Mass Spectrometry Reveals Abundant Non-K48 Linkages in PolyUb Chains
To analyze the level of polyUb linkages by mass spectrometry (MS), we employed an isotope dilution method (Kirkpatrick et al., 2006
; Xu et al., 2006
), which uses heavy isotope-labeled peptides as internal standards for lighter versions of unlabeled, native linkage-specific peptides that are generated during trypsin digestion of Ub polymers (, Table S1
). Ubiquitinated proteins are usually isolated from yeast cells by affinity purification and digested by trypsin, during which Ub is trimmed to a tag of two Gly residues (GG, monoisotopic mass of 114.0429 Da, ) attached to the modified Lys side chains of remnant peptides of Ub itself or non-Ub proteins (Peng et al., 2003b
). Therefore, the abundance of specific Ub-Ub linkages is represented by the level of the corresponding GG-tagged tryptic peptides. The chemical properties of the labeled standards are indistinguishable from those of native peptides, but, being different in mass, the labeled and unlabeled peptides are resolved by MS, allowing for quantitation of the native peptides. Using chemically synthesized, heavy isotope-labeled, GG-tagged peptides as references, we found that the concentration of K48 linkages is 7.3 ± 0.3 picomoles per milligram protein in log-phase yeast cell lysate. There is a striking and unexpectedly high abundance of the unconventional non-K48 linkages, especially K11 linkages. Based on the absolute quantification of all seven linkages (a sum of 100%), the percent abundance of individual linkages is 10.9 ± 1.9% (K6), 28.0 ± 1.4% (K11), 9.0 ± 0.1% (K27), 3.2 ± 0.1% (K29), 3.5 ± 0.1% (K33), 29.1 ± 1.9% (K48) and 16.3 ± 0.2% (K63). Thus, unconventional Ub chains form a major component of the conjugated Ub pool.
Inhibition of the Ub-proteasome system increases the level of all non-K63 linked polyUb chains.
We further developed a more precise method to quantify polyUb linkages using stable-isotope labeled cells/proteins instead of peptides (), and found that levels of monoUb and all seven Ub-Ub linkages are essentially unchanged as a function of cell growth stage (Table S2
). When monoUb pool was augmented ~10-fold by induction in yeast, all seven polyUb linkages were increased, but to varying levels (3- to 12-fold, Figure S1, supplemental Data
), suggesting that the seven polyUb linkages display different kinetics of synthesis and disassembly.
Proteasomal Degradation is Mediated by Non-K63 PolyUb Linkages
To investigate the potential physiological roles of the abundant unconventional polyUb chains compared with K48-linked chains, we analyzed the change in these linkages during proteasomal inhibition. After treatment with MG132 proteasome inhibitor, bulk Ub conjugates accumulated, leveling off after ~2 hr (). When the MG132 concentration was increased from 10 to 1000 μM, ubiquitinated species accumulated in a dose-dependent manner, almost reaching saturation at ~100 μM. The Ub conjugate levels also markedly increased in the presence of 30 μM of PS341, a more potent and specific proteasome inhibitor. MS analyses indicated that MG132 (100 μM) and PS341 (30 μM) had similar effects on the level of polyUb linkages (, Figure S2
, Table S2
). After 2 hr treatment, K48 linkages increased ~8-fold; K6, K11, and K29 linkages 4–5-fold; and K27 and K33 linkages ~2-fold. In contrast, K63 linkages did not change significantly. When MG132 was administrated at a level (30 μM) causing incomplete proteasomal inhibition, all non-K63 linkages also accumulated, though to a lesser degree (Table S2
). These data indicate that the levels of all non-K63 polyUb chains are inversely correlated with the proteolytic activity of the proteasome. Substrates modified by such chains might therefore be targeted to the proteasome.
