTD probe. The pattern of possible phosphoCTD-interacting proteins does not mirror the profile of proteins eluted from the ion-exchange column (not all lanes have putative PCAPs, and not all proteins in any given lane interact with the probe), arguing against the simple possibility that the PCTD probe acts merely as a polyanion (see also discussion in ref 39
FIGURE 2 PCAP purification by method 1 (details in Materials and Methods). HiTrap S fractions subjected to SDS–PAGE were stained with Coomassie Blue (A) or analyzed by far-western blotting (B) with β-gal-[32P]PCTD fusion protein as probe [OP )onput, (more ...)
Since there were many other proteins in the fractions that contain putative PCAPs and it was not possible to correlate a band in the far-western assay with a given Coomassiestained band, HiTrap S fractions were subjected to GST-PCTD affinity chromatography to better enrich for PCAPs. The staining profile of the elutions in , which illustrates one such purification of PCAPs from HiTrap S fraction 23, shows that proteins were indeed fractionated on the basis of PCTD association. Most PCAPs were retained on the column (, compare onput and flow-through), and these constitute only a small fraction of the total proteins in the onput (, compare onput and flow-through). All of these proteins were recovered in a few well-defined fractions (, lanes corresponding to 0.5 M elution). To determine which of these proteins associates with the phosphoCTD by binding directly to it, we performed a far-western assay using hyperphosphorylated GST-PCTD as probe (). The bands in the far-western assay correspond to five distinct bands in the stained gel. These stained bands were excised from the gel and identified by mass spectrometry as Ssd1, Hrk1, Ssd1, Set2, Rgd1, and Hrr25. Again, note that several proteins seen by staining do not bind the PCTD in the far-western analysis (). The binding of Ssd1, Hrr25, and Set2 to the PCTD was further characterized as described below.
Fractions 19–22, 24 + 25, 26–28, 29–33, and 34–38 from the HiTrap S column were also subjected to a similar analysis. As it was not possible in all cases to equate a stained band with a far-western band, all major stained bands were excised from the gel and analyzed by mass spectrometry. Hrk1, Rgd1, and Hrr25 were identified again (data not shown). In most cases, however, the sample was insufficient for unambiguous identification.
In an effort to improve the yield of PCAPs, the above general approach was applied to a new, different extract, and a different ion-exchange column was used for the initial chromatography step (method 2). These modifications did not dramatically change the overall protein pattern and PCAP profile for the ion-exchange column (not shown). We then passed the ion-exchange column fractions through a control (GST) column before applying them to a GST-PCTD matrix. Bound proteins from both columns were eluted with salt steps as before, concentrated (see Materials and Methods), subjected to SDS–PAGE, and stained with Coomassie (one example is shown in ). All stained bands (marked with asterisks) were excised from the gel and identified by mass spectrometry. As indicated in , several proteins were identified by method 2 that had also previously been identified by method 1 (Hrk1, Set2, and Hrr25). Method 2 also led to the purification of Ess1 [which had previously been shown to bind the PCTD (36
)]. The similarity of results obtained by both methods 1 and 2 attests to the validity of the overall approach as a general PCAP purification strategy.
FIGURE 3 Example of PCAP purification by method 2 (details in Materials and Methods). CM fractions were passed through a control GST column and then applied to a GST-PCTD affinity matrix. Both columns were eluted with salt steps, and the eluted proteins were analyzed (more ...)
However, results in (and data not shown) indicate that a number of potential PCAPs remain unidentified, implying that further refinements to the purification strategy would be required to optimize recovery and identification of PCAPs. We reasoned that we could refine our purification scheme if we understood in more detail the PCTD binding properties of individual PCAPs. Toward this end, we characterized recombinant versions of five distinctly different PCAPs [Ssd1, Set2, Hrr25, Ess1, and the previously identified PCTD-interacting splicing factor Prp40 (34
)] in terms of their phosphoCTD-interacting domains (PCIDs) and/ or their phosphoepitope binding specificities, as described in the following sections.
