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Edited by Jasper Rine, University of California, Berkeley, CA, and approved November 27, 2009 (received for review September 15, 2009)
Eukaryotic cells maintain proteostasis by quality control (QC) destruction of misfolded nuclear proteins (2–4). The mechanism
degradation. These pathways can specifically target a wide variety used by the QC ligases to detect misfolded substrates can vary,
of distinct misfolded proteins, and so are important for manage- with some employing chaperones (5) and others not. The details
ment of cellular stress. Although a number of conserved QC of substrate recognition are key to understanding the envelope of
pathways have been described in yeast, the E3 ligases responsible structures subject to destruction by a given pathway.
for cytoplasmic QC are unknown. We now show that Ubr1 and So far, no widely conserved ubiquitination pathway has been
San1 mediate chaperone-dependent ubiquitination of numerous described for cytoplasmic QC. Metazoans express the CHIP
misfolded cytoplasmic proteins. This action of Ubr1 is distinct from ubiquitin ligase that mediates cytoplasmic QC, using Hsp70
its role in the “N-end rule.” In this capacity, Ubr1 functions to chaperones to detect misfolded proteins (6, 7). However, CHIP
protect cells from proteotoxic stresses. Our phenotypic and bio- is not conserved in all eukaryotes. For example, no CHIP is
chemical studies of Ubr1 and San1 indicate that two strategies are encoded in yeast. Nevertheless, chaperone-dependent ubiquiti-
employed for cytoplasmic QC: chaperone-assisted ubiquitination nation of misfolded proteins has been observed in yeast (8),
by Ubr1 and chaperone-dependent delivery to nuclear San1. The indicating that previously undescribed, and probably highly
broad conservation of Ubr ligases and the relevant chaperones conserved, cytoplasmic QC pathways remain to be discovered.
indicates that these mechanisms will be important in understand- To that end, we have investigated the E3 ligases involved in
ing both basic and biomedical aspects of cellular proteostasis. ubiquitination of misfolded cytoplasmic proteins in yeast. We
have discovered that two E3 ligases collaborate in ubiquitination
chaperone | proteostasis | misfolding of a diverse set of misfolded proteins, including full-length sub-
strates with point mutations and truncated proteins. The two E3s
Doa10 involved in endoplasmic reticulum-associated degrada- This article contains supporting information online at www.pnas.org/cgi/content/full/
tion (ERAD) and the San1 ubiquitin ligase that mediates 0910591107/DCSupplemental.
ground with either SAN1, empty vector (EV), or SAN1-NLS expressed from
D). Importantly, the addition of GFP to tGnd1 created a full-
plasmids. (E) Ubiquitination of CPY‡-GFP in the SAN1 or SAN1-NLS strain in D.
(F) Cycloheximide chase of CPY‡-GFP from ubr1Δsan1Δ strains expressing UBR1 length fusion protein with a folded C-terminal domain. Thus, the
or the UBR1MR1 ring mutant compared with the EV strain. (G) Ubiquitination pathway defined by the CPY‡-GFP reporter is involved in the
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of CPY‡-GFP from the Ubr1-expressing strains in F. (H) Cycloheximide chase of degradation of a variety of unrelated cytoplasmic proteins,
cytoplasm-restricted CPY‡-GFP+NES in the strains used in A. including both truncated and misfolded full-length proteins.
Heck et al. PNAS | January 19, 2010 | vol. 107 | no. 3 | 1107
QC pathways selectively degrade misfolded proteins, sparing We tested a variety of growth conditions to evaluate physio-
the fully folded forms (e.g., 9). We evaluated this specificity with logical roles for these ligases, including ethanol, which has been
Gnd1. The original CPY‡-GFP reporter cannot be used in this test, employed as a proteotoxic stress (21). We found that 8% (vol/vol)
because secretory proteins such as BSA (7) and nonmutant CPY- ethanol in solid medium caused a significant cold-sensitive growth
GFP do not correctly fold in the cytosol. Conversely, the full- phenotype in the ubr1Δ null. The ubr1Δ strains showed a strong
length cytoplasmic Gnd1 is normally folded in the cytosol. 3HA- growth defect on the ethanol plates at 23°C (∼600-fold; Fig. 3B).