To test the apparent involvement of unconventional polyUb chains in proteasomal targeting, we further measured levels of polyUb linkages in yeast strains carrying mutations in components of the Ub-proteasome system. We first developed a method that used total cell lysate directly, rather than Ub conjugates purified by nickel affinity chromatography, because these strains only express untagged Ub (). With this method, we were able to detect only the three most abundant linkages (K11, K48, and K63), the other linkages being undetectable because of their lower concentrations and the greater protein complexity in total cell lysate versus purified Ub conjugates. Single-gene deletions of proteasomal subunits (sem1
Δ or pre9
Δ) or proteasome-assembly factors (pba3
Δ or pba4
Δ) caused comparable increases of K11 linkages (~1.8-fold) and K48 linkages (~2.4-fold, , Table S2
). Similarly, deletion of Ub-receptors Dsk2 or Rad23, both of which deliver Ub-conjugates to the proteasome, raised the level of K11 (~2.0-fold) and K48 (~1.8-fold) linkages, and the double deletion had a more dramatic effect (K11, 2.9-fold; K48, 2.8-fold; ). In all these mutant strains, K63 linkages remained constant. The data strongly support the possibility that both K11-and K48-linked chains are targeting signals for the proteasome.
In addition, we analyzed the abundance of K11, K48 and K63 linkages in yeast strains containing mutations in DUB genes (). Consistent with previous results that deletion of proteasome-associated Ubp6 caused little accumulation of high molecular weight ubiquitinated proteins (Amerik et al., 2000a
), we did not observe large changes in the three linkages. In contrast, in doa4
Δ strain, K11 and K48 linkages were markedly decreased (5-fold and 2.5-fold, respectively), probably due to Ub depletion (Swaminathan et al., 1999
), whereas K63 linkages showed a small increase (1.3-fold) despite the decrease of overall Ub levels, in agreement with the role of Doa4 in membrane trafficking that involves K63-linked polyUb chains (Amerik et al., 2000b
). Loss of the UBP2
gene, encoding another DUB that has preference for K63 linkages and regulates protein sorting efficiency (Kee et al., 2006
), raised the level of K63 linkages (1.8-fold), but also increased K48 linkages (1.9-fold). Loss of UBP14
, a DUB that cleaves unanchored polyUb chains with various linkages (Amerik et al., 2000a
), resulted in higher amount of all three linkages (1.2 to 1.7-fold). These results suggest that distinct DUBs have specific Ub-Ub linkage preferences in vivo. The patterns of polyUb linkages in the DUB mutants are also different from that in the above proteasome-defective cells, supporting the nondegradative role for K63-linked chains and the function of other linkages in degradation.
PolyUb chains with distinct linkages are processed by the 26S proteasome.
If the unconventional polyUb chains direct proteins for proteasomal degradation, the Ub in these chains could be recycled by deubiquitinating activities associated with the proteasome complex. To test this idea, we set up an in vitro deubiquitinating assay using purified native Ub conjugates () and 26S proteasome (). The purified Ub-conjugates were not disassembled without the addition of proteasome, indicating that the Ub-conjugates had little contamination by active DUBs or other proteases (). When the conjugates were incubated with the 26S in the presence of ATP, Ub-conjugate levels were dramatically decreased (), suggesting that polyUb chains were disassembled by the action of the 26S proteasome. This process was obstructed by the addition of MG132 (), probably because it inhibits the proteasome core particle and indirectly interferes with the deubiquitinating activity of a proteasome subunit Rpn11 (Jin et al., 2008
). In addition, this deubiquitination was also sensitive to high-salt treatment (), which inactivates the 26S-associated DUBs, such as Rpn11 (Verma et al., 2002
) and USP14 (a mammalian homolog of yeast Ubp6) (Leggett et al., 2002
). Moreover, we found that all seven linkages were cleaved by proteasome-associated DUBs, but to different extents, with K48 linkages hydrolyzed most readily (). All these data further confirm that unconventional polyUb linkages can be recognized and disassembled in the 26S proteasome.