Characterization of PCAPs. (A) Identification of the PCTD-Interacting Domain in Ssd1
Ssd1, a member of the ribonuclease II family of proteins (), has been implicated in diverse pathways regulating cell growth and differentiation (e.g., refs 46
). It has been shown to interact genetically with many different proteins, but very little is known about its biochemistry. Despite its being in the RNase II family, no RNase activity has been demonstrated for Ssd1; however, it can bind RNA and is thought to be RNA-associated in vivo (49
is polymorphic, and four different alleles having been identified in laboratory strains: SSD1-υ1
, and ssd1-d2
. The d alleles are recessive to the v alleles and encode a C-terminal truncation of the protein (46
). The presence of the d allele affects the penetrance of mutations in many different genes, and in fact, we had previously found that a deletion of CTK1
(the catalytic subunit of CTDK-I) was synthetically lethal at 37 °C in the presence of the ssd1-d
allele (J. M. Lee and A. L. Greenleaf, unpublished data).
FIGURE 4 A) Schematic representation of domain architecture of PCAPs Ssd1, Set2, and Hrr25. (B) Confirmation of the Ssd1– phosphoCTD interaction and identification of the binding domain. Fragments of Ssd1 and Set2 were expressed as recombinant MBP fusion (more ...)
With a view to determining the PCTD binding region of Ssd1, we probed various recombinant MBP–Ssd1 fusion proteins in a far-western assay. confirms that Ssd1 is a PCAP and demonstrates that the p
omain (PCID) lies within the first 240 amino acids of the protein (, lane 2). Although this is a region with no obvious homology to known PCIDs, it is essential for the function of Ssd1 in vivo (47
). Deletions from either end of this 240 residue–fragment appear to decrease the strength of PCTD binding (, lanes 3–5). Although complete characterization of this domain awaits future experiments, we have determined that binding of the N-terminal domain to CTD repeats depends on their pattern
of phosphorylation (; see below).
(B) PCTD Binding Domains in Set2
The SET domain-containing protein Set2 () has recently been shown by several groups to interact with the phosphorylated form of RNA polymerase II both in vitro and in vivo (37
). Set2 is a histone methyltransferase that methylates lysine 36 of histone H3; its catalytic activity resides in its SET domain. In addition to the SET domain, Set2 also has a WW domain and a predicted coiled-coil domain (SMART domain database, http://smart.embl-heidelberg.de/
). To identify the domain in Set2 that mediates PCTD binding, we employed the far-western assay as before to screen various recombinant MBP–Set2 fusion proteins. This approach mapped the PCTD binding activity to a location C-terminal to the SET domain (, lane 7, and data not shown). Dissecting the non-SET domain part of the protein led to finding a region at the extreme carboxyl-terminal end of Set2 (amino acids 619–733) that binds directly to the PCTD in vitro [this region, termed the SRI (65
), is not homologous to any previously described PCID]. Correspondingly, a deletion of this region abolishes the ability of Set2 to immunoprecipitate Pol IIO, and it eliminates methylation uniquely of lysine 36 of histone H3 (65
).These data support a functional role for the C-terminal 115 amino acids in PCTD binding in vivo.
However, in the process of dissecting Set2 to find its PCTD-interacting domain (PCID), we observed that an MBP–Set2(1–618) construct lacking the SRI region is also able to interact directly with the PCTD when probed in a far-western assay, albeit weakly (data not shown). Since this region of Set2 contained a WW domain, and because certain WW domains bind directly to the PCTD (34
), we wished to establish whether it was the WW domain-containing region of Set2 that mediates interaction of this region of Set2 with the PCTD. To this end, a recombinant MBP fusion protein carrying the WW domain of Set2 flanked by 50 amino acids on either side [MBP–Set2(425–551)] was used as a probe in a “reverse” far-western assay. GST-CTD and GST-PCTD fusion proteins were blotted to nitrocellulose and probed with MBP–Set2(425–551), which was then detected with an anti-MBP antibody. As shown in , this region of Set2 (containing the WW domain) does indeed bind directly to the PCTD; moreover, it requires the CTD to be phosphorylated in order to bind. In contrast, purified recombinant MBP alone does not bind either the GST-CTD or GST-PCTD (). Western blotting using an anti-GST antibody on the same blot as in demonstrates the presence of the GST-CTD fusion proteins in both lanes (). Although there are several slower mobility bands in the PCTD lane [corresponding to intermediates of the phosphorylation reaction (cf., e.g., ref 41
)], the MBP–Set2-(425–551) fusion protein selectively binds to the slowest migrating (most highly phosphorylated) form of the PCTD (compare panels C and E of ). Set2 thus appears to have at least two regions which can bind directly to the PCTD. An appreciation of their functional significance can only attend detailed future comparisons of these two PCIDs in Set2.
