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INTRODUCTION |
The endoplasmic reticulum
(ER)1 contains a highly
effective protein quality control system, which guarantees delivery of
only properly folded proteins to their site of action (1). Proteins that pass through the folding process but are unable to acquire their
proper conformation or cannot assemble with a respective binding
partner are degraded rapidly. This process is known as ER-associated
degradation or simply ER degradation (2-4). Recent studies have
revealed that degradation of misfolded or unassembled secretory
proteins requires retrograde transport from the ER to the cytoplasm of
the cell and the ubiquitin-proteasome system (2-4). The degradation of
a multitude of misfolded and unassembled secretory proteins has been
followed in mammalian cells (2, 3). Most prominent are the degradation
of unassembled T-cell receptor
-chains (5, 6) and a mutated version
of the human cystic fibrosis transmembrane conductance
regulator, which leads to the severe disease cystic fibrosis (7,
8). The yeast Saccharomyces cerevisiae has turned out to be
an excellent model system to study eukaryotic cell function. Here,
mutant versions of the vacuolar carboxypeptidase yscY (CPY*)
(9), the plasma membrane ATP-binding cassette transporter Pdr5p (Pdr5*)
(10), the ER membrane-located Sec61p (11), the mating type pheromone
-factor (12), and down-regulated hydroxymethylglutaryl-CoA reductase
(13) were found to be subject to ER degradation. As found for the
mammalian proteins destined for ER degradation, removal of these
proteins requires the proteasome and in most cases an intact ubiquitin
conjugation system (9-11, 14). Proteasomal substrates are usually
tagged by repeated attachment of ubiquitin moieties through isopeptide
bonds between the carboxyl-terminal glycine of ubiquitin and
-amino
groups of lysine residues within the substrate molecules (15). A
sequence of enzymatic reactions is necessary to link ubiquitin to
protein substrates: after activation through an E1 enzyme
via an ATP-requiring step, which leads to a thiol ester intermediate,
ubiquitin is transferred to a cysteine thiol group of a
ubiquitin-conjugating enzyme E2. In a third step involving a
ubiquitin-protein ligase (E3), ubiquitin is linked by its
carboxyl terminus to an
-amino group of the target protein, forming
an isopeptide linkage (15). When studying the fate of mutated, soluble
vacuolar CPY* and the mutated plasma membrane protein Pdr5*, we found
both proteins to be polyubiquitinated via the ubiquitin-conjugating
enzymes Ubc6p and Ubc7p, whereby Ubc7p appeared to be the most
prominent catalyst (9, 10). Similar results have been obtained for
mutated Sec61p and down-regulated hydroxymethylglutaryl-CoA-reductase
(11, 14). The ubiquitin-protein ligase E3 participating in
this process has remained a mystery. Genetic screens have uncovered a
variety of new gene products embedded in the ER membrane which are
necessary for ubiquitination and degradation of the respective proteins
(13, 16-18). One of these proteins, Der1p, was shown up until now to
be necessary only for ER-degradation of soluble, fully translocated
CPY* (16, 19), whereas Cue1p, Der3/Hrd1p, and Hrd3p have more general functions in the ER degradation process. Cue1p was discovered as to be
a receptor for Ubc7p in the ER membrane, activating this ubiquitin-conjugating enzyme (18). The function of Der3/Hrd1p and Hrd3p
have remained unknown so far. Recently, a new family of
ubiquitin-protein ligases (E3) has been discovered, which
all contain a RING finger domain as a common motif (20-27). Der3/Hrd1p is a RING-H2 domain-containing protein (13, 17) that resides in the ER
membrane (17). We have found previously that deletion of the RING-H2
domain or exchange of a single cysteine residue at position 399 against
serine in the Der3/Hrd1 protein completely abolishes degradation of
CPY* and Pdr5*, indicating the essentiality of this domain (10, 17,
28). We have furthermore shown that Der3/Hrd1p, Hrd3p, and Sec61p
interact genetically, which led us to the proposal that these proteins
formed both the retrotranslocon and an E3 complex (19). Here
we examine the topology of Der3/Hrd1p in the ER membrane via
N-glycosylation scanning and fusion of a topology-sensitive
reporter protein domain to carboxyl-terminally truncated versions of
the Der3/Hrd1 protein. We show that the carboxyl-terminally located
RING-H2 domain of Der3/Hrd1p faces the cytoplasm. Altogether, RING
finger-dependent ubiquitination of CPY* in vivo
as well as self-ubiquitination of Der3/Hrd1p in vitro, and
RING finger-dependent binding of Ubc7p identify Der3/Hrd1p as the ubiquitin-protein ligase (E3) of the ER degradation process.