Gnd1-GFP, with full-length normal Gnd1 in the fusion, was quite In contrast, the san1Δ null showed no defect of growth, nor did it
stable in cells compared with the identically expressed 3HA- enhance the sensitivity of the ubr1Δ (Fig. 3B, san1Δ vs. WT and
tGnd1-GFP (Fig. 2E). QC pathways are selectively able to degrade san1Δubr1Δ vs. ubr1Δ). Thus, full-length San1 was not involved in
a large variety of misfolded proteins. This flexibility includes this ubr1Δ-sensitive stress. However, the growth phenotype of the
diverse substrates, or distinct misfolded versions of the same ubr1Δsan1Δ double null was suppressed by cytoplasmic SAN1-
protein, as seen with the many unstable “type II” CFTR mutants NLS expressed in single copy from its native promoter (Fig. 3B,
(18). As a separate test of the generality of this cytoplasmic QC +SAN1-NLS). This strongly implied that the ethanol stress
pathway, we used the x-ray structure of yeast Gnd1 protein to imposed in this test occurred in the cytosol and that it was
generate a unique QC substrate (19). The original tGnd1 substrate remediated by destruction of misfolded proteins, either by nor-
is truncated in the middle of the second folded domain (Fig. 2F, mally present Ubr1 or by the cytoplasmic -NLS version of San1.
gray arrow; residues 1–368, 75%). The first domain is a widely The ethyl alcohol (EtOH) growth phenotype was also
observed structure that allows NADP+ binding with a Rossman observed in the absence of the E2 Rad6 (Fig. 3C, rad6Δ), which
fold. We tested a version of Gnd1p with a truncation in the first is implicated in many actions of Ubr1, including QC (Fig. 2G).
domain (Fig. 2F, black arrow; residues 1–150, 30%), calling the The decreased survival in rad6Δ was more pronounced than in
substrate stGnd1 (short truncated Gnd1). Indeed, 3HA-stGnd1 ubr1Δ, which may be attributable to Rad6 having numerous roles
was rapidly degraded (Fig. 2G), showing a 10-min half-life in the in cell function. Alternatively, the Ubr1 homolog Ubr2 (∼50%
presence of Ubr1 and strong stabilization in the ubr1Δ null. identical to Ubr1) also uses Rad6 (22) and could also have been
Interestingly, this substrate was entirely dependent on Ubr1; the involved in cytoplasmic QC. However, the ubr2Δ null showed no
presence of san1Δ alone or in combination with ubr1Δ had no growth defect in the 8% (vol/vol) EtOH test (Fig. 3C), nor did
effect on the stability of stGnd1. Identically tagged full-length the presence of the ubr2Δ null make the ubr1Δ sensitivity any
Gnd1 was completely stable (Fig. 2G, Bottom). Thus, Ubr-medi- greater (ubr1Δubr2Δ). Furthermore, the ubr2Δ null had no effect
ated QC is specific for misfolded proteins but is broadly inclusive in on degradation of our substrates, and overexpression of Ubr2 did
selection of substrates. not increase the degradation rate of any tested QC substrates.
We employed the Ubr1 specificity of stGnd1 degradation to Thus, QC function is unique to the Ubr1 isozyme.
evaluate the E2 enzymes involved in Ubr1-mediated QC. As The observation that San1 can recognize a variety of misfolded
observed in other actions of this E3 (20), both Rad6/Ubc2 and substrates is consistent with its known role as a QC E3 (9). How-
Ubc4 could mediate degradation and the double E2 null phe- ever, a role for Ubr1 in QC was unique and unexplored. Accord-
nocopied the ubr1Δ strain (Fig. 2H). ingly, we next examined mechanistic features of Ubr1-mediated
These studies show that both Ubr1 and San1 are required for QC in greater detail. We used the substrate tGnd1p-GFP to test
degradation of a wide variety of misfolded proteins that originate more fully the idea that Ubr1-mediated QC was distinct from its
in the cytoplasm. We next tested each ligase for roles in manage- well-studied role in the N-end rule, in which the N terminus of a
ment of proteotoxic stress. We initially tested stress caused by protein determines its rate of Ubr1-mediated degradation (11, 23,
overexpression of CPY‡-GFP. Although this protein can be 24). Ubr1 is the E3 ligase, or “N-recognin” of the N-end rule
expressed in any of the nulls without consequence from the TDH3 pathway, binding to and catalyzing ubiquitination of proteins with
promoter in glucose medium, expression of CPY‡-GFP from the appropriate N-terminal amino acids present by cleavage or enzy-
stronger galactose promoter caused growth sensitivity that was matic addition (16). It was formally possible that the Ubr1-
exacerbated by either the ubr1Δ or the san1Δ and greatly worsened dependent ubiquitination of our misfolded substrates was attrib-
in the double null (Fig. 3A, Top four rows). Thus, as expected from utable to cleavage of a small amount of the protein to reveal a fast-
the CPY‡-GFP degradation data, both Ubr1 and San1 can lessen degradation N-terminal residue, followed by traditional N-end
the stress caused by strong expression of this model substrate. recognition by Ubr1. We addressed this issue in several ways. We
Identical expression of the cytoplasm-restricted CPY‡-GFP+NES expressed and immunoprecipitated FLAG-tGnd1-GFP, bearing a
resulted in stress that was only affected by ubr1Δ; the presence of single N-terminal tag, with anti-FLAG antibody to evaluate the
san1Δ alone or in combination with ubr1Δ had no additional effect ability of Ubr1 to ubiquitinate tGnd1-GFP with an intact N ter-
on growth (Fig. 3A, Bottom four rows). This result highlights minus. The resulting ubiquitination in the suite of four strains was
Ubr1’s direct role in survival of cytoplasmic proteotoxic stress. identical to that seen above with anti-GFP antibodies (Fig. 2D and
in the indicated strains. Detection was with anti-HA antibodies. (H) Cyclo-
heximide chase of stGnd1 in E2 UBC strains rad6Δ and ubc4Δ as indicated.