(C) Further Characterization of Hrr25: Confirmation of the Interaction and Specificity for the Phosphorylated Form of the CTD
Hrr25, a casein kinase I isoform, physically interacts with and phosphorylates Swi6 and is involved in mediating the transcriptional response to DNA damage caused by methyl methanesulfonate (MMS) or treatment with hydroxyurea (HU); specifically, hrr25
Δ cells were shown to be defective in the transcriptional induction of RNR
gene expression upon exposure to HU (66
). Recombinant MBP– Hrr25 was used in a (reverse) far-western assay to confirm its in vitro interaction with the phosphoCTD. shows that Hrr25 binds directly to the PCTD and that this binding requires the CTD to be phosphorylated. As for the Set2 WW domain-containing construct, it can be seen that the MBP–Hrr25 fusion protein binds to only the slowest migrating form of the PCTD (compare panels E and F of ).
(D) SPR (BIACORE) Analysis: Specificity of PCAPs for Doubly Phosphorylated CTD Repeats
We recently demonstrated that CTDK-I can efficiently generate doubly phosphorylated CTD repeats in vitro (32
). We were hence intrigued by the results in , where it appeared that the WW domain of Set2, as well as full-length Hrr25, bound selectively and specifically to the most highly phosphorylated form of the PCTD generated by CTDK-I. Therefore, with the objective of further characterizing the phosphoCTD-epitope binding specificity of these and other PCAPs, we used surface plasmon resonance (SPR) measurements (BIA-CORE) to examine the binding of a subset of PCAPs to a set of chemically synthesized CTD peptides with phosphorylated serines in exactly known positions (), which presumably mimic phosphoCTD forms likely encountered by these proteins in vivo.
Purified recombinant proteins, carrying known or presumed PCIDs of different PCAPs, were interacted with biotinylated CTD peptides immobilized on the surface of a streptavidin sensor chip (). Binding of the PCAPs to the peptides was recorded in BIACORE sensorgrams, where binding is represented by response units (RU, on the y
-axis) as a function of time (on the x
-axis). The sensorgrams in reveal several interesting facts. First, all proteins tested exhibit virtually no binding to the non-phosphoCTD peptide under these conditions, corroborating the serine phosphate dependence of the binding observed in far-western analysis. Second, confirms that the WW domain-containing construct of Set2 can indeed bind directly to the PCTD. There are thus at least two independent phosphoCTD binding domains in Set2, one encompassed by amino acids 425–551 and the other by amino acids 619– 733 (the SRI region; see ref 65
). Third, shows that the N-terminal 160 amino acids in Ssd1 are sufficient for phosphoCTD binding. In light of the far-western analysis in , it will be interesting to see whether the various fragments of the Ssd1 PCID differ at all in their specificity and whether this domain can be dissected further. Fourth, , in addition to confirming the interaction between the C-terminal FF domains of splicing factor Prp40 and the phosphoCTD (amino acids 267–583; see ref 34
), shows that this region of the protein can also associate specifically with the 5P peptide. Fifth, a comparison of the binding sensorgrams of the interaction between various PCAPs and the 2,5P peptide () illustrates that these proteins vary widely in terms of their binding kinetics. The WW domain of Ess1, for example, displays very fast association and dissociation rates (); in contrast, Hrr25 has both a slower association as well as a slower dissociation rate (). Finally, the binding sensorgrams in also demonstrate that all PCAPs tested bind with the highest RU level to the 2,5P peptide, consistent with the positional specificity of CTDK-I in vitro (32