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EXPERIMENTAL PROCEDURES |
Construction of Plasmids
Genetic experiments and molecular biological methods were
carried out using standard protocols (29).
DER3-SUC2-HIS4C Fusion Constructs--
The plasmid
pRS315-DER3 was generated by subcloning a 3,570-base pair
BamHI/HindIII fragment from plasmid
YEpDER3 (17), containing the DER3 gene under its
native promoter, into plasmid pRS315 (30). Fragments of the
DER3 gene were PCR amplified from this plasmid using the
oligonucleotide 5/6R (5'-gacggccagtgaattgtaat-3') as the upstream
primer and one each of the oligonucleotides DER3-Q37 (5'-cggcggctcgagttgcaaaaaggaaacgcttgtc-3'), DER3-E78
(5'-cggcggctcgagctcaataagcctcagttcacc-3'), DER3-R103
(5'-cggcggctcgagccgttcgtggaacagtg agg-3'), DER3-Q134 (5'-cggcggctcgagctgtaataaggcctccagcc-3'), DER3-Q176
(5'-cggcggctcgagttggtttgtatatatggaggag-3'), or DER3-D345
(5'-cggcggctcgagatcatttgcagaat tttgtagc-3') as the downstream primer (XhoI sites are underlined). The PCR
products were digested with SacI/XhoI and ligated
with the SacI/XhoI fragment of plasmid pR90 (31)
containing the SUC2-HIS4C fusion to yield the plasmids pQ37,
pE78, pR103, pQ134, pQ176, and pD345, respectively.
Insertion of an N-Glycosylation Site at the Carboxyl Terminus of
Der3p--
A carboxyl-terminal region of DER3 was PCR
amplified from YEpDER3 using the primers DER3-G1
(5'-cccaaaaggttaccttgtggc-3') and DER3-G2
(5'-gccgcctctagatagtactattatgctggataaatttatctgg-3'). The XbaI site containing the stop codon of DER3
is underlined, the bases encoding the N-glycosylation
sequence Asn-Ser-Thr are in bold letters. The PCR product was digested
with BglII/XbaI and inserted into
YEpDER3 linearized with the same enzymes, yielding plasmid
YEpDER3-G. For Western blot analysis of the protein, the plasmid was transformed into yeast strains W303-1C (16) and YJB009
(17).
Construction of GST-DER3 Fusions--
The carboxyl-terminal part
of DER3 starting with cysteine 208 was PCR amplified using
the primers DER3-GST1
(5'-ttcctagaattctgtttgaatttctgggaattt-3') and DER3-GST2
(5'-taagtcgacacatgcaatctagatatgctggat-3'), digested with
EcoRI/SalI (sites are underlined), and inserted
into the GST expression vector pGEX-4T-1 (Amersham Pharmacia Biotech), yielding plasmid pGEX-DER3. A 645-base pair
NcoI/BglII fragment of this plasmid was replaced
by the corresponding fragment from plasmid pRS426-DER3C399S
(17) to yield plasmid pGEX-DER3C399S.
Split Ubiquitin Constructs--
A carboxyl-terminal fragment of
HRD3 was amplified using the oligonucleotides HRD3-CUB1
(5'-ccgccgcggccgccaagaaccacagatcagag-3') and HRD3-CUB2
(5'-gccgccgctcgagcccgaacctatggcgaatatctgaacattg-3'), digested with EagI/XhoI (sites are underlined),
and ligated upstream of the CUB-RURA3 fusion construct in
pRS305 (32). To express the Hrd3-Cub fusion protein chromosomally, the
resulting plasmid was linearized using the MamI site within
the carboxyl-terminal HRD3 fragment and transformed into
yeast strain JD53 (32), yielding strain YPD41. For fusion of
DER3 with NUB, the complete DER3 gene was amplified with the primers DER-NUB1
(5'-cggcggcggatccctggtgacgtgccagaaaatagaaggaaac-3') and
DER-NUB2 (5'-ccgccggaattcctgattaacagggggac-3'), digested
with BamHI/EcoRI (sites are underlined), and
inserted downstream of the NUB fragment in pRS314 which is
under the control of the CUP1 promoter (32), resulting in
plasmid pNub-Der3.
