Degradation of misfolded protein in the cytoplasm is mediated
by the ubiquitin ligase Ubr1
Frederik Eisele, Dieter H. Wolf*
Institut fu ¨r Biochemie, Universita ¨t Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany
Received 6 October 2008; revised 3 November 2008; accepted 10 November 2008
Available online 27 November 2008
Edited by Noboru Mizushima
terminally misfolded proteins occurs via the ubiquitin–protea-
some system. Tagging of misfolded proteins with ubiquitin for
degradation depends on a cascade of reactions involving an ubiq-
uitin activating enzyme (E1), ubiquitin conjugating enzymes (E2)
and ubiquitin ligases (E3). While ubiquitin ligases responsible for
targeting misfolded secretory proteins to proteasomal degrada-
tion (ERAD) have been uncovered, no such E3 enzymes have
been found for elimination of misfolded cytoplasmic proteins in
yeast. Here we report on the discovery of Ubr1, the E3 ligase
of the N-end rule pathway, to be responsible for targeting mis-
folded cytosoplasmic protein to proteasomal degradation.
? ? 2008 Federation of European Biochemical Societies. Pub-
lished by Elsevier B.V. All rights reserved.
Protein quality control and subsequent elimination of
Keywords: Protein quality control; Misfolded protein;
Ubiquitin ligase; Ubr1; Proteasome; Protein degradation
Proper protein folding is essential for cellular well-being and
survival. Very sophisticated mechanisms including the action
of chaperones help the proteins fold into their native confor-
mation. Stresses like heat, heavy metal ions, oxidation or sim-
ply mutations might prevent folding of a protein into its native
state. Sensing of the folding process and recognition of mis-
folded proteins is summarized as a process called protein qual-
ity control. Conformational aberrant proteins which in many
cases are toxic for the cell have to be eliminated. The impor-
tance of protein quality control and degradation of terminally
misfolded proteins for cellular well-being is underscored by the
many examples of disease, as are for instance Parkinson-, Alz-
heimer- or Creutzfeldt–Jakob-disease. Protein quality control
and degradation has been extensively studied for secretory
proteins (ERQD). A multitude of components required for
folding, folding control, recognition and delivery of misfolded
secretory proteins to the proteolytic system for elimination has
been uncovered [1–7]. Recently, advances in our understanding
of the quality control of misfolded cytoplasmic proteins and
their degradation (CQD) has been published [8,9]. It is a com-
mon feature of the protein quality control pathways of the ER
and the cytosol, that Hsp70-type chaperones bind and sense
misfolded proteins and finally deliver them for degradation
by the ubiquitin–proteasome pathway of the cytosol [7,10].
This major proteolytic pathway of all eukaryotic cells requires
tagging of the misfolded protein by the 76 amino acid polypep-
tide ubiquitin, which is brought about by a cascade of reac-
tions catalyzed by an ubiquitin activating enzyme (E1),
ubiquitin conjugating enzymes (E2) and ubiquitin ligases
(E3). The tagging reaction ends up in the formation of a poly-
ubiquitin chain at intrinsic lysine residues or the amino termi-
nus of the protein to be degraded. This process finally targets
the protein for degradation via the proteasome, a proteolytic
nanomachine [11,12]. The concerted action of ubiquitin conju-
gating enzymes and ubiquitin ligases determines the specificity
of the polyubiquitination process of a selected protein. While
degradation of misfolded secretory proteins mainly depends
on the ubiquitin conjugating enzymes Ubc6 and Ubc7
[3,13,14] misfolded cytoplasmic proteins are targeted by the
ubiquitin conjugating enzymes Ubc4 and Ubc5 for degrada-
tion [8,9]. The involvement of the ubiquitin ligase in the ubiq-
uitin targeting reaction of misfolded cytoplasmic proteins in
yeast cells remained elusive: Even though the ubiquitin ligases
Der3/Hrd1 and Doa10 required for polyubiquitination of mis-
folded secretory proteins carry the specificity for recognition of
unfolded protein patches, they do not function in polyubiqui-
tination of the misfolded cytoplasmic proteins tested . In
mammalian cells the E3 enzymes CHIP and Parkin have been
reported to be responsible for ubiquitination of misfolded or
aggregation-prone protein substrates of the cytoplasm (re-
viewed in ). However, no orthologous E3 enzymes have
been found in yeast. Here we report on the discovery of the
RING-finger ubiquitin ligase Ubr1 as an essential E3-enzyme
for delivering misfolded protein of the yeast cytoplasm to pro-
2. Materials and methods
2.1. Yeast strains and plasmids
Media preparation, genetic and molecular biology techniques were
carried out using standard methods [16,17]. All experiments were done
in the genetic background of Saccharomyces cerevisiae strain W303
prc1-1 (MATa ade2-1ocre can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-
1 prc1-1) . The UBR1 gene was deleted via homologous recombina-
tion with a KanMX deletion module .