The binding of PCAPs to the three-repeat 2,5P peptide, which contains six phosphoserine residues, is presumably influenced both by the number of negatively charged phosphate groups and by their sequence context. To assess how much of the binding to the 2,5P peptide might be due to its six phosphoserines alone, purified recombinant PCAPs were tested against a scrambled CTD-like peptide (6PC, shown in ), which also carried six phosphoserines but not in the context of the CTD repeat sequence. PCAPs were interacted with this peptide and with three CTD consensus repeat 21-mers with either six phosphoserines (2,5P) or three phosphoserines (2P and 5P), as shown in , immobilized on a second streptavidin sensor chip. As the results of indicate, even after subtracting the binding to 6PC, all proteins tested show the highest RU level on the 2,5P peptide. Interestingly, the PCID of Ssd1 as well as the FF domains of Prp40 appears to bind either one or both of the 2P and 5P peptides more strongly than the 6PC peptide (). Thus, although the six phosphoserines on the 2,5P peptide make a contribution to PCAP binding, they clearly do not account for all of the binding to this peptide; the phosphoserines in the context of the CTD heptad sequence are critical determinants of binding specificity.
Optimizing Methods for PCAP Purification and Identification. (A) Affinity Columns Utilizing Synthetic Phosphopeptides
In light of the data in , as well as our recently published results (32
), we asked whether a three-repeat CTD peptide phosphorylated at serines 2 and 5 of each heptad repeat could substitute for a CTDK-I-generated PCTD as an affinity matrix for the purification of PCAPs. Such a replacement would not only conserve CTDK-I [preparing each PCTD column required expenditure of a relatively large amount of CTDK-I, which is purified in only low amounts from yeast extract (41
)] but would also allow preparing columns with higher densities of binding determinants. Thus we coupled the biotinylated 2,5P peptide used in the BIACORE analysis (shown in ) to streptavidin– agarose beads and used this material in a test purification of PCAPs from a fraction eluted from the CM column (Materials and Methods). Results showed () that the far-western profile of the 2,5P column recapitulates that of the PCTD column; one slight difference is that PCAPs are eluted from the peptide column at lower salt than from the PCTD column. Studies described below indicate that yields are improved by coupling more peptide to the affinity matrix. Also, we found that a different column matrix significantly reduced nonspecific binding of contaminant proteins (below and data not shown). Thus, a doubly phosphorylated 2,5P peptide column can indeed serve as a convenient substitute for the CTDK-I-generated PCTD column.
FIGURE 6 Synthetic 2,5P peptide column mimics PCTD column. Each half of a CM fraction was subjected to affinity chromatography on either a PCTD matrix or a 2,5P peptide matrix. Both columns were eluted with salt steps and the eluted proteins analyzed by far-western (more ...)
(B) Method 3: Combining New Extraction Procedures with Different Column Materials
In further efforts to improve efficiency and increase the yield of PCAPs, we also modified the initial extraction protocol and changed the ionexchange column fractionation (Materials and Methods). Ionexchange column (P11) fractions were stained and probed as before and are shown in . Comparing the far-western pattern of the onput and flow-through of the P11 column (, lanes 1 and 2) shows that PCAPs were indeed retained on the P11 column, and a comparison of the far-western profiles of the elutions from the P11 column () with the elutions from the HiTrap S column () shows that PCAPs are better enriched on the P11 column. Note that proteins that might have bound to the DEAE column were eluted with a step of 0.35 M NaCl and were probed in a far-western assay (, lane 3). No PCAPs were seen to be retained on the DEAE column by this assay. The PCAP profile of the P11 column differs greatly from the overall protein profile; that is, only a minority of proteins that bind the P11 cellulose phosphate resin (stained gel) also bind the PCTD in the far-western assay. Furthermore, strong PCAP bands (far-western) mostly do not correspond to strong stained bands.