Analysis of the Der3-His4C Fusion Proteins
Yeast strain STY50 (31) was separately transformed with plasmids
pQ37, pE78, pR103, pQ134, pQ176, pD345, and YEp352. Transformants growing on SD plates supplemented with histidine but lacking uracil were tested for their ability to grow on minimal medium lacking uracil
and containing 6 mM histidinol instead of histidine. Plates were incubated at 30 °C for 3-5 days.
Immunoprecipitation and deglycosylation of the Der3-His4C fusion
proteins were carried out as described (31) using polyclonal anti-invertase antiserum (R. Kölling, Düsseldorf).
Split Ubiquitin Assay
Yeast strain YPD41 (see above) was transformed with plasmid
pNub-Der3 and a plasmid encoding a Nub-Sed5 fusion (32). Transformants were grown overnight in SD medium lacking tryptophan, diluted with
water, and spotted on minimal medium containing 1 mg/ml 5-FOA and 0.4 mM uracil. Plates were incubated at 30 °C for 3-4 days.
GST Pull-down Assays
The GST-Der3 fusion proteins were expressed from plasmids
pGEX-DER3 and pGEX-DER3C399S in Escherichia
coli strain BL21 (Amersham Pharmacia Biotech). GST alone was
expressed from plasmid pGEX-4T-1. Culturing of the cells and
preparation of the bacterial protein extracts were carried out
according to the manufacturer's instructions. The extracts were
incubated with 100 µl (bead volume) of gluthathione Sepharose 4B
(Amersham Pharmacia Biotech) for 3 h at 4 °C. Sepharose beads
were collected (5 min at 3,000 rpm), washed three times with 1 ml of
phosphate-buffered saline (140 mM NaCl, 2.7 mM
KCl, 10 mM Na 2HPO4, and 1.8 mM KH 2PO4, pH 7.3), and incubated
at 4 °C overnight with whole cell extracts prepared from yeast
strain W303-1C, carrying the plasmid pTX146 (18) encoding a Myc-tagged version of Ubc7p. Sepharose beads were collected and washed for 5 min
with 1 ml of phosphate-buffered saline containing 500 mM NaCl. Proteins from the supernatant and the wash fraction were precipitated with trichloroacetic acid and dissolved in urea buffer. Proteins bound to the Sepharose matrix were then eluted with urea buffer by incubating at 95 °C for 5 min. Samples were subjected to
Western blot analysis using monoclonal anti-Myc antibody (Roche).
In Vitro Ubiquitination Experiments
Ubiquitination reactions were performed in 25 mM
Tris/HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl
2, 1 mM dithiothreitol, 10 mM ATP, and
0.5 µg/µl ubiquitin (yeast, Sigma) and contained one or more of the
following components as indicated in Fig. 6: 0.5 µg of E1
(yeast, Affiniti), 1 µg of E2 (human UBCH1, Affiniti), and approximately 1 µg of GST-Der3 fusion protein (see above). Incubation of the reactions for 60 min at 30 °C was followed by dilution with
phosphate-buffered saline and precipitation of GST-Der3 species with
Sepharose beads (3 h at 4 °C). The beads were collected, washed
three times with phosphate-buffered saline, and eluted with urea
buffer. Samples were subjected to Western blot analysis using
monoclonal anti-ubiquitin antibody (Calbiochem) or polyclonal anti-Der3
antiserum (17), respectively.
Immunoprecipitation of Ubiquitinated CPY*
For in vivo ubiquitination of CPY*, HA-tagged
ubiquitin was expressed from plasmid YEp112 (33) by induction with
copper as indicated in Fig. 5. The experiment was performed in a
DER3 wild type strain (W303-1C) and a der3
deletion strain (YJB009) expressing the C399S mutant of Der3p from
plasmid YCpDER3C399S (17). Immunoprecipitation and detection
of ubiquitinated CPY* species were done as described previously
(9).
Western Blot Analysis
For Western blot analysis of complete cellular protein, 3 A 600 units of cells were subjected to alkaline
lysis, and proteins were precipitated with trichloroacetic acid and
resuspended in urea buffer. Protein samples were separated on 6-12%
SDS-polyacrylamide gels and subjected to immunoblotting as described
before (19).
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RESULTS |
Der3/Hrd1p Spans the ER Membrane Six Times; Its Amino and Carboxyl
Termini Face the Cytoplasm--
Sequence analysis of Der3/Hrd1p had
indicated that the protein contains a hydrophobic amino-terminal region
with five putative membrane domains and a long hydrophilic
carboxyl-terminal tail containing the RING-H2 motif (13, 17). Protease
protection and immunolocalization experiments had localized the
carboxyl-terminal RING-H2 finger domain to the lumen of the ER.