Plasmid pFE15 encoding the cytoplasmic fusion protein DssCL*myc
(pRS316-PPRC1-prc1-1Dss, lacking base pairs 2–57 encoding the signal
sequence (ss) and the last 39 base pairs of PRC1), LEU2-myc13(bps
1813–3453 of CTL*myc encoded by pSK7 ) was constructed by
PCR amplification of the LEU2-myc13encoding region of pSK7 using
the oligonucleotides TCCGCGGCAGTTAACTCTGCCCCTAA-
thereby introducing the restriction enzyme sites of HpaI and Hind3.
The HpaI and Hind3 digested fragment was ligated with digested plas-
mid pZK116m (pRS316-PPRC1prc1-1Dss) .
*Corresponding author. Fax: +49 0711 685 64392.
E-mail address: firstname.lastname@example.org (D.H. Wolf).
0014-5793/$34.00 ? 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 582 (2008) 4143–4146
A high-copy plasmid expressing N-terminally flag tagged UBR1
from the ADH1 promoter and a respective control plasmid pRB where
the ORF of flagUbr1 had been removed were a kind gift from Du et al.
For precipitation of DssCL*myc and detection in immunoblots
monoclonal c-myc antibodies (Santa Cruz, clone 9E10) were used.
For precipitation and immunoblots of flag tagged Ubr1 polyclonal flag
antibodies (Sigma) were used.
2.3. Pulse chase analysis
Pulse chase experiments using cells expressing DssCL*myc were per-
formed as described [22,23].
Briefly, cells were grown in selective media (CM without uracil and
leucine) and shifted to starvation media (CM without uracil, leucine
and sulfate) for 50 min. Eight OD600of cells were labelled with nine
MBq of35S-Met for 20 min. Cells were chased with unlabelled media
containing an excess of non-radioactive methionine. Samples were ta-
ken at the time points indicated in the respective figure legends and ex-
tracts were prepared.
Diagrams represent data of three independent experiments. Error
bars indicate the respective standard error of the mean.
One-hundred and fifty OD600of logarithmically grown cells were
harvested and washed once in ice-cold destilled water containing
30 mM NaN3. Cells were resuspended in 2 ml IP buffer (50 mM Tris
(pH 7.5), 200 mM NaAcetate, 10% glycerol). Complete inhibitor mix
(Roche), 1 mM PMSF, 1 lg/ml each of benzamidin, pepstatin A,
and chymostatin were added shortly before use of the buffer. Cells were
lysed with glass beads . Lysates were pre-cleared by centrifugation
at 500 · g for 5 min at 4 ?C. Lysates were centrifuged at 100000 · g for
1 h at 4 ?C. DssCL*myc and flag tagged Ubr1 were immunoprecipi-
tated from the supernatant using 5 ll of anti-myc or 5 ll of anti-flag,
respectively, and incubating for 1 h at room temperature. Five milli-
grams of Protein A sepharose, blocked with 10% BSA, was added
for antibody precipitation for an additional hour. After washing with
IP buffer the proteins were eluted with 60 ll urea loading buffer (8 M
urea, 200 mM Tris/HCL (pH 6.8), 0.1 mM EDTA, 5% (w/v) SDS,
0.03% (w/v) bromophenol blue, 1% b-mercaptoethanol). Fifteen
microliters of each sample were used for immunoblot analysis.
