Deletion of the acidic tail of human Cdc34 results in a severe loss of Cdc34-SCF activity in an “autoubiquitylation” assay (22
). This loss of activity can be explained by one of three possible hypotheses. First, deletion of the tail may affect binding of Cdc34 to SCF. Second, tail deletion may result in a loss of catalytic activity of the Cdc34-SCF complex. Finally, the acidic tail of Cdc34 may confer both binding and catalytic properties to the conjugating enzyme. Prior pulldown studies in yeast have pointed to a role for the acidic tail in binding to SCF (21
), and this conclusion was supported by a biochemical study of human Cdc34 that utilized autoubiquitylation as a functional read-out (22
). However, a subsequent analysis of p27 ubiquitylation by human Cdc34 concluded that the acidic tail is dispensable for binding to SCF and instead appears to play a catalytic function (23
). To deconvolve the functions of the acidic tail, we sought to measure its quantitative contribution to the kinetic parameters of ubiquitylation with an authentic substrate.
We began our investigation by comparing the activities of yeast Cdc34-Δ270 and Cdc34-Δ190. Cdc34-Δ270 lacks the distal 25 amino acids of the tail but retains multiple highly acidic stretches, is fully active in vitro
), and supplies CDC34
function in vivo
. Cdc34-Δ190 lacks ~85% of the tail (which starts approximately at residue 171), including the major acidic stretches, and is unable to supply Cdc34 function in vivo
. The assay we employed was a steady-state di-ubiquitin synthesis assay (28
), a key advantage of which is that it can be used to assess both SCF-independent and SCF-dependent activities for Cdc34. Radiolabeled K48R ubiquitin was added to reactions containing E1, Cdc34, and ATP in either the presence or the absence of SCF. Because Cdc34 forms Lys48
ubiquitin chains with high specificity, K48R ubiquitin can form thioesters with Cdc34 but will not attack Cdc34·ubiquitin thioesters. Once Cdc34 was charged fully with the K48R ubiquitin, the reactions were initiated by the addition of unlabeled D77 ubiquitin, which cannot form thioesters with E2 because the terminal glycine residue is blocked but is nevertheless able to attack Cdc34·ubiquitin thioesters because Lys48
remains intact. The product appears in the form of radiolabeled D77-K48R di-ubiquitin.
Using a concentration of both Cdc34-Δ270 and Cdc34-Δ190 (2 μm) that was sufficient for Δ270 to saturate SCF, we measured the rate of di-ubiquitin synthesis in both the presence and the absence of SCFCdc4 (for simplicity, we will refer to SCFCdc4 hereafter as SCF unless noted otherwise). Both Cdc34-Δ270 and Cdc34-Δ190 formed di-ubiquitin with nearly identical rates (, A and B). The addition of SCF stimulated Cdc34-Δ270 activity by ~30-fold (C). In stark contrast, the rate of di-ubiquitin formed by Cdc34-Δ190 was not enhanced at all by SCF (D). These results demonstrate that although deletion of the acidic tail had no effect on the SCF-independent catalytic activity of Cdc34, it completely vitiated the SCF-enhanced boost in activity that was observed for Cdc34-Δ270.
FIGURE 1. Yeast Cdc34-Δ190 is functional but cannot be activated by SCF. Di-ubiquitin synthesis assays containing 250 nm E1, 2 μm Cdc34-Δ270 or Cdc34-Δ190, 6 μm 32P-labeled K48R ubiquitin, and 50 μm D77 ubiquitin (more ...)
Prior work has demonstrated that the C-terminal tail of Cdc34 is both necessary and sufficient for binding SCF in a pulldown assay (21
). We next sought to test whether this binding is sufficient to inhibit SCF activity. Titration of GST yeast tail into Sic1 ubiquitylation reactions resulted in the nearly complete inhibition of product formation at 200 μm
GST-tail () with an IC50
of 16 ± 6 μm
. Note that inhibition was not an artifact of the high concentration of protein because 200 μm
GST had no obvious effect on Sic1 ubiquitylation. This result is consistent with the observation that the acidic tail is important for Cdc34 function (21
) and furthermore indicates that the function of the tail cannot be supplied in trans
FIGURE 2. The acidic tail inhibits Sic1 ubiquitylation by Cdc34-SCF. Reactions containing 1.6 μm yeast E1, 100 nm yeast SCF, 1 μm 32P-labeled Sic1, 2 μm yeast Cdc34-Δ270, and 150 μm ubiquitin were incubated for 1 min at 20–22 (more ...)
