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Vol. 14, Issue 8, 3437-3448, August 2003
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* Department of Biological Sciences, University of Pittsburgh, Pittsburgh,
Pennsylvania 15260;
Departments of Biochemistry and Molecular Biology and Chemistry, University of
Massachusetts, Amherst, Massachusetts 01003; and
Department of Molecular Biology, Princeton University, Princeton, New Jersey
08544
Submitted December 28, 2002;
Revised March 3, 2003;
Accepted March 28, 2003
Monitoring Editor: Reid Gilmore
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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); aberrant polypeptides may be retro-translocated from the ER
to the cytoplasm and destroyed by the proteasome in a process termed
ER-associated degradation (ERAD; McCracken
and Brodsky, 1996). The importance of defining the molecular
mechanism of ERAD is underscored by the fact that several human diseases arise
from the accumulation or accelerated degradation of ERAD substrates and
because some bacterial toxins and viruses coopt the ERAD pathway to exert
their effects (reviewed in Thomas et
al., 1995; Brodsky and
McCracken, 1999; Aridor and
Hannan, 2000; Fewell et
al., 2001).
ERAD may result from inefficient protein folding, so it is not surprising
that molecular chaperones are required for this process. Chaperones prevent
the formation of off-pathway intermediates or directly catalyze folding and
have been proposed to "judge" whether a nascent protein will
ultimately fold or whether it should be targeted for degradation (reviewed by
Hayes and Dice, 1996;
Hartl, 1996;
Horwich et al., 1999;
Plemper and Wolf, 1999;
Römisch, 1999;
Wickner et al., 1999;
Fewell et al., 2001;
Höhfeld et al.,
2001). Hsp70 (heat shock proteins with a molecular mass of 
70
kDa) molecular chaperones hydrolyze ATP concomitant with the binding of
peptides with overall hydrophobic character
(Flynn et al., 1991;
Blond-Elguindi et al.,
1993; Rüdiger et
al., 1997); therefore, Hsp70s might retain the solubility of
unfolded, retro-translocating polypeptides during their voyage from the ER to
the cytoplasm via the Sec61p translocation channel or might "gate"
this channel (Wiertz et al.,
1996; Pilon et al.,
1997; Plemper et al.,
1997; Johnson and Haigh,
2000; Tsai et al.,
2002).
In accordance with these hypotheses, several lines of evidence support a
role for the lumenal Hsp70, BiP, in ERAD in both mammals and yeast. First, the
proteolysis of ERAD substrates coincides with the rate at which they are
released from BiP in the mammalian ER
(Knittler et al.,
1995; Beggah et al.,
1996; Skowronek et
al., 1998; Chillaron and
Haas, 2000), and BiP associates preferentially with exposed
regions of an unfolded ERAD substrate
(Schmitz et al.,
1995). Second, the degradation of an ERAD substrate is slowed when
yeast contain a mutant allele in the gene encoding BiP, KAR2
(kar2-113; Plemper et
al., 1997), and two ERAD substrates in yeast, CPY* and
p
F, aggregate in microsomes prepared from another kar2 mutant
shifted to the nonpermissive temperature (kar2–203;
Nishikawa et al.,
2001). The delivery of an ERAD substrate to the proteasome is also
reduced in microsomes prepared from kar2-1 and kar2-133
mutants; however, in contrast to the kar2 mutants used in these other
studies, the kar2-1 and kar2-133 alleles compromise ERAD
efficiency but do not affect protein translocation (import) into the ER,
suggesting that the roles for BiP during protein translocation and
retro-translocation are distinct (Brodsky
et al., 1999) and that a biochemical analysis of the
corresponding Kar2-1 and Kar2-133 mutant proteins might elucidate which
biochemical activities of BiP are required for ERAD.