3. Results and discussion
For elucidation of ubiquitin ligases involved in the degrada-
tion of the cytoplasmic misfolded protein DssCL*myc, a deriv-
ative of signal sequence deleted mutated carboxypeptidase
yscY, we tested yeast strains of the EUROSCARF collection
deleted in the genes of proteins predicted to be ubiquitin ligases
. We had previously shown that signal sequence deleted
carboxypepdidase yscY derivatives locate to the cytoplasm of
cells . Plasmids expressing the cytoplasmic misfolded protein
DssCL*myc (Fig. 1A) were transformed into these strains
which are defective in the LEU2 gene encoding 3-isopropylm-
Strains wild type for DssCL*myc degradation are unable to
grow on media without leucine because the misfolded protein
including the Leu2 moiety is rapidly eliminated, thus being un-
able to complement the leucine auxotrophy. In contrast,
strains defective in a component of the degradation pathway
of DssCL*myc are able to grow due to stabilization of the
Leu2 containing substrate and by this complementing the
LEU2 deficiency [23,25,26].
As can be seen in Fig. 1B a promising candidate of the screen
is a strain deleted in the gene of the ubiquitin ligase Ubr1. This
strain exhibited strong growth when compared to wild type on
medium lacking leucine, indicating stabilization of the sub-
To elucidate whether degradation of DssCL*myc is indeed
disturbed in the Dubr1 strain, pulse chase analysis was per-
formed to follow the fate of the substrate.
As can be seen in Fig. 2A and B degradation of DssCL*myc
is considerably delayed in the Dubr1 mutant. Expression of a
flag tagged Ubr1 protein in the Dubr1 deletion strain led to
complementation of the degradation defect. As flagUbr1 is ex-
pressed from a multi-copy plasmid, degradation kinetics in
strains expressing this construct is even faster than in the wild
type strain expressing Ubr1 from its chromosomal locus
(Fig. 2B). These data indicate that Ubr1 is indeed involved
in the degradation process of DssCL*myc. As degradation of
the substrate is not completely blocked in Dubr1 cells we pre-
dict additional ubiquitin ligase activities to be involved in the
elimination of misfolded cytoplasmic proteins.
Involvement of Ubr1 in degradation of DssCL*myc predicts
physical interaction of the E3 ligase with its substrate. This
interaction is expected, however, to be rather quick. For a
co-immunoprecipitation experiment we transformed a plasmid
expressing flag tagged Ubr1 into wild type cells expressing
DssCL*myc at the same time. When pulling down the sub-
strate using myc antibodies we were able to coprecipitate flag-
Ubr1 (Fig. 3, Lane 8). When pulling down flag tagged Ubr1 no
substrate was coprecipitated (Lane 10). Obviously, when pull-
ing down the substrate, part of the precipitated DssCL*myc
molecules are complexed with Ubr1 by this selectively enrich-
ing the ligase in this sample. In contrast, when pulling down
flagUbr1 the precipitated molecules should contain a multi-
tude of interacting substrates of which DssCL*myc is only a
minority and therefore cannot be visualized.
We noted that the input levels of DssCL*myc are lower when
flagUbr1 is expressed in cells (Fig. 3, Lane 2 and 4). This is
most likely due the short half-life of DssCL*myc in the pres-
ence of flagUbr1. Whether the rather low steady state level
of flagUbr1 in the presence of substrate is due to degradation
has to be explored in future studies.