However, because the ubiquitin-conjugating enzyme in charge, Ubc7p, as
well as the proteasome reside in the cytoplasm, it was hard to
reconcile the fact that the RING-H2 domain of the protein resided in
the ER lumen with a function for Der3/Hrd1p as a ubiquitin-protein ligase, unless one assumed a process by which the carboxyl terminus with the RING-H2 domain and bound substrate reached back through the
translocon into the cytoplasm of the cell (19).
We therefore undertook a new set of experiments to localize the
carboxyl-terminal RING finger domain of Der3/Hrd1p and to uncover the
membrane topology of the protein. First we undertook an
N-glycosylation scanning experiment. Der3/Hrd1p contains two N-glycosylation sites at positions 58 and 137 of its
sequence (17). However, these sites are not accessible to the
glycosylation machinery because they are predicted to be located in a
transmembrane span and on the cytosolic side of the ER membrane,
respectively. Therefore, we introduced a new potential
N-glycosylation site between the penultimate and last amino
acid of Der3/Hrd1p as done with CPY* (34). When expressing this
modified Der3/Hrd1 protein either in a DER3 wild type or a
der3 deletion background, no shift of the molecule to higher
molecular mass was visible although a similarly modified CPY* molecule
was shifted to higher molecular mass due to additional glycosylation
(Fig. 1). This may indicate that the
carboxyl terminus of the ER-localized Der3/Hrd1 protein does not reach
the lumen of the organelle where core glycosylation occurs. However,
this experiment does not give final proof for cytoplasmic localization
of the carboxyl terminus of Der3/Hrd1p.

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Fig. 1.
An N-glycosylation site
introduced carboxyl-terminally into Der3/Hrd1p fails to be
glycosylated. The amino acids Asn-Ser-Thr were introduced between
the last two amino acids of Der3/Hrd1p, and the resulting protein
(Der3-G) was expressed in a DER3 wild type and a
der3 deletion strain and subjected to Western
blot analysis. In contrast to a CPY* species modified in the same
manner (Prc1-G5) (34), no increase of the molecular mass resulting from
N-glycosylation is observed.
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We set up further experiments to prove this result and to elucidate the
exact membrane topology of Der3/Hrd1p. The hydropathy profile of
Der3/Hrd1p predicts the existence of five or six transmembrane domains
(Fig. 2A). We created fusion
constructs of Der3/Hrd1p with a truncated version of the His4 protein
(His4C) containing a fragment of invertase (Suc2p) which we used as
topology-sensitive reporters. His4C harbors histidinol dehydrogenase
activity and is translocated through the ER membrane when fused to a
signal sequence (35). Yeast his4 mutant strains expressing a
His4C fusion are able to grow on minimal medium containing histidinol when the catalytic domain is present on the cytoplasmic side of the ER
membrane. In this case, histidinol is metabolized to histidine resulting in a His+ phenotype. When the catalytic domain is
targeted to the ER lumen, histidinol cannot be converted to histidine,
leading to a His
phenotype. However, when located in the
ER lumen, the protein becomes extensively glycosylated because of the
presence of the SUC2 sequence and thus several glycosylation
sites (31). We constructed a series of fusion proteins consisting of
carboxyl-terminally truncated versions of Der3/Hrd1p and the His4C
protein domain (Fig. 2A and Table
I), which allowed us to identify the
orientation of the transmembrane domains of Der3/Hrd3p and, in
addition, the location of the carboxyl-terminal RING-H2 finger domain.
The invertase sequence contained within these constructs also
introduced an epitope for immunodetection, thus making it possible to
visualize the fusion proteins using anti-invertase antibodies.

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Fig. 2.
Growth phenotypes and glycosylation analysis
of Der3/Hrd1-His4C reporter fusions identify Der3/Hrd1p
topology. A, the hydropathy profile of Der3/Hrd1p using
a window of 17 amino acids is shown (41). Potential
transmembrane-spanning domains are marked with Roman
numerals. The terminal amino acids of the respective Der3/Hrd1p
portions in the individual Der3/Hrd1-His4C fusions are indicated.