Phenotypic Effects of Combining Multiple Substitutions in Chain-forming Lys Residues
Although these unconventional polyUb chains are abundant in the cell and appear to function as signals for proteasomal degradation, only the K48R substitution in Ub is lethal in yeast, and the K27R mutation causes a small growth defect (Spence et al., 1995
). Therefore, we asked whether polyUb chains with unconventional linkages have redundant functions by combining mutations in non-K48 lysine residues and testing yeast survival. We generated a series of yeast Ub-mutant strains, including single (R11 or R27), double (R11R63), triple (R11R27R63), and quadruple (R6R11R27R63) lysine substitutions (Figure S3A
). We also attempted to make a strain expressing a sextuple Ub mutant (R6R11R27R29R33R63) with K48 as the single lysine but failed in spite of extensive screening (Figure S3B
), suggesting that Ub with K48 alone cannot support viability in yeast.
To confirm that Ub with K48 as the lone lysine is not sufficient for yeast survival, we used a strategy to switch expression from wild-type to mutant Ub genes in yeast and then examined cell growth (). We constructed yeast strains that maintained two plasmids: one expressing wild-type Ub from galactose-inducible GAL10
promoter, and the other expressing individual Ub mutants under the constitutive CUP1
promoter (). In synthetic galactose media, Ub genes on both plasmids were actively transcribed. Switching to glucose media suppressed the expression of wild-type Ub, whereas the mutant gene was still expressed as the only source of ubiquitin. When Ub-K48R was introduced into the strain, it could not support growth (), confirming that Ub-K48 is an essential residue (Finley et al., 1994
). More importantly, expressing the single-lysine Ub (K48 alone) also resulted in a complete deficiency in cell growth (), and the culture’s growth curve was indistinguishable from that of the Ub-K48R strain (Figure S3D
). These data indicate that Ub with K48 as its only lysine is not sufficient to sustain yeast growth, underscoring the physiological significance of non-K48 lysine residues.
Ub with K48 alone cannot support yeast viability and cumulative K to R substitutions lead to growth defects.
To rescue viability of the K48-only Ub mutant, restitution of K29 and K33 (R6R11R27R63 strain) is sufficient. However, this mutant exhibited severely retarded growth (). The growth curves of various lysine mutant strains in rich medium (YPD) were further compared (). The order of growth rates was WT = R11 = R11R63 > R27 > R11R27R63 > R6R11R27R63, suggesting that the mutations have a cumulative effect on cell proliferation. To eliminate the possibility that the mutations affected the stability of ubiquitin, thereby reducing the availability of Ub for conjugation, we examined the level of Ub and its conjugates in all strains by Western blotting. In spite of a slight increase in Ub-conjugates in the quadruple mutant, all strains showed comparable levels of Ub monomer and conjugates (). Thus, the lethal effect of mutating combinations of non-K48 lysines is unlikely resulted from a general defect in conjugate formation. However, this does not rule out subtle functional defects not related to the formation of specific chains.
To examine potential interdependence of the different Ub-Ub linkages, we used MS to measure the changes in the polyUb linkage levels in the Ub mutants (). Neither R11 nor R11R63 affected cell growth, and they had little effect on the abundance of other linkages (< 2-fold). K27R replacement influenced linkages only at the nearby lysines K29 (1.9-fold) and K33 (4.7-fold). The triple substitution mutant (R11R27R63) dramatically reduced cell growth and increased K48 (1.6-fold), K29 (3.8-fold), and K33 (7.0-fold) linkages. Additional mutation of K6 resulted in a further increase in the utilization of the remaining sites (K48, 3.9-fold; K29, 6.1-fold; and K33, 22.2-fold). These results indicate that the physiological roles of these lysine residues are partially redundant, and thus the growth defects are cumulative when more lysine residues are mutated. Conversely, concomitant increases of polyUb linkages at the remaining lysines occur but do not support wild-type growth rate. This suggests that different polyUb linkages may modify specific substrates and have unique functions (unnaturally high levels of alternative Ub-Ub linkages might be deleterious as well; see Discussion).