Ubr1 was shown to be an ubiquitin ligase, which is able to
recognize N-end rule substrates. Recognition of these N-end
CM -Ura CM -Ura -Leu
Fig. 1. A strain expressing DssCL*myc and deleted in the ubiquitin
ligase Ubr1 grows on medium lacking leucine. (A) Schematic drawing
of the chimeric protein DssCL*myc, consisting of cytoplasmically
misfolded CPY*C-terminally fused to Leu2 and a 13myc tag. (B)
Growth of a W303 prc1-1 wild type (WT) strain and a Dubr1 strain,
both defective in the LEU2 and URA3 genes, harbouring a plasmid
with the URA3 selection marker expressing DssCL*myc under the
control of the PRC1 promoter. Cells were spotted in a five fold dilution
series on solid CM medium lacking leucine and uracil, or solely uracil,
F. Eisele, D.H. Wolf / FEBS Letters 582 (2008) 4143–4146
rule substrates occurs at two sites, type-1 and type-2. The type-
1 site is specific for recognition of basic N-terminal amino acid
residues, the type-2 site is responsible for recognition of bulky
hydrophobic amino acid residues of proteins. In addition Ubr1
contains a third substrate-binding site, which targets an inter-
nal degron of Cup9, a transcriptional repressor of peptide im-
port [27–29]. Removal of the signal sequence from misfolded
carboxypeptidase yscY and construction of the DssCL*myc
substrate resulted in a novel amino terminus starting with
Met-Ile-Ser as the first three amino acids. According to the
‘‘Sherman-rule’’ the amino terminal methionine is only cleaved
off a polypeptide chain in yeast when it is followed by an ami-
no acid with a radius of gyration of 1.29 A˚or less . The sec-
ond amino acid in DssCL*myc is an isoleucine which
according to the ‘‘Sherman-rule’’ does not allow cleavage of
methionine from the amino terminus of this substrate. As
methionine is a stabilizing N-terminal amino acid, DssCL*myc
cannot be recruited to Ubr1 via the type-1 or type-2 binding
sites. DssCL*myc might be recruited to Ubr1 by the third bind-
ing site which was demonstrated to bind Cup9. Targeting of
Cup9 to Ubr1 was shown to be dependent on the binding of
cognate dipeptides to the type-1/2 sites of Ubr1 [21,31,32]. Spe-
cific binding of Cup9 to Ubr1 can also occur by a chaperone
such as yeast EF1A or through macromolecular crowding,
conditions which are present in vivo . We have shown that
degradation of all tested misfolded DssCPY*variants in the
cytoplasm require the Hsp70 chaperone Ssa1 . It is therefore
most likely that also degradation of DssCL*myc is dependent
on Ssa1. This chaperone might target DssCL*myc to ubiquiti-
nation via Ubr1. This process may involve the Cup9 binding
site or some other yet unknown site of Ubr1 which may detect
the chaperone bound substrate or, alternatively, hydrophobic
patches of the substrate. We cannot however completely ex-
clude the generation of a destablizing N-end rule amino termi-
nus on DssCL*myc by some unrecognized proteolytic cut.
However, our pulse chase experiments do not show the occur-
rence of a cleaved intermediate product of DssCL*myc. Only if
a few amino acids were taken off the substrate, this event
would escape unrecognized. For the moment we consider this
to be rather unlikely.
Acknowledgements: We thank Alex Varshavsky for the plasmid encod-
ing flag tagged Ubr1 and Mario Scazzari for discussions. The work
was supported by a grant from the Deutsche Forschungsgemeinschaft,
 Sommer, T. and Wolf, D.H. (1997) Endoplasmic reticulum
degradation: reverse protein flow of no return. Faseb J. 11,
 Brodsky, J.L. and McCracken, A.A. (1999) ER protein quality
control and proteasome-mediated protein degradation. Semin.
Cell Dev. Biol. 10, 507–513.