The QC function of Ubr1 can be further distinguished from its arginyl transferase (25). Accordingly, we tested Ubr1-dependent
role in the N-end rule through its clear chaperone dependence. A degradation in ate1Δ and ate1Δsan1Δ nulls (Fig. S4). No effect
defining feature of Ubr1-mediated QC in our studies is depend- was observed in ate1Δ strains, indicating that the Ubr1-dependent
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ence on molecular chaperones for both degradation and ubiq- degradation of these misfolded proteins did not require addition
uitination (see below). In particular, all our QC substrates reveal a of arginine at the N terminus. Taken together, these experiments
Heck et al. PNAS | January 19, 2010 | vol. 107 | no. 3 | 1109
demonstrated that Ubr1-mediated recognition of QC substrates The previously unknown QC function for Ubr1 was distinct from
was independent of its well-described function in the N-end rule. its elegantly described action in the N-end rule and was restricted to
We used the in vitro assay to evaluate the role of chaperones in the Ubr1 isozyme. Both E2s reported to work with Ubr1 could
Ubr1- and San1-mediated QC. In vivo, loss of either Sse1 or support ubiquitination of a Ubr1-selective QC substrate. Intrigu-
Hsp70 caused a strong defect in ubiquitination, implying that they ingly, Ubr1 has a number of reported substrates that are not rec-
were involved in the action of each ligase. However, our in vitro ognized by the N-end rule, including some that bear degradation
analysis indicated that there are clear differences in the role of determinants also found in misfolded proteins (26–28). It will be
chaperones with each ligase. In vivo, FLAG-tGnd1-GFP ubiq- important to reevaluate these proteins as possible QC substrates. In
uitination was mediated by either Ubr1 or San1 (Fig. 4A). How- addition, a variety of phenotypes have been observed in murine nulls
ever, in vitro, the same substrate was ubiquitinated almost entirely of several Ubr isoforms (29–32); it may be that these effects are the
by Ubr1. A ubr1Δ null cytosol was incapable of supporting tGnd1- result of deficient QC control in the affected tissues.
GFP ubiquitination, whereas the reaction in a san1Δ cytosol was Although the San1 ubiquitin ligase was originally reported as a
identical to that in WT (Fig. 5A, lanes 1–3). What is the cause of QC E3 (9), our studies significantly expand the range of substrates
this difference in vitro? We discovered that the strong in vitro bias to include misfolded proteins of cytoplasmic origin. Because San1
toward Ubr1-dependent tGnd1 ubiquitination was attributable to originates in the cytosol, it was possible that the cytoplasmic QC
a surprising inhibition of San1 by Sse1. An sse1Δ cytosol sup- function was attributable to the subset of molecules present in
ported robust ubiquitination of the substrate (Fig. 5A, lane 4). that compartment at steady state. Indeed, San1 restricted to the
This sse1Δ-stimulated ubiquitination was strongly dependent on cytosol was fully capable of mediating degradation of CPY‡-GFP.
San1 (Fig. 5A, lane 6), showing a large decrease in the However, the other studies in this work imply that San1 and Ubr1
sse1Δsan1Δ double null. This is in striking contrast to the case in function in distinct compartments to mediate destruction of
the intact cell, in which the San1-dependent component of tGnd1- misfolded cytoplasmic proteins (see below).
GFP ubiquitination (measured in a ubr1Δ null) was strongly It has been previously reported that degradation of cytoplas-
inhibited by the presence of the sse1Δ null (Fig. 5B). mic proteins requires chaperones (5, 14), and the Ubr1/San1-
Our in vivo studies indicate that Ubr1-mediated QC is Hsp70- dependent substrates appropriately showed a strong dependence
dependent. The in vitro assays above (Fig. 4E) show that Ubr1- on Hsp70 and the Sse1 (Hsp110) cochaperone. However, the
mediated ubiquitination depends on the Sse1 cochaperone. role of chaperones in the action of each was distinguishable.