To better define the action of BiP during ERAD we sought kar2 mutants in which the unfolded protein response (UPR) was induced and identified the kar2-1 allele. We also present a biochemical analysis of the Kar2-1 and Kar2-133 mutant proteins and the physiological consequences of Kar2-1/133p expression in yeast.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) and the
kar2-133 mutant was obtained by plasmid shuffle, as outlined in Vogel
et al. (1990). DNA
sequence analysis identified the mutations in the kar2-1 and
kar2-133 open reading frames. To obtain kar2 mutants in
which the UPR was activated, the following strains were constructed. First, a
KAR2 disruption cassette was obtained by amplifying the HIS3
gene from pRS303 with primers BiPDel1 (5'-G G A C A C G A A A A G G G T
T C T C T G G A A G A T A T A A A T A T G G C T A T G C T C T T G G C C T C C
T C T A G-3') and BiPDel2 (5' C A C T G T T A C T G A G T A C C T
T A A C C C C A G T C T C TATACTCTTCTCGTTCAGAATGACACG-3') and that
contain a region of homology to the KAR2 gene on each end. This
disruption cassette was used to transform the yeast wild-type diploid strain
YPH501 (a/,
his3-
200/his3-200,
leu2-1/leu2-1, ura3-52/ura3-52,
trp1-63/trp1-63) and His+ transformants
in which one KAR2 allele was disrupted were confirmed by PCR using
primers HISend (5'-GTGATTAACGTCCACACAGG-3') and BiPend
(5'-GGGATGAGATGAGATGAGATG-3'). The resulting strain was
transformed with the single-copy, KAR2-containing plasmid, pMR397,
which is marked with URA3. The transformed diploid strain was starved
for nitrogen for 5 d to induce sporulation and tetrads were dissected and
grown on rich medium. After replica plating the resulting colonies on various
selective media to screen for auxotrophies, the MMY8–2 strain (,
kar2::HIS3, his3-200, leu2-1, ura3-52,
trp1-63, pMR397) was isolated. MMY8–2 was then
transformed with pMZ11 (CEN4, TRP1, UPRE::LacZ) to monitor UPR
activation through 
-galactosidase activity
(Zhou and Schekman, 1999). The
resulting strain is called MMY9 and was used for the screen described
below.
BiP was overproduced in wild-type and kar2-1 yeast from the 2µ
URA-marked plasmid, pMR109, which contained the
ClaI-SalI (KAR2) fragment from pMR48
(Rose et al., 1989).
Cells with pMR109 were selected on synthetic complete medium lacking uracil
(SC-ura) and supplemented with glucose to a final concentration of 2%. All
manipulations involving yeast were performed using standard protocols
(Rose et al.,
1990).
Isolation of kar2 Mutants Exhibiting an Enhanced UPR
Plasmid pMR713 (CEN4, LEU2, KAR2) was mutagenized with
hydroxylamine (Rose et al.,
1990) and transformed into MMY9 (see above). The resulting
transformants, which grew on medium lacking tryptophan, uracil, and leucine,
were replica-plated on 5-FOA at 30°C to select for yeast lacking pMR397.
Approximately 35,000 transformants were screened for constitutive activation
of the UPR by monitoring -galactosidase activity at 30°C with an
agarose overlay containing X-Gal (100 µg/ml) and 0.2% Sarkosyl as a
permeating agent (Kabani et al.,
2000a). Candidate colonies were restruck to reconfirm UPR
activation, and the corresponding pMR713 mutagenized plasmids from the
rescreened colonies (120) were isolated and individually transformed back
into MMY9. After replica-plating on 5-FOA and testing for -galactosidase
activity to confirm that UPR activation was plasmid-based, 20 mutants were
retained. These resulting pMR713 plasmids were also introduced into
MMY8–2 and the transformants were used for further analysis (see below).
As a control, MMY8–2 was transformed with untreated pMR713 and
replica-plated on 5-FOA to cure yeast of the pMR397 plasmid and is referred to
as wild type where indicated.
ERAD, Translocation, and UPR Assays
The degradation of unglycosylated proalpha factor (pF) in yeast
ER-derived microsomes was measured as described
(McCracken and Brodsky, 1996).