Kinetic Analysis of Cdc34 Tail Truncations Reveals Defects in Both Binding to and Catalysis with SCF
Our next goal was to characterize Cdc34-Δ270 and Cdc34-Δ190 by measuring the Michaelis-Menten kinetics for these E2s. To accomplish this, E2 was titrated into multi-turnover Sic1 ubiquitylation reactions. The initial rate of Sic1 ubiquitylation (defined as molecules of substrate modified with one or more ubiquitins as a function of time) was plotted against Cdc34 concentration, and data were fit to the Michaelis-Menten equation. If Cdc34 is in rapid equilibrium with SCF relative to the rate of ubiquitin transfer, the Km may be interpreted as a pseudo equilibrium binding constant between E2 and enzyme. Because numerous ubiquitin molecules may be transferred to substrate during the time course of a reaction, it is important to note that kcat, the maximum rate of Sic1 ubiquitylation, is a function of all the first order rate constants, including the ubiquitin transfer rate and product dissociation, and cannot be simply interpreted as the rate of the chemical step. However, it seems very unlikely that tail truncation would affect the dissociation of ubiquitylated product from SCF, and thus any reduction in kcat (as well as a reduction in the number of ubiquitins attached per substrate when E2 is saturating SCF) is likely to be indicative of a defect in catalysis.
We first measured Km
for Cdc34-Δ230, which has nearly the same activity for Sic1 ubiquitylation as Cdc34-Δ270 (supplemental Fig. S2
). Cdc34-Δ230 had a Km
of ~160 nm
and a kcat
of ~3 min−1
). Saturating concentrations of Cdc34-Δ230 resulted in highly ubiquitylated products that barely migrated into the SDS-PAGE gel. To complement the Km
determination, we also measured the intracellular concentration of Cdc34 by a combination of quantitative immunoblotting and quantitative fluorescence (supplemental Fig. S3
). Cdc34 was present at ~10 μm
in the nucleus and 2.9 μm
in the cytoplasm. It should be noted that Km
was measured at 50 mm
NaCl, and interaction of human Cdc34 with human SCF is sensitive to salt (45
). Nevertheless, it would appear that a significant fraction of SCF is bound to Cdc34 in vivo
and in the nucleus is likely to be saturated.
FIGURE 3. Progressive deletion of the Cdc34 acidic tail results in defects in both Cdc34 binding to SCF and catalysis. All of the reactions contained 1.6 μm yeast E1, 100 nm yeast SCF, 1.2 μm 32P-labeled Sic1, 300 μm ubiquitin, and Cdc34 (more ...)
By contrast with the efficient ubiquitylation of Sic1 by Cdc34-Δ230, titration of Cdc34-Δ190 into Sic1 ubiquitylation reactions yielded dramatically different kinetic parameters. The Km
for Cdc34-Δ190 (16 μm
) was ~100-fold greater, which together with the GST tail inhibition experiment solidifies the notion that the acidic tail of Cdc34 is an important determinant of functional E2 binding to SCF (, B
and H, and supplemental Fig. S4
). We note that Km
for Cdc34-Δ190 and the IC50
for the GST tail were nearly identical, suggesting that the catalytic domain and the acidic tail of Cdc34 alone have comparable weak affinities for SCF. Direct binding measurements confirmed the kinetic observations reported here that the acidic tail stabilized association of the catalytic domain with SCF (45
In addition to the increase in Km, we also observed that the kcat for Cdc34-Δ190 was ~10-fold less than kcat for Cdc34-Δ230. Even when Cdc34-Δ190 was close to saturating SCF, chain elongation on Sic1 was substantially attenuated (B). For example, notice that unlike Cdc34-Δ230, there were no high molecular weight products formed with Cdc34-Δ190 that migrated at the top of the gel. Thus, in addition to promoting binding to SCF, the acidic tail also promotes ubiquitin transfer within the Cdc34·Ub-SCF-substrate complex.
To dissect more finely the different activities of the acidic tail, we created a truncation series in which 5 or 10 residue blocks were progressively deleted from the C terminus of the Cdc34-Δ230 sequence. The first mutant in the truncation series, Cdc34-Δ225, resulted in an activity that was indistinguishable from Cdc34-Δ230 (, C
, and supplemental Fig. S4
). The next mutant, Cdc34-Δ220, exhibited a modest 3-fold increase in Km
but no change in either kcat
or ubiquitin chain elongation on substrate (, D
, and supplemental Fig. S4
). Deletion of an additional 10 residues created a mutant (Cdc34-Δ210) that was severely defective in Km
, and supplemental Fig. S4
). The Km
for Cdc34-Δ210 was comparable with the Km
for Cdc34-Δ190, indicating that residues in between amino acids 211 and 220, in the sequence DDENGSVILQ, can mediate binding of Cdc34 to SCF. In addition to the Km
defect, there was a noticeable decrease in ubiquitin chain elongation. Finally, Cdc34-Δ205 exhibited a Km
similar to Cdc34-Δ210, but unlike Cdc34-Δ210, Cdc34-Δ205 showed a decrease in kcat
in addition to a noticeable defect in ubiquitin chain elongation (, F
, and supplemental Fig. S4
). In conclusion, we found that residues 211–220 in the Cdc34 acidic tail contribute to binding to SCF, whereas the more proximal segment 191–210 appears to affect catalysis.