B, growth phenotypes of the Der3/Hrd1-His4C fusions. Yeast
strain STY50 was transformed with the control vector YEp352 or plasmids
encoding the different Der3/Hrd1-His4 fusions. Transformants were
streaked on selective media supplemented with histidine (left
panel) or histidinol (right panel) and incubated for
3-5 days at 30 °C. C, analysis of the
N-glycosylation state of the Der3/Hrd1-His4C fusion
proteins. The proteins were immunoprecipitated from whole cell extracts
of STY50 transformed with plasmids pQ37 to pD345 (lanes
1-12) or plasmid YEp352 (lane 13) using polyclonal
anti-invertase antibody. Immunoprecipitates were treated with
endoglycosidase H (Endo H) as indicated and separated on 6%
SDS-polyacrylamide gels. Western blot analysis was performed using
anti-invertase antibody.
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Table I
Der3/Hrd1-His4C fusion constructs
Der3/Hrd1-His4C fusion proteins were constructed as described. The
invertase and His4C portion accounts for 122,084 Da of the total
molecular mass of each Der3/Hrd1 fusion protein. The constructs carry
14 potential glycosylation sites, which increase the mass by 35,000 Da
when the protein becomes N-glycosylated (31). Growth on
histidinol-containing medium and lack of N-glycosylation
indicate that His4C faces the cytoplasm, whereas the absence of growth
on histidinol and extensive glycosylation indicate an ER luminal
orientation.
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We first determined the orientation of the amino terminus of Der3/Hrd1p
using the fusion construct Der3/Hrd1Q37 containing the
first transmembrane domain (Fig. 2A and Table I). As can be
seen in Fig. 2B, Der3/Hrd1Q37 does not support
growth on histidinol. Instead, the construct is heavily glycosylated;
endoglycosidase H treatment reduces the molecular mass of the fusion
protein considerably, leading to its calculated value (Fig.
2C, lanes 1 and 2). These results
indicate that the amino terminus of Der3/Hrd1Q37 resides on
the cytoplasmic side of the ER membrane. However, there is still the
possibility that the first transmembrane region functions as a
cleavable signal sequence or that the entire construct fully
translocates into the ER lumen. The latter possibility is ruled out on
the basis that Der3/Hrd1Q37 is detected in the fraction
corresponding to the membrane pellet when anti-invertase antibodies are
used. Der3/Hrd1Q37 cannot be solubilized with urea, high
salt, or carbonate. Only after treatment with Triton X-100 was
Der3/Hrd1Q37 transferred into the soluble fraction (data
not shown). Furthermore, we had shown previously that Der3/Hrd1p
genetically interacts with another ER membrane protein necessary for ER
degradation, Hrd3p (19). Hrd3p spans the membrane once, its carboxyl
terminus being exposed to the cytosol (19). Thus it was feasible that the amino terminus of Der3/Hrd1p was in close proximity to the carboxyl
terminus of Hrd3p. To elucidate this possibility and to show that the
amino terminus of Der3/Hrd1p indeed faces the cytoplasm, we made use of
the split ubiquitin technique (32, 36). This technique monitors
interactions between proteins in vivo and is based on the
self-reassembly of the amino- and carboxyl-terminal halves (Nub and
Cub) of ubiquitin. The reassembled ubiquitin is recognized by
ubiquitin-specific proteases that cleave any carboxyl-terminally attached polypeptide from Cub (32, 36). Cub, extended with an
amino-terminally modified version of the enzyme Ura3p (RUra3p), was
linked to the cytosolic carboxyl terminus of Hrd3p. Nub was linked to
the amino terminus of Der3/Hrd1p. If Cub and Nub interacted, RUra3p
would be cleaved off by the ubiquitin-specific proteases. Because the
amino-terminal residue of RUra3p is an arginine, rapid degradation of
RUra3p by the enzymes of the N-end rule pathway leads to a
Ura
phenotype. 5-FOA is converted by Ura3p into
5-fluorouracil, which is toxic for the cells. However, degradation of
RUra3p rescues cells and allows growth on medium containing 5-FOA (32).
As can be seen in Fig. 3, cells
expressing the carboxyl-terminally modified Hrd3-Cub-RUra3 and the
amino-terminally modified Nub-Der3/Hrd1 can grow on 5-FOA, indicating
close proximity or interaction between both proteins and at the same
time a cytoplasmically exposed amino terminus of Der3/Hrd1p.

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Fig. 3.