In brief, 35S-labeled prepro-alpha factor (ppF) lacking the
core consensus glycosylation sites was translocated into microsomes, after
which the microsomes were harvested, washed twice, and resuspended in a chase
reaction either containing or lacking an ATP-regenerating mix and yeast
cytosol at a final concentration of 5 mg/ml. Reactions were incubated at
30°C and were quenched after 20 min by the addition of trichloroacetic
acid (TCA) to a final concentration of 20%. The percentage of pF
remaining was determined by phosphorimage analysis after SDS-PAGE of
electrophoresed reaction products.
In vitro translocation assays were prepared as above except that after a
40-min translocation assay the reactions were divided and incubated in the
presence or absence of trypsin in order to determine the amount of protected,
and thus translocated, pF. Samples were processed and translocation
efficiency was calculated as described
(Brodsky and Schekman, 1993).
Translocation of preBiP and ppF was assayed in vivo after incorporation
of 35S-methionine into total cellular protein and
immunoprecipitation of BiP and ppF using specific antiserum and Protein
A-sepharose, as previously published
(Morrow and Brodsky,
2001).
The UPR was assayed quantitatively by measuring -galactosidase
activity in extracts (Rose and Botstein,
1983) prepared from yeast transformed with pJC104 (kindly provided
by Dr. Peter Walter, University of California, San Francisco). Plasmid pJC104
encodes the lacZ gene behind four repeats of the unfolded protein
response element (UPRE; Mori et
al., 1992; Cox et
al., 1993).
Protein Purification and Other Biochemical Assays
Hexahistidine-tagged derivatives of wild-type, Kar2-1p, and Kar2-133p were
generated and purified by nickel affinity and conventional chromatography as
described for other mutant forms of Kar2p
(McClellan et al.,
1998). The proteins were estimated to be >95% pure as
determined by SDS-PAGE and Coomassie Brilliant Blue staining. Single-turnover
ATPase assays (Sullivan et al.,
2000) of [-32P]ATP-BiP complexes indicated that
the rate of ATP hydrolysis by hexahistidine-tagged yeast BiP (0.12–0.21
min–1) was similar to that reported for
hexahistidine-tagged mammalian BiP (0.30 min–1;
Chevalier et al.,
1998). Steady state ATPase assays were performed as previously
published (McClellan et al.,
1998).
The glutathione-S-transferase (GST)-Sec63p-J fusion protein was
purified as described (Corsi and Schekman,
1997), and a GST-Jem1p-J was purified from the Escherichia
coli TG1 strain transformed with pSNJ20 (S. Nishikawa S. and T. Endo,
unpublished data). The pSNJ20 plasmid allows the expression of the C-terminal
115 residues of Jem1p fused at the C terminus of GST. The fusion protein also
contains a hexahistidine-tag at the C terminus. Bacteria were grown in Luria
broth (LB) containing 50 µg/ml ampicillin at 26°C to midlog phase and
isopropyl--D-thiogalactopyranoside (IPTG) was added to a
final concentration of 1 mM, and the cells were incubated further for 1 h. The
bacteria were harvested, washed with water, and resuspended in Buffer A (50 mM
HEPES-KOH, pH 7.4, 5 mM -mercaptoethanol, 200 mM NaCl, 10 mM imidazole).