WT + control vector
Δubr1 + flagUbr1
WT + flagUbr1
Δubr1 + control vector
WT + control vector
Δubr1 + control vector
WT + flagUbr1
Δubr1 + flagUbr1
Remaining ΔssCL*myc [%]
Fig. 2. Deletion of UBR1 results in prolonged half-life of DssCL*myc
while over-expression of UBR1 accelerates its degradation. (A) Pulse
chase analysis of WT and Dubr1 cells expressig DssCL*myc. Where
indicated a flag tagged Ubr1 from a high-copy vector under the control
of the ADH1 promoter or a respective empty control vector was
transformed into cells. Cells were harvested at the indicted time points,
lysed and subjected to immunoprecipitation with myc antibodies and
separated by SDS–PAGE (B). Pulse chase experiments were quantified
by using a PhosphorImager and ImageQuaNT. Data represent the
mean values of three independent experiments.
pRS316 (empty vector)
pRB (empty vector)
myc myc flag flag myc myc
Lysate (1% input)
9 1011 12
Fig. 3. DssCL*myc co-immunoprecipitates with flag tagged Ubr1. Yeast cells expressing DssCL*myc and flag tagged Ubr1, or harbouring empty
vectors (pRS316 and/or pRB, respectively) were lysed. One percent of total cell extract was analysed by Western blotting using antibodies as
indicated. Samples obtained after co-immunoprecipitation (CoIP) were separated by SDS–PAGE and analysed by Western blotting using anti-myc
F. Eisele, D.H. Wolf / FEBS Letters 582 (2008) 4143–4146
 Kostova, Z. and Wolf, D.H. (2003) For whom the bell tolls: Download full-text
protein quality control of the endoplasmic reticulum and the
ubiquitin–proteasome connection. EMBO J. 22, 2309–2317.
 Ellgaard, L., Molinari, M. and Helenius, A. (1999) Setting the
standards: quality control in the secretory pathway. Science 286,
 Ellgaard, L. and Helenius, A. (2003) Quality control in the
endoplasmic reticulum. Nat. Rev. Mol. Cell Biol. 4, 181–191.
 Raasi, S. and Wolf, D.H. (2007) Ubiquitin receptors and ERAD:
a network of pathways to the proteasome. Semin. Cell Dev. Biol.
 Scha ¨fer, A., Kostova, Z. and Wolf, D.H. (2008) Endoplasmic
reticulum protein quality control and degradation in: Protein
Degradation (Mayer, R.J., Ciechanover, A. and Rechsteiner, M.,
Eds.), pp. 123–143, Wiley-VCH Verlag, Weinheim.
 McClellan, A.J., Scott, M.D. and Frydman, J. (2005) Folding and
quality control of the VHL tumor suppressor proceed through
distinct chaperone pathways. Cell 121, 739–748.
 Park, S.H., Bolender, N., Eisele, F., Kostova, Z., Takeuchi, J.,
Coffino, P. and Wolf, D.H. (2007) The cytoplasmic Hsp70
chaperone machinery subjects misfolded and endoplasmic retic-
ulum import-incompetent proteins to degradation via the ubiq-
uitin–proteasome system. Mol. Biol. Cell 18, 153–165.
 McClellan, A.J., Tam, S., Kaganovich, D. and Frydman, J. (2005)
Protein quality control: chaperones culling corrupt conforma-
tions. Nat. Cell Biol. 7, 736–741.
 Glickman, M.H. and Ciechanover, A. (2002) The ubiquitin–
proteasome proteolytic pathway: destruction for the sake of
construction. Physiol. Rev. 82, 373–428.
 Wolf, D.H. and Hilt, W. (2004) The proteasome: a proteolytic
nanomachine of cell regulation and waste disposal. Biochim.
Biophys. Acta 1695, 19–31.
 Biederer, T., Volkwein, C. and Sommer, T. (1996) Degradation of
subunits of the Sec61p complex, an integral component of the ER
membrane, by the ubiquitin-proteasome pathway. EMBO J. 15,
 Hiller, M.M., Finger, A., Schweiger, M. and Wolf, D.H. (1996)
ER degradation of a misfolded luminal protein by the cytosolic
ubiquitin–proteasome pathway. Science 273, 1725–1728.
 Esser, C., Alberti, S. and Ho ¨hfeld, J. (2004) Cooperation of
molecular chaperones with the ubiquitin–proteasome system.