Although it is clear that the tail is essential for Cdc34 function and interaction with SCF, it is largely unknown exactly what property of the tail comprises its essential function. Our fine scale deletion mapping data cast doubt on the assumption that the acidity of the tail is its critical feature, because the sequence in the region spanning amino acids 211–220 (DDENGSVILQ) contains only three acidic residues, but its deletion increases Km by 36-fold. Meanwhile, both the segments immediately upstream (residues 201–210) and downstream (residues 221–230) are more enriched for Glu and Asp, but deletion of either segment has only a modest effect on Km. To resolve this apparent discrepancy, we sought to address directly the issue of whether the acidity of the tail is in fact relevant to its biological function by replacing the entire stretch of residues from 211 to 230 with acidic amino acids. Remarkably, this mutant, Cdc34-Δ210ED-230, was capable of ubiquitylating Sic1 with essentially identical kinetics as compared with Cdc34-Δ230 (). In addition to the highly similar values of Km and kcat for the two proteins, chain elongation on Sic1 was also very similar. This result provides compelling evidence that electrostatic interactions involving tail residues C-terminal to residue Asp210 play a key role in mediating contacts between Cdc34 and SCF.
FIGURE 4. The distal segment of the acidic tail can be replaced entirely by poly(Glu-Asp). Reactions containing 1.6 μm yeast E1, 100 nm yeast SCF, 1.2 μm 32P-labeled Sic1, 300 μm ubiquitin, and a 2-fold linearly decreasing titration of Cdc34-Δ210 (more ...)
Fusion of Cdc34 to Cul1 Partially Rescues Cdc34-Δ190 Activity
If a key function of the acidic tail is indeed to help tether Cdc34 to SCF, then it should be possible to at least partially compensate for deletion of the tail by directly fusing Cdc34 to SCF. To test this hypothesis, we sought to construct fusions between human Cul1 and human Cdc34. The rationale for using the human proteins for this experiment is 2-fold. First, there are crystal structures for human Cul1 and Cdc34 but not the yeast proteins. Second, the human Cul1 can be expressed at high levels in E. coli
as two separate fragments, which would facilitate the expression and analysis of chimeric constructs. It is important to note that the acidic tail of human Cdc34 is important for its function in SCF-dependent substrate ubiquitylation (22
). Moreover, we confirmed that the tail domain of human Cdc34, like that of yeast Cdc34 (21
) bound Cul1-Rbx1 in a GST pulldown assay (supplemental Fig. S5
To construct an SCF-Cdc34 chimera, we settled on a design in which the N terminus of Cdc34 was fused to the C terminus of Cul1 using a flexible 16-residue glycine-rich linker that was predicted by computational analysis to be long enough to span the two termini in an E2-SCF complex. We anticipated that the fusion should keep SCF and Cdc34 in close proximity, such that SCF would be continuously saturated with Cdc34.
For the experiments shown in , wild type Cdc34 and Cdc34-Δ190 fused to Cul1 were co-expressed with Rbx1 in E. coli
and purified. To avoid confusion, the noncovalent complex formed by co-expressed Cul1 and Rbx1 is referred to as Rbx1+Cul1. Fused complexes were either assayed as is or assembled with Skp1-βTrCP expressed in baculovirus-infected insect cells. In a direct comparison, unfused wild type Cdc34 and Rbx1+Cul1 (A
) and wild type Rbx1+Cul1-Cdc34 fusion (B
) yielded similar activities in the di-ubiquitin synthesis assay. Because it is known that the same interface on E2 binds to both RING and E1 (i.e.