Lysozyme was added to a final concentration of 0.1 mg/ml and the cells were
incubated on ice for 30 min. Protease inhibitors
(phenylmethylsulfonylfluoride, 1 mM; leupeptin, 1 µg/ml; pepsatin A, 1
µg/ml) were added and the cells were disrupted six times by sonication for
30 s with a 1-min incubation on ice between each sonication. Triton X-100 was
added to a final concentration of 0.1%, the broken cells were centrifuged for
10 min at 13,000 rpm in a Sorvall SA600 rotor, and the resulting supernatant
was centrifuged at 20,000 rpm in a Sorvall SA600 rotor for 20 min. The cleared
lysate was loaded onto a 5 ml Ni-NTA agarose column (Qiagen, Valencia, CA)
that had been equilibrated in buffer A, and the column was washed with 30 ml
of the following: (1) Buffer A; (2) Buffer A containing 1% Triton X-100 and 5%
glycerol; (3) Buffer A containing 1 M NaCl and 5% glycerol; (4) Buffer A
containing 5 mM MgCl2, 5 mM ATP, and 300 mM NaCl; (5) Buffer A
containing 0.5 M Tris-HCl, pH 7.4, and 300 mM NaCl; (6) Buffer A containing
300 mM NaCl, 5% glycerol, and 25 mM imidazole; and (7) Buffer A containing 300
mM NaCl, 5% glycerol, and 50 mM imidazole. The GST-Jem1p protein was eluted
with a 15 x 15-ml linear gradient of Buffer A containing 300 mM NaCl, 5%
glycerol, and imidazole from 50–250 mM; 1-ml fractions were collected
and analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. Peak fractions
were pooled and dialyzed against 20 mM HEPES-KOH, pH 7.4, 50 mM KCl, and 5%
glycerol, and then snap-frozen in liquid nitrogen and stored at
–80°C. The GST-Jem1p was >95% pure as assessed by Coomassie
Brilliant Blue staining.
GST-fusion protein pull-down assays were performed as published (Kabani
et al., 2000b,
2002). Cell extracts for
Western blots were prepared as reported
(Morrow and Brodsky, 2001),
and the proteins were resolved by SDS-PAGE and transferred to nitrocellulose
membranes (0.22-µM pore diameter; Schleicher and Schuell, Keene, NH).
Quantitative immunoblots were performed using anti-BiP rabbit antiserum
(Brodsky and Schekman, 1993)
and anti-Sec61p rabbit antiserum (Stirling
et al., 1992), used as a loading control, both of which
were decorated with 125I-Protein A (Amersham Biosciences,
Piscataway, NJ). Images were obtained and quantified using a Fuji
phosphorimager and MacBas software (v. 2.4; Fuji Medical Systems, Stamford,
CT). Carbonate extraction on 1 equivalent of ER-derived microsomes was
accomplished as described in Fujiki et al.
(1982), and the pellet and
supernatant fractions were analyzed by SDS-PAGE and immunoblot analysis using
Enhanced Chemiluminescence (Pierce, Rockford, IL). The Sec63p-BiP complex was
purified from octylglucoside-solubilized yeast microsomes by
diethylaminoethane, Superose-6, and hydroxylapatite column chromatography as
previously published (Brodsky and Schekman,
1993).
The oligomeric state of pF was assessed as described
(Nishikawa et al.,
2001). In brief, washed microsomes containing pF were
obtained after an in vitro translocation assay (see above), the membranes were
solubilized with Triton X-100, and the clarified extract was loaded onto a
5–40% sucrose gradient in 0.1% Triton X-100 and centrifuged at 145,000
x g for 20 h at 4°C. After the gradient was fractionated
pF was detected and quantified by SDS-PAGE and phosphorimager
analysis.
Spectroscopy
The CALLQSRLLLSAPRRAAATARY (APPY) peptide was synthesized, purified,
labeled, and analyzed by mass spectrometry as described in Montgomery et
al. (1999). The CLLLSAPRR
(p5) peptide (Pierpaoli et al.,
1998) was synthesized by solid phase methods on PerSeptive
Biosystems (Framingham, MA) automated peptide synthesizer and purified by
reverse-phase chromatography on a C18 column and analyzed by mass
spectrometry. The affinity of yeast BiP for F-APPY (fluorescein-labeled APPY)
and the binding of AEDANS-labeled p5 to BiP were measured by fluorescence
anisotropy as described by Montgomery et al.