Biochim. Biophys. Acta 1695, 171–188.
 Sambrook, J., Maniatis, T. and Fritsch, E.F.s. (1989) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
 Guthrie, C. and Fink, G.R.s. (2002) Guide to Yeast Genetics and
Molecular and Cell Biology, Academic Press, San Diego,
 Knop, M., Hauser, N. and Wolf, D.H. (1996) N-Glycosylation
affects endoplasmic reticulum degradation of a mutated derivative
of carboxypeptidase yscY in yeast. Yeast 12, 1229–1238.
 Gueldener, U., Heinisch, J., Koehler, G.J., Voss, D. and
Hegemann, J.H. (2002) A second set of loxP marker cassettes
for Cre-mediated multiple gene knockouts in budding yeast. Nucl.
Acids Res. 30, e23.
 Kohlmann, S., Scha ¨fer, A. and Wolf, D.H. (2008) Ubiquitin ligase
Hul5 is required for fragment-specific substrate degradation in
endoplasmic reticulum-associated degradation. J. Biol. Chem.
 Du, F., Navarro-Garcia, F., Xia, Z., Tasaki, T. and Varshavsky,
A. (2002) Pairs of dipeptides synergistically activate the binding of
substrate by ubiquitin ligase through dissociation of its autoin-
hibitory domain. Proc. Natl. Acad. Sci. USA 99, 14110–14115.
 Taxis, C., Vogel, F. and Wolf, D.H. (2002) ER-golgi traffic is a
prerequisite for efficient ER degradation. Mol. Biol. Cell 13,
 Medicherla, B., Kostova, Z., Schaefer, A. and Wolf, D.H. (2004)
A genomic screen identifies Dsk2p and Rad23p as essential
components of ER-associated degradation. EMBO Rep. 5, 692–
 Scheel, H. (2005) Comparative Analysis of the Ubiquitin–
Proteasome System in Homo sapiens and Saccharomyces cerevi-
siae. Ph.D. Thesis, University of Cologne.
 Buschhorn, B.A., Kostova, Z., Medicherla, B. and Wolf, D.H.
(2004) A genome-wide screen identifies Yos9p as essential for ER-
associated degradation of glycoproteins. FEBS Lett. 577, 422–
 Schafer, A. and Wolf, D.H. (2005) Yeast genomics in the
elucidation of endoplasmic reticulum (ER) quality control and
associated protein degradation (ERQD). Methods Enzymol. 399,
 Xia, Z., Webster, A., Du, F., Piatkov, K., Ghislain, M. and
Varshavsky, A. (2008) Substrate-binding sites of UBR1, the
ubiquitin ligase of the N-end rule pathway. J. Biol. Chem. 283,
 Bartel, B., Wunning, I. and Varshavsky, A. (1990) The recogni-
tion component of the N-end rule pathway. EMBO J. 9, 3179–
 Xie, Y. and Varshavsky, A. (1999) The E2–E3 interaction in the
N-end rule pathway: the RING-H2 finger of E3 is required for the
synthesis of multiubiquitin chain. EMBO J. 18, 6832–6844.
 Moerschell, R.P., Hosokawa, Y., Tsunasawa, S. and Sherman, F.
(1990) The specificities of yeast methionine aminopeptidase and
acetylation of amino-terminal methionine in vivo. Processing of
altered iso-1-cytochromes c created by oligonucleotide transfor-
mation. J. Biol. Chem. 265, 19638–19643.
 Byrd, C., Turner, G.C. and Varshavsky, A. (1998) The N-end rule
pathway controls the import of peptides through degradation of a
transcriptional repressor. EMBO J. 17, 269–277.
 Turner, G.C., Du, F. and Varshavsky, A. (2000) Peptides
accelerate their uptake by activating a ubiquitin-dependent
proteolytic pathway. Nature 405, 579–583.
F. Eisele, D.H. Wolf / FEBS Letters 582 (2008) 4143–4146