Split ubiquitin assay confirms the
cytoplasmic orientation of the Der3/Hrd1p amino terminus. The
carboxyl-terminal half of ubiquitin (Cub), followed by an
arginine residue and the Ura3 protein (RUra3), was fused to
the cytosolic carboxyl terminus of Hrd3p. The amino-terminal half of
ubiquitin (Nub) was attached to the amino terminus of
Der3/Hrd1p. Cells coexpressing these two constructs were spotted on
uracil-containing minimal medium conditioned with 5-FOA in 1:10
dilution steps. Growth of cells is promoted by reassembly of ubiquitin,
leading to cleavage and degradation of the Ura3 protein. To show the
specificity of the Der3/Hrd1p-Hrd3p interaction, the Nub fusion of the
Golgi protein Sed5p was expressed together with the Cub fusion of
Hrd3p.
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N-Glycosylation scanning with a newly introduced
N-glycosylation site at the carboxyl terminus of Der3/Hrd1p
had indicated that this part of the protein does not reach the ER lumen
and is localized in the cytoplasm. When the construct
Der3/Hrd1D345 was tested, which contains His4C in the
carboxyl-terminal region, growth of cells on histidinol (Fig.
2B) and the absence of any glycosylation (Fig.
2C, lanes 11 and 12) were observed. A
protein band of calculated molecular mass was detected upon Western
blot analysis. This strongly suggests that the carboxyl terminus of Der3/Hrd1p is located on the cytoplasmic side of the ER. This again
indicates that the RING-H2 domain of Der3/Hrd1p is cytoplasmic. Orientation of the amino and carboxyl terminus of Der3/Hrd1p to the
same side, the cytoplasm, suggests the presence of an even number of
transmembrane domains in Der3/Hrd1p. We therefore analyzed the fusion
proteins Der3/Hrd1E78, Der3/Hrd1R103,
Der3/Hrd1Q134, and Der3/Hrd1Q176. The
constructs Der3/Hrd1E78 and Der3/Hrd1Q134
promoted growth of cells on histidinol (Fig. 2B) and
exhibited a weight comparable to their calculated molecular masses with no glycosylation occurring (Fig. 2C, lanes 3 and
4 and lanes 7 and 8). This points to a
cytoplasmic localization of both His4C domains arguing for an identical
orientation of both transmembrane helices II and IV. This indicates
that the hydrophobic sequence III (Fig. 2A) also constitutes
a short helix, which dips into the ER membrane in the opposite
direction. Indeed, the construct Der3/Hrd1R103 did not
promote growth on histidinol (Fig. 2B) and was heavily glycosylated (Fig. 2C, lanes 5 and 6).
As expected, the fusion construct Der3/Hrd1Q176 did not
promote growth on histidinol either (Fig. 2B), and it was
heavily glycosylated (Fig. 2C, lanes 9 and
10), supporting the view of ER luminal orientation of the
His4C portion and of helix V spanning the ER membrane in the opposite
direction to transmembrane helices II, IV, and VI. From these studies,
we predict Der3/Hrd1p to be a protein with six membrane-spanning
helices, whereby the amino and carboxyl termini of the protein face the cytoplasm (Fig. 4).
Der3/Hrd1p Is a Ubiquitin-Protein Ligase (E3)--
We had shown
that Der3/Hrd1p is required for degradation of CPY* and the polytopic
membrane protein Pdr5*. We furthermore provided evidence for the
RING-H2 domain as being crucially involved: deletion of the RING-H2
domain or mutation of one of the cysteine residues (Cys-399) to serine
within this domain abolished the function of the protein (10, 17, 28).
Recent studies have uncovered a new family of ubiquitin-protein ligases
(E3 enzymes) which contain a RING-H2 finger domain that is
necessary for function (20-27). It was thus very likely that
Der3/Hrd1p constituted the ubiquitin-protein ligase involved in the
degradation of the ER proteins tested so far (13, 17). As shown in Fig.
5, the C399S mutation in Der3/Hrd1p
indeed abolishes ubiquitination of CPY*. It is an outstanding feature
of ubiquitin-protein ligases to self-ubiquitinate in vitro
in a RING finger-dependent manner in the absence of other substrate proteins (20, 23, 27). We tested Der3/Hrd1p for this
property. The soluble RING-H2 finger-containing carboxyl-terminal domain of Der3/Hrd1p (amino acids 208-551) was expressed as a GST
fusion protein and incubated with ubiquitin, E1, and
E2 enzymes and Mg 2+-ATP in vitro.
Indeed, the GST-Der3/Hrd1 protein ubiquitinated itself to some extent
in an E1- and E2-dependent fashion.
As expected, formation of the ubiquitin conjugates was completely
abolished in the Der3/Hrd1p C399S mutant (Fig.
6).