E2 cannot bind to both E1 and RING simultaneously) (29
), this result indicated that fusion of Cul1 to Cdc34 did not impede the access of Cdc34 to either Rbx1 or E1 enzyme and that the catalytic domain of the fused Cdc34 can dissociate from and reassociate with SCF to engage in multiple rounds of di-ubiquitin synthesis. As expected, assaying unfused human Cdc34-Δ190 (using the same concentration as wild type Cdc34) with Rbx1+Cul1 produced no detectable product during the reaction time course (A
). However, the Rbx1+Cul1-Cdc34-Δ190 fusion was active and produced di-ubiquitin, albeit at a reduced rate compared with the wild type fusion (B
FIGURE 5. Fusion of human Cdc34-Δ190 to Cul1 can partially rescue the defect of Cdc34 tail deletion in ubiquitylation reactions. A, di-ubiquitin synthesis assay comparing wild type and Cdc34-Δ190 in the presence of Rbx1+Cul1. B, comparison of wild (more ...)
Human SCF activity is stimulated by covalent modification of a conserved lysine residue on Cul1 with the ubiquitin-like protein Nedd8 (30
). We tested whether neddylation of the Cul1 fusion proteins would stimulate activity in a manner similar to the unfused proteins. Indeed, neddylation of both the wild type and Cdc34-Δ190 fusions resulted in a substantial increase in activity (supplemental Fig. S6
), further demonstrating that fusion of Cul1 and Cdc34 did not corrupt normal SCF function. Given that the stimulatory effect of neddylation was modest under the multi-turnover reaction conditions used here, we chose to work with unmodified SCF for the remainder of the experiments with the fusion constructs.
Given the qualitative rescue effect of the fusion, the time courses for wild type and Δ190 fusion were repeated with more time points and longer reaction times to quantify the difference in activity, which was ~10-fold (, C and D). Because the Cul1+Rbx1 module within the fusion complex should be saturated with Cdc34, the 10-fold reduction in the rate of di-ubiquitin formation most likely represents a defect in catalysis, which compares favorably with our kinetic analysis of unfused yeast Cdc34-Δ190 (B).
We next compared the activities of the wild type and Cdc34-Δ190 fusion SCFβTrcp
complexes using the SCFβTrcp
substrate, mono-ubiquitylated β-catenin peptide (Ub-β-catenin) (25
). We chose to use Ub-β-catenin as substrate because it is conjugated with ubiquitin more rapidly than the unmodified peptide, thereby simplifying quantitative analysis. Remarkably, the activity of the Cdc34-Δ190 fusion was within 2-fold of the wild type fusion (E
). Thus, the acidic tail of Cdc34 was almost entirely dispensable for ubiquitylation of Ub-β-catenin when Cdc34 was fused to Cul1. Interestingly, the activity of the wild type Cul1-Cdc34 fusion toward an authentic substrate was substantially reduced compared with unfused proteins (~15-fold reduction of substrate consumption). Recall that the fused proteins had approximately normal activity in a di-ubiquitin synthesis assay (, A
). We suggest that Cdc34 in the fusion complex is overconstrained by having three separate attachments to SCF (the fusion joint, the catalytic domain-RING interaction, and the acidic tail-SCF interaction). Together, these interactions may restrict rotational and conformational flexibility required for juxtaposition of Cdc34~Ub and substrate within the SCF complex.
Acidic stretches in protein sequences may be a general strategy for promoting protein-protein interactions. Given the critical role of the acidic tail in Cdc34 function, we wondered whether there are additional proteins in the yeast proteome whose function might also depend on highly acidic peptides. To gain insight into this question, the yeast proteome was systematically searched for proteins that contain acidic stretches. Surprisingly there were numerous proteins that contained at least one highly acidic stretch of residues of significant length.
For example, consider that there is an acidic stretch of 25 contiguous residues in the yeast Cdc34 amino acid sequence (residues 208–232) where 72% of those residues are aspartates or glutamates. We found 83 protein sequences in the yeast proteome that each contain at least one acidic stretch spanning 25 contiguous residues in which at least 70% of the residues were Glu or Asp (B
). When protein sequences from the entire yeast proteome were shuffled and reanalyzed for the presence of equivalent acidic stretches, essentially no proteins were identified (supplemental Table S1
), eliminating the trivial possibility that our analysis revealed protein sequences loaded with acidic residues.
FIGURE 6. Proteins that contain long acidic stretches are abundant in the yeast proteome, suggesting that the paradigm of Cdc34-SCF represents a common mechanistic solution for facilitating protein assembly. Each protein sequence in the yeast proteome was searched (more ...)
Careful inspection of the Cdc34-Δ230 acidic tail sequence (as well as the human ones) identifies a smaller stretch of residues that is even more acidic. These concentrated stretches may be important for biological function. Searching the yeast proteome for acidic stretches of 15 residues where at least 80% of the residues are acidic yielded 146 proteins (C), hinting that the binding mechanism utilized by Cdc34 and SCF may be a general phenomenon.