Because the in vitro assay is almost entirely Ubr1-dependent in Ubr1 appeared to use both Sse1 and Hsp70 directly, as indicated
these conditions, we used this assay to examine the role of Hsp70 by the in vitro assays. In contrast, although San1-dependent
in Ubr1 QC directly. Indeed, loss of two of the four redundant degradation (observed in a ubr1Δ null) was strongly Sse1-
Hsp70 genes (ssa1Δssa2Δ) resulted in a strong decrease in Ubr1- dependent in vivo, its action was inhibited by Sse1 in vitro.
mediated tGnd1-GFP ubiquitination in vitro (Fig. 5C), indicat- Taken together, our data suggest a two-compartment model for
ing a direct role for Hsp70 in this unique function of Ubr1. the roles of Ubr1 and San1 in cytoplasmic QC. The degradation and
Taken together, these studies indicate that Ubr1 directly phenotypic studies with the cytoplasm-restricted CPY‡-GFP+NES
employs chaperones for ubiquitination of substrates, whereas were consistent with distinct actions of these ligases. Ubr1 main-
San1 has a more complex relation with chaperones that appears tained its ability to degrade this purely cytoplasmic substrate,
to operate separately from substrate ubiquitination (see below). whereas San1 had no role in its degradation. Similarly, the stress
These studies have defined Ubr1 and San1 as E3 ligases phenotype caused by this cytoplasmic substrate was only sensitive
involved in chaperone-dependent cytoplasmic QC. Degradation to the ubr1Δ and not to the san1Δ, again indicating a direct cyto-
mediated by these E3s meets the criteria for physiologically plasmic role for Ubr1 but not for San1. Taken together, these data
relevant protein QC: ubiquitination is selective for misfolded suggest the following model. A misfolded cytoplasmic protein can
proteins, a broad range of misfolded substrates are recognized by undergo ubiquitination by either Ubr1 or San1. The Ubr1-
either ligase, and both ligases participate in management of dependent branch occurs in the cytosol, using chaperones in con-
junction with Ubr1 to recognize and ubiquitinate a substrate. In
proteotoxic stress. Each ligase has been studied in other
contrast, San1-dependent ubiquitination of the same substrate
capacities. Ubr1 is known for degradation of N-end rule sub-
requires chaperone-dependent transport into the nucleus, where
strates as well as numerous proteins recognized by distinct
San1 ubiquitinates the substrate. The dichotomous requirements
mechanisms (11, 12). San1 is a recently described nuclear QC E3
for Sse1 observed for San1 in vivo and in vitro are consistent with
(9). Our work thus significantly extends the functional range of
this model: in vivo, Sse1, in conjunction with Hsp70, promotes
each ligase.
delivery of misfolded substrate to the nuclear pool of San1. In vitro,
Sse1 gains access to San1 and inhibits it, perhaps by interacting with
San1 features that interact with misfolded proteins. In support of
this idea, we have observed that a fraction of CPY‡-GFP builds up
in the nucleus in a san1Δ null and that this buildup is dependent on
Sse1 (Fig. S5): In an sse1Δ san1Δ double null, CPY‡-GFP accu-
mulates in the cytoplasm only. This dependency appeared to be
strong, because addition of the NES construct to the CPY‡-GFP
had almost the same effect as sse1Δ in this optical experiment (Fig.
S5). Direct tests of this model will be an important avenue of
future inquiry.
Fig. 5. Differential chaperone requirement for Ubr1- and San1-mediated Our studies with nulls indicate that the two pathways function
ubiquitination. (A) In vitro ubiquitination of tGnd1-GFP bound to anti-FLAG independently: Each E3 appears to contribute a component of
beads after incubation with cytosol from WT, ubr1Δ, san1Δ, sse1Δ, ubiquitination that is independent of the presence of the other
ubr1Δsse1Δ, or san1Δsse1Δ strains. Ub, ubiquitin. (B) In vivo ubiquitination of
ligase. However, there was phenotypic synergy in the stress
FLAG-tGnd1-GFP in ubr1Δ or ubr1Δsse1Δ strains. FLAG-tGnd1-GFP was
immunoprecipitated from cell lysates with anti-FLAG beads and immuno-
experiments involving CPY‡-GFP, because loss of both E3s had
blotted for GFP or Ub. (C) In vitro ubiquitination of FLAG-tGnd1-GFP. tGnd1- a much more drastic effect than that of either single null (Fig.
GFP bound to anti-FLAG beads was incubated with cytosol from WT or 3A). Thus, the issue of regulatory or mechanistic crosstalk
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ssa1Δssa2Δ strains for 1 h at 30°C and assayed for ubiquitination by immu- between these two modes of cytoplasmic QC is an open and
noblotting for GFP or Ub. interesting one. It is clear that Ubr1 plays the principal role in
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