FIGURE 7 PCAP purification by method 3. P11 fractions subjected to SDS-PAGE were stained with Coomassie Blue (A) or analyzed by far-western blotting (B) with GST-PCTD fusion protein as probe [OP ) onput, FT ) flow-through, DE ) proteins stepped off DEAE column (more ...)
(C) Further Purification of PCAPs from the P11 Column
On the basis of the distribution of PCAPs (as determined by the far-western assay), sets of P11 fractions (representing early, middle, and late parts of the salt gradient elution) were pooled, and PCAPs therein were further purified by affinity chromatography using a biotinylated 2,5P peptide (see Materials and Methods).
To determine the optimal amount of peptide to immobilize to the avidin matrix, P11 middle fractions were applied to columns containing different amounts of 2,5P peptide, and the columns were developed as described in Materials and Methods (). First, it can be seen that the control column (lanes C) bound no PCAPs (far-western blot) and no detectable protein (stained gel), in contrast to the different matrix material used earlier (cf. and data not shown). Second, by comparison of lanes P1 and P2 (which represent elutions from two different peptide columns with approximately 25 and 50 µM 2,5P peptide, respectively) to each other and to lane C (which represents the elution from the control column), it is apparent not only that proteins (PCAPs) are selectively enriched on the experimental columns but that this enrichment is dependent on the amount of peptide immobilized on the matrix. All further purifications with a 2,5P peptide column were therefore carried out at a peptide concentration of 50 µM (as defined by moles of peptide per volume of beads; see Materials and Methods) on the matrix.
(D) PCAPs Are Enriched on the 2,5P Peptide Column
We next wished to determine whether the enrichment of proteins on the experimental columns was dependent on the pattern of phosphorylation of the CTD peptide coupled to the matrix. To this end, we prepared additional affinity columns comprising biotinylated three-repeat CTD peptides phosphorylated at either serine 2 of each repeat (2P) or serine 5 of each repeat (5P) (). Both 2P and 5P matrices had a peptide concentration of 75 µM. Pooled P11 early fractions were passed through a control avidin column and divided into three aliquots; each aliquot was then subjected to affinity chromatography on a different CTD peptide matrix. The three columns were eluted with salt steps as described (Materials and Methods). An examination of the 2P, 5P, and 2,5P lanes in (which represent elutions from the 2P, 5P, and 2,5P columns, respectively) reveals that the selective enrichment of PCAPs on the experimental column is highly dependent on the phosphorylation pattern of the peptide coupled to the matrix; in this case, virtually all of the proteins that bind to a peptide affinity column bind to the 2,5P matrix but not to the 2P or 5P matrices.
Late fractions from the P11 column were also pooled and, after preclearing through a control column, subjected to affinity chromatography using a 2,5P peptide column. The stained gel () reveals that, once again, proteins are greatly enriched on the 2,5P peptide column as compared to the control column (compare lanes C and P, which represent elutions from the control and peptide columns, respectively). In addition, far-western blotting demonstrates that the 2,5P peptide column dramatically enriches for PCAPs (). It is worth noting that although a few faint bands are evident in the control lanes by Coomassie staining, these bands do not bind to the PCTD in the far-western assay (compare lanes C in panels E and F of ). Moreover, bands that do bind the PCTD in the far-western assay are sometimes too faint to be visualized by Coomassie staining (compare lanes P in panels E and F of ).
Taken together, our results show that PCAPs (proteins that bind the CTDK-I-phosphorylated CTD) are greatly enriched on the 2,5P peptide column. We therefore excised each of the Coomassie-stained bands from the gels shown in and identified them by mass spectrometry; lists all of the approximately 100 proteins that were so identified, grouped into functional categories based on extant information. Note that Ssd1, Hrr25, Set2, and Ess1 all bound to the 2,5P column, consistent with their preference for the doubly phosphorylated peptide in the BIACORE analysis.
Proteins, from pooled P11 early, middle, and late fractions, affinity-purified on 2,5P peptide columns (see and Materials and Methods).