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Fig. 5.
Intact Der3/Hrd1p is required for
ubiquitination of CPY* in vivo. In cells expressing either
wild type (WT) Der3/Hrd1p or the Der3/Hrd1 C399S mutant
protein, HA-tagged ubiquitin was overexpressed from the CUP1 promoter
by induction with copper as indicated. After cell lysis under alkaline
conditions, CPY* species were immunoprecipitated, separated by gel
electrophoresis, and subjected to Western blot analysis using anti-HA
antibodies to visualize CPY*(HA-Ub)x conjugates.
No conjugates are detectable in the Der3/Hrd1p C399S mutant or when
overexpression of HA-Ub is not induced. Efficiency of the
immunoprecipitation is proved by Western blot analysis of the samples
using anti-CPY antibody.
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Fig. 6.
Der3/Hrd1p promotes its self-ubiquitination
in vitro. A GST fusion protein with the carboxyl-terminal,
soluble part of Der3/Hrd1p, containing the RING-H2 domain, was
incubated in the presence of ubiquitin, Mg 2+-ATP, and
purified E1 and E2. Gluthatione-Sepharose was
added to the reaction mixtures, and bound proteins were subjected to
gel electrophoresis. Subsequent Western blot analysis using an
anti-ubiquitin antibody shows a series of multiubiquitinated species of
higher molecular weight than the GST-Der3 fusion protein itself.
Formation of these conjugates was completely abolished when the C399S
mutant of Der3/Hrd1p was used or when either Der 3/Hrd1p or
E1 and E2 were omitted. To visualize the amounts
of GST-Der3 fusion protein in the different reactions, the samples were
subjected to Western blot analysis using anti-Der3/Hrd1p
antibodies.
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It has been demonstrated that the RING domain mediates the recruitment
of the respective ubiquitin-conjugating enzyme E2 involved in the degradation reaction (25, 37). In the case of the
ubiquitin-protein ligase c-Cbl, the RING finger motif has been shown to
be essential for binding of the E2 enzyme (37, 38). We
tested the ability of Der3/Hrd1p to bind the ubiquitin-conjugating
enzyme Ubc7p, which is known to be necessary for all ubiquitin
conjugation reactions dependent also on Der3/Hrd1p (10, 13, 14, 17).
The soluble GST-fused RING-H2 finger-containing domain of Der3/Hrd1p
was bound to glutathione-Sepharose. Application of extracts of cells
expressing a functional Myc-tagged Ubc7p (18) to the GST-Der3/Hrd1
Sepharose beads resulted in specific binding of Ubc7p to the protein
(Fig. 7A). Interestingly, the
inactive C399S mutant of Der3/Hrd1p is also defective in binding of
Ubc7p (Fig. 7B). Taken together, the lack of CPY*
ubiquitination in cells carrying a mutated Der3/Hrd1 C399S protein, the
ability to ubiquitinate itself, and its interaction with Ubc7p clearly
identify Der3/Hrd1p as the ubiquitin-protein ligase (E3) of
the ER degradation process. The results also show that the RING-H2
domain of the ligase is crucial for recruitment of the
ubiquitin-conjugating enzyme Ubc7p.

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Fig. 7.
The Der3/Hrd1p RING-H2 domain binds
Ubc7p. A, GST pull-down assay confirms protein-protein
interaction between Der3/Hrd1p and Ubc7p. A fusion protein of the
soluble carboxyl-terminal part of Der3/Hrd1p containing the RING-H2
domain with GST was bound to glutathione-Sepharose and incubated with a
whole cell extract prepared from a strain expressing a functional
Myc-tagged version of Ubc7p. The nonbound supernatant (S)
was removed and the gel beads washed with incubation buffer containing
500 mM NaCl (W). Bound proteins were eluted with
urea buffer (E), and samples were analyzed by Western blot
analysis using monoclonal anti-Myc antibodies. As a control, GST alone
was attached to the Sepharose beads. B, when the same
experiment was performed using the mutated C399S species of Der3/Hrd1p,
specific binding of Myc-Ubc7p to the GST fusion protein was completely
abolished.