K11 Linkages Modify Specific Substrates Revealed by Quantitative MS
Since K11 Ub-Ub isopeptide bonds are as abundant as K48 linkages in vivo, but are not nearly as well characterized, we sought to identify K11 polyUb chain-linked substrates by comparing R11 and wild-type strains using a quantitative MS strategy, termed stable isotope labeling with amino acids in cell culture (SILAC) (Ong et al., 2002
). R11 and wild-type cells were differentially labeled in light and heavy stable isotope media, mixed after harvesting, and then used for the purification of Ub conjugates under denaturing conditions (). Proteins from total cell lysate and Ub conjugates were both resolved on an SDS gel, excised from the gel, digested by trypsin and analyzed by reverse phase liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS, ). This resulted in the quantification of 1,576 proteins in the total lysate (Table S3
), and 347 Ub conjugate candidates with at least two assigned peptides. Because samples enriched for Ub conjugates can still be contaminated by unmodified proteins, we developed an algorithm to construct “virtual Western blot” images of every identified protein () to evaluate whether the protein is likely to be ubiquitinated based on the large mobility shift expected from the polyUb tag (Seyfried et al., 2008
). Ultimately, out of the 347 candidates, we accepted 75 Ub conjugates, 37 (49%) of which were also quantified in the total cell lysate (Table S4
). Comparing the accepted Ub conjugates to the proteins in the total cell lysate revealed that the K11R substitution caused a small, global decrease in ubiquitination (main peak for the Ub conjugates in ) and markedly inhibited the ubiquitination of a subset of proteins (shoulder in the histogram of Ub conjugates; ). Moreover, out of the 1,576 proteins in the cell lysate, 91 proteins showed statistically significant changes in abundance (absolute log2
ratio at least 0.8 and signal-to-noise ratio > 1.5, Figure S4E
, Table S3
). Gene ontology classification (Ashburner et al., 2000
) indicates that the K11R mutation perturbs the yeast proteome in a profound manner, suggesting that K11 linkages are involved in a broad range of cellular processes (, table S5
Large-scale protein profiling of the wild-type and Ub-R11 strains to identify linkage-specific substrates.
Validation of K11 linkage-specific substrates by virtual Western blots and protein turnover analyses.
Among the 37 proteins identified in both the total cell lysate and the ubiquitinated proteome, we searched for potential K11 linkage targets using two criteria: (i) the protein’s level was elevated in the total cell lysate, and (ii) the level of the ubiquitinated forms was reduced. Two potential substrates were found: Ubc6, an E2 Ub-conjugating enzyme (Chen et al., 1993
; Sommer and Jentsch, 1993
), and Rbl2, a tubulin-folding cofactor (Lopez-Fanarraga et al., 2001
). In protein half-life assays, the lack of K11 in Ub significantly reduced the turnover rate of these two proteins, but did not stabilize two negative control proteins (Ub and Cdc48, ). The reduced degradation of Ubc6 by Ub-K11R mutation was further validated in different yeast strains expressing untagged Ub (). These data support the inference that the degradation of a specific subset of proteins is dependent on K11-linked polyUb chains.
To confirm that Ubc6 is a genuine substrate modified by K11-linked polyUb chains, we reconstituted an in vitro ubiquitination reaction using recombinant yeast E1 and Ubc6. The recombinant Ubc6 lacked the C-terminal transmembrane domain (M233-K250) to promote its solubility. In this reaction, Ubc6 modified itself with multiple Ub molecules, causing a mass shift of up to 40 kDa (~4–5 Ub tags), whereas no free Ub polymers were generated during the reaction (). The autoubiquitination of Ubc6 was abolished by substituting Cys87 with Ser in the catalytic site (). After a 30-min reaction (a time point prior to the reaction plateau, ), the K11R mutation in Ub caused a small, but visible defect in the formation of higher order Ubc6-linked Ub-conjugates (). Further MS analysis () indicated that Ubc6 was modified mainly by K11-linked polyUb (53%), but also by other polyUb chains assembled through K33 (14%), K48 (20%), and K63 (14%). Ub-K11R could still form polyUb chains on Ubc6 in vitro through increased use of other lysines: K33 (18%), K48 (42%) and K63 (40%) sites (). More interestingly, deletion of the UBC6 gene in yeast cells reduced the total cellular K11 polyUb linkages by ~40%, but had no impact on K48 or K63 chains (). These results reveal that Ubc6 is not only a substrate modified by K11-linked polyUb chains, but is also one of the primary E2s contributing to the synthesis of K11 linkages in vivo.