(1999) after incubating
0.02–0.05 µM of F-APPY or p5-AEDANS with the indicated concentrations
of BiP overnight at 4°C. The BiP used for these experiments was dialyzed
for 16 h against 20 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2, 0.8
mM DTT and concentrated using Centricon microconcentrators (Amicon, Beverly,
MA). Circular Dichroism (CD) spectroscopy of yeast BiP (final concentration
1.6 µM) was performed on an Aviv CD Spectrometer (Model 202; Lakewood, NJ)
using a 0.1-cm path-length. The temperature of the cuvette was maintained at
26°C.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Therefore, we desired allele-specific
kar2 mutants to more specifically define the action of BiP during
ERAD. Our screen for such alleles is based on that used by Zhou and Schekman
(1999) to isolate
sec61 mutants that were ERAD-defective but translocation-proficient,
and the premise for the screen derives from the fact that defects in ERAD
result in UPR induction (Cassagrande
et al., 2000;
Friedlander et al.,
2000; Ng et al.,
2000; Travers et al.,
2000).
The MMY9 strain bears a chromosomal deletion of the KAR2 gene and
is viable because it contains pMR397, a single-copy uracil-selectable plasmid
that expresses KAR2 under the control of its own promoter
(Figure 1). MMY9 also contains
plasmid pMZ11 that can be used to report on the UPR by measurements of
-galactosidase activity (Cox et
al., 1993; Zhou and
Schekman, 1999). A library of mutagenized kar2 alleles
(kar2*) was then transformed into this strain and pMR397 was
counterselected on medium containing 5-FOA. After -galactosidase
activity was determined using an agar-overlay (see MATERIALS AND METHODS),
kar2*-containing plasmids from candidate colonies were purified and
their ability to induce the UPR was reconfirmed. Twenty of the retransformed
mutants exhibited UPR activation and were retained for further analysis.
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We next assayed the growth properties of the kar2 mutants at various temperatures. Although each mutant exhibited wild-type growth at 30°C (Figure 2) and 35°C (our unpublished data), several strains were thermosensitive at 37°C (e.g., kar2-51), cryosensitive at 15°C (e.g., kar2-40), or both (e.g., kar2-68). Also shown in Figure 2 is the relative strength of the UPR activation in each strain, although no obvious correlation between growth phenotype and UPR was observed. For example, both kar2-49 and kar2-51 exhibit high UPR activation, whereas only kar2-51 is thermosensitive (Figure 2).
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To assay protein translocation, we examined whether cytoplasmic precursors
of two secreted proteins, prepro--Factor (ppF) and preBiP,
accumulated in each strain at 26°C or after a 5-min shift at 37°C.
ppF is translocated posttranslationally into the ER, whereas BiP uses
both the co- and posttranslational pathways
(Ng et al., 1996). As
shown in Figure 3 the
kar2 mutants displayed varying translocation defects, ranging from no
defect at 26°C and a very mild defect after temperature shift (e.g.,
kar2–51) to a strong block at 26 and 37°C (e.g.,
kar2-40). Interestingly, a band corresponding only to preBiP was
observed in the kar2-65 and kar2-78 mutants; a similar
result was obtained when the molecular mass of BiP was examined in ER-derived
microsomes prepared from these strains by immunoblot analysis (our unpublished
data). This result suggested that the Kar2-78 and Kar2-65 mutant proteins
might possess an uncleaved signal sequence. Indeed, DNA sequence analysis
revealed that both kar2-65 and kar2-78 bear a mutation of
glycine 42 to an aspartic acid at the predicted signal sequence cleavage site
(Rose et al., 1989;
Figure 2).
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To determine whether retention of a signal sequence affected BiP
solubility, we performed sodium carbonate extraction on microsomes obtained
from the wild-type and kar2-01 strains (in which signal sequence
cleavage was apparent; Figure
3) and the kar2-65 and kar2-78 strains.
Wild-type BiP and Kar2-01p were exclusively or predominantly found in the
soluble fraction, as expected; in contrast, Kar2-65p and Kar2-78p were
primarily membrane-associated. Moreover, we noted that 50% of Kar2-65p
and Kar2-78p were sensitive to exogenous protease when microsomes were
prepared from strains expressing these mutant alleles (our unpublished data).