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DISCUSSION |
This study presents a first analysis of the complete membrane
topology of Der3/Hrd1p, an essential component of the ER degradation machinery. We furthermore provide evidence that Der3/Hrd1p is the
ubiquitin-protein ligase (E3) of the ER degradation process. Using a topology-sensitive reporter protein domain, we show that Der3/Hrd1p contains six transmembrane domains. The amino terminus and
the carboxyl terminus of the protein face the cytoplasmic side of the
ER membrane. The newly established cytoplasmic localization of the
carboxyl-terminal domain of Der3/Hrd1p corrects our previous preliminary finding, in which upon protease protection experiments and
subsequent immunolocalization studies with an antibody directed against
the carboxyl terminus of Der3/Hrd1p, an ER luminal localization of the
carboxyl-terminal domain of the protein had been suggested (17). This
may have been the result of conditions leading to an artifactual
burying of the carboxyl terminus of Der3/Hrd1p because of membrane
aggregation and occlusion of membrane surfaces (39) or a carboxyl
terminus resistant to protease attack. While our work was nearly
completed, a paper appeared which also reported a cytoplasmic
localization for the carboxyl-terminal domain of Der3/Hrd1p (40).
The carboxyl-terminal domain of Der3/Hrd1p contains a RING-H2 finger
motif that is crucial for its function: deletion of the motif or
exchange of one of the cysteine residues (Cys-399) of the RING-H2
domain to serine completely abolishes degradation of mutated and
malfolded ER proteins (17, 28). RING finger-containing proteins have
been identified as a new class of ubiquitin-protein ligases
(E3 enzymes) necessary for ubiquitination of substrates destined for degradation via the proteasome (20-27). We show here that
cells carrying a mutated Der3/Hrd1p C399S version are unable to
ubiquitinate CPY* in vivo (Fig. 5). A common feature of RING finger-containing ubiquitin-protein ligases is their ability to ubiquitinate themselves in vitro in the absence of
substrates (20, 23, 27). We found that this also holds true for
Der3/Hrd1p (Fig. 6). Function of a protein as an E3 requires
recruitment of the respective ubiquitin-conjugating enzyme
(E2). Protein-protein interaction studies using a GST-bound
carboxyl-terminal fragment of Der3/Hrd1p containing the RING-H2 domain
demonstrated that Ubc7p, the major ubiquitin-conjugating enzyme of the
ER degradation process, selectively bound to the protein. Altogether,
these properties identify Der3/Hrd1p as the ubiquitin-protein ligase
(E3) of ER degradation as suggested previously (19).
Mutation of a crucial cysteine (C399S) in the RING-H2 finger domain
completely abolished binding of Ubc7p, suggesting that the RING-H2
domain is directly involved in the binding of E2. Cue1p,
another ER membrane protein, had been shown to bind Ubc7p and to be
necessary for Ubc7p activity (18). Our pull-down experiment clearly
demonstrates that Der3/Hrd1p carries an intrinsic binding activity
toward Ubc7p. The additional binding of Ubc7p by Cue1p might result in
a tightening of Ubc7p binding to the membrane and by this inducing the
formation of the "active" structure of Ubc7p.
We had shown previously that Der3/Hrd1p and Hrd3p, another essential ER
membrane protein involved in ER degradation, interact genetically,
indicating complex formation between these two proteins (19). Using the
split ubiquitin system, we could show that the cytoplasmically
localized amino terminus of Der3/Hrd1p and the carboxyl terminus of
Hrd3p are in close proximity, which might indicate that they physically
interact (Fig. 3). An interaction between both proteins has also been
presented recently by others (40). The fact that the carboxyl terminus
of Der3/Hrd1p with its RING-H2 domain is localized to the cytoplasm and
never sees the ER lumen makes the previous proposal of Der3/Hrd1p as
being a binding partner of the substrate in the ER lumen, which
delivers it to the cytoplasmic degradation machinery, unlikely. It also makes a function of Hrd3p as a recycling molecule for the carboxyl terminus of Der3/Hrd1p from the cytoplasm back into the ER lumen improbable (19). A complex formation of Der3/Hrd1p and Hrd3p must have
different functions. As induction of Ubc7p-dependent Der3/Hrd1p degradation upon Hrd3p deletion indicates the presence of a
fully competent ubiquitination and degradation machinery in
hrd3 cells, the defective degradation of ER substrates in the absence of Hrd3p must have other reasons. It would be plausible if
Hrd3p had substrate recognition and signaling functions, which lead to
integration of processes such as substrate delivery via the Sec61
translocon, ubiquitination via Der3/Hrd1p and Ubc7p, and degradation
via the proteasome in a fashion that makes the overall process highly
regulated and processive. Similar functions for the Der3/Hrd1p-Hrd3p
interaction are also discussed by Gardner et al. (40).
Future experiments will uncover the functional relationship of both proteins.