Functional Implications for CTD Phosphorylation by CTDK-I
In addition to expected functions, such as transcription elongation and RNA processing, many of the proteins we identified have been previously assigned other functions, including roles in the processes of DNA metabolism, establishment and maintenance of chromatin structure, regulation of intracellular transport, RNA degradation, sn-RNA modification, and snoRNP biogenesis. In view of the above, we hypothesize that these processes are connected to CTD phosphorylation by CTDK-I. Recent results lend credence to this hypothesis. For example, as mentioned earlier, deletion of either CTDK-I catalytic activity or the SRI region of Set2 leads to a specific loss of H3 K36 methylation, suggesting that binding of Set2 to the PCTD made by CTDK-I is central to its activity in vivo (37
). Analogously, the identification of Hrr25 as a PCAP suggests a role for CTDK-I in modulating DNA damage responses. Indeed, like hrr25
Δ cells, ctk1
Δ cells are unable to efficiently induce RNR
gene transcription upon exposure to DNA damaging agents (67
). Do the splicing factors found in the current or previous work to be PCTD-associated (Prp40, Snu56, Ssd1) therefore entail a role for CTDK-I in splicing?
(A) ctk1 Δ Cells Are Defective in Splicing
To test whether ctk1
Δ cells were defective in splicing, we assayed the splicing of the endogenous RPS17A
transcript [which codes for a ribosomal protein (RP)] at various intervals after returning starved ctk1
Δ cells to rich medium. Under these conditions, RP gene transcription is greatly upregulated (68
), and splicing of newly synthesized transcripts can be assayed. In contrast to wild type, in which unspliced precursor is never observed (e.g., , lanes 2 and 3), ctk1
Δ cells consistently contain significant amounts of the unspliced RPS17A
transcript (lanes 5 and 6). These results provide strong support for the idea that interactions between one or more of the above listed splicing factors and the CTDK-I-generated PCTD are important for splicing in vivo.
FIGURE 9 Cells lacking CTDK-I are defective in splicing. (A) Diagram of the nuclease protection assay components; the asterisk indicates radiolabeled phosphate at the 5′ end of the probe. (B) Nuclease-protected RNA fragments were analyzed by denaturing (more ...)
Functional Organization of PCAPs and Associated Proteins
The far-western analysis on the 2,5P elutions in indicates that not all proteins present bind the PCTD probe directly, implying that some of the proteins associate with the peptide column indirectly. We therefore asked whether any of the proteins eluting from the 2,5P columns have been previously known to copurify or otherwise associate with each other. A search of recent proteomic analyses from yeast (69
) revealed that the majority of proteins that we found on the peptide columns (listed in ) have indeed been shown previously to copurify with others on the columns. PCAPs and associated proteins that have previously been found together in a complex are listed in Table 1 of the Supporting Information
, categorized by either the complex number from ref 70
or the “bait” protein from ref 69
. Interestingly, not all proteins from any given complex were found on our columns; for example, of the 46 proteins comprising complex number 132 as described in ref 70
, we found only 9 (Brx1, Cbf5, Has1, Mis1, Mrp7, Upt20, Cic1, Fun12, Noc2); some were purified from “middle” P11 fractions and some from “late” P11 fractions. Similar profiles were observed for almost all such complexes as described in either ref 70
or ref 69
(see Table 1 of Supporting Information
and Discussion). Intriguingly, in addition to such physical interactions, these proteins also interact with each other genetically [SGD, Saccharomyces
Genome Database, http://www.yeastgenome.org/
)], indicating that the observed physical interactions also have functional significance in vivo (Table 2 of Supporting Information
). In addition, several of the proteins have been found to interact genetically with genes encoding transcription elongation-related functions, such as CDC73
). Moreover, such genes encoding transcription elongation-related functions (e.g., CDC73
) in their turn interact genetically with CTK1
). Taken together, the new and extant data support the idea that the physical interactions between these proteins are functionally significant in the context of CTD phosphorylation and/or transcription elongation.