Ubc6 and Doa10 contribute to the synthesis of K11 linkages.
K11 Linkages Function in Endoplasmic Reticulum Associated Degradation (ERAD)
Ubc6 is a component of the ERAD pathway, a quality control pathway in which misfolded or improperly assembled proteins of the ER are ubiquitinated, translocated to the cytosol if necessary, and degraded by the proteasome (Ravid et al., 2006
; Sommer and Jentsch, 1993
). Thus we tested whether polyUb chains with K11 linkages impact the ERAD in any way. There are two main E3 ligases involved in the ERAD: Doa10, which requires two different E2s, Ubc6 and Ubc7 (Ravid et al., 2006
), and Hrd1, which acts primarily with Ubc7, an E2 that assembles K48-linked polyUb chains (Bazirgan and Hampton, 2008
). Consistent with the connection between Doa10 and Ubc6, we found a selective decrease of K11 linkages in doa10
Δ and ubc6
Δ double mutant cells, an effect comparable to that measured in the ubc6
Δ strain; in contrast, ubc7
Δ or hrd1
Δ did not affect K11 linkages (). Thus, Ubc6 and Doa10 define a pathway for synthesizing K11-linked polyUb chains in vivo.
Given the link between K11 polyUb chains and ERAD, we examined whether the R11 mutant had a higher sensitivity to ER stress-inducing reagents. Indeed, the R11 strain grew more poorly than the wild-type in the presence of high levels of dithiothreitol (30 mM DTT, a thiol reductant that interferes with disulfide bond formation) or tunicamycin (an antibiotic that blocks the synthesis of N-linked glycoproteins in the ER) (). In contrast, the Ub-R63 mutation had no obvious effect on cell proliferation under the same conditions, and a strain expressing the doubly mutated Ub (R11R63) displayed defects similar to those of the R11 single mutant (). However, no hypersensitivity to ER stress was observed in ubc6Δ or ubc7Δ cells (), indicating that ubc6Δ is not as sensitive to ER stress as the Ub-K11R mutant. These findings are consistent with the fact that Ubc6 only catalyzes formation of ~40% of cellular K11 Ub-Ub linkages.
Ub-K11 linkages function in the ER stress response.
Furthermore, we measured the level of all seven polyUb linkages in wild-type yeast treated with DTT or tunicamycin. The treatment selectively stimulated formation of K11 linkages, and DTT had a stronger effect than tunicamycin (2.5-fold vs. 1.7-fold after 2 hr, ), consistent with their relative potency in growth inhibition (). There was no detectable change in the levels of K48 linkages, either because formation of K48-linked polyUb chains, such as those made by Ubc7, is already occurring at maximal rates in unstressed cells, or because K48 chains are used more widely than K11 chains in processes other than ER homeostasis. The global levels of other polyUb linkages did not change either (). The selective accumulation of K11 Ub-Ub linkages during ER stress strongly supports the role for such polyUb chains in the ER stress response.
ER stress induces the unfolded protein response (UPR), a homeostatic mechanism initiated by activation of the ER-resident Ire1 kinase (Ron and Walter, 2007
). We measured the UPR induction in the WT, R11 and R63 strains using a UPR reporter, consisting of the E. coli lacZ
gene under the control of a promoter containing 4 copies of the UPR element. Indeed, the R11 but not R63 mutation caused a weak but reproducible increase in both the basal and DTT-induced levels of the UPR (, p
<0.01). Notably, when we deleted IRE1
gene in our yeast strains, we observed a striking synthetic growth defect in combination with Ub-R11, but not Ub-R63, in the presence of low concentrations of DTT (5 mM, ). These results demonstrate that K11-linked polyUb chains function in ubiquitination events related to the ER stress pathway.