Because a strong ppF translocation defect was noted in kar2-65
and kar2-78 yeast (Figure
3), we suggest that a proportion of the signal sequence-containing
BiP mutant proteins are incompletely translocated or fail to translocate
and/or that BiP activity is reduced when its signal sequence is retained. In
either case, both translocation and retro-translocation (ERAD) would be
compromised and the UPR would be activated.
The kar2-51 Mutant Bears the Same Amino Acid Substitution as the
ERAD-specific kar2-1 Mutant
On the basis of the results presented in Figures
2 and
3, we identified several
mutants that were translocation-defective and might have exhibited an induced
UPR because of further effects on ERAD and/or protein folding. For example,
defects in ERAD, protein folding, and translocation were previously noted for
kar2 mutant strains containing amino acid substitutions in the ATPase
domain (e.g., kar2-113 and kar2-159;
Brodsky et al., 1995;
Simons et al., 1995;
Plemper et al., 1997;
see below). Consistent with this result, DNA sequence analysis uncovered an
A203V mutation in kar2-62 and an A194T mutation in kar2-40
(Figure 2), both of which
affect amino acids in BiP's ATPase domain.
We also identified a few strains that were translocation-proficient at
permissive temperatures but exhibited an increased UPR, two criteria expected
for ERAD-specific kar2 mutants. Several of these candidates possess
>1 amino acid changes as determined by DNA sequence analysis of the inserts
in the corresponding kar2-containing plasmids (our unpublished data).
However, we identified a point mutation of proline 515 to a leucine in
kar2-51, which is the same mutation found on the chromosome in
kar2-1 yeast, a previously identified ERAD-specific mutant
(Brodsky et al.,
1999). To confirm that yeast containing the plasmid-borne
kar2-51 mutation were also ERAD-defective but proficient for
translocation, we performed in vitro translocation and ERAD assays with
microsomes from the wild-type and kar2-51 strains. As a control, we
assayed these processes in microsomes prepared from the kar2-159
strain, which contains a mutation in the ATPase domain, severely affecting the
ability of Kar2-159p to bind ATP, and which consequently is defective for both
ERAD and translocation (Brodsky et
al., 1995, and our unpublished data; see MATERIALS AND
METHODS). We found that the ppF translocation efficiency in microsomes
derived from wild-type and kar2-51 yeast were identical (30%;
Figure 4A), whereas the
translocation efficiency in kar2-159–derived microsomes was
9%. Microsomes obtained from either kar2-51 or kar2-159
yeast exhibited reduced retro-translocation and degradation of unglycosylated
pF, an in vitro ERAD-substrate
(Figure 4B;
McCracken and Brodsky, 1996).
We conclude that kar2-51 is ERAD-defective but
translocation-proficient, thus supporting the premise of our screen.
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For the remainder of this study, we chose to focus on the
kar2-1/kar2-51 mutant and on the kar2-133 mutant, which is
also ERAD-defective but translocation-proficient
(Brodsky et al.,
1999). A further analysis of the other mutants will be reported
elsewhere.
The kar2-1 and kar2-133 Mutations Reside in the
Peptide-binding Domain
DNA sequence analysis of the kar2-1 and kar2-133
mutations indicates that a proline at position 515 (P-470 in DnaK) is
converted to a leucine and a threonine at position 473 (T-428 in DnaK) is
replaced by a phenylalanine, respectively. These residues are highly conserved
among Hsp70s and lie near the tips of loops that connect -sheets and
that in turn forge the platform of the Hsp70 substrate-binding domain
(Figure 5;
Zhu et al., 1996; our
unpublished data). The mutation in kar2-1 is in loop 6, whereas in
kar2-133 the mutation is in loop 3. Notably, the side chain of the
threonine mutated in kar2-133 is predicted to form a hydrogen bond to
the polypeptide backbone at the tip of loop 5,6. Thus, we propose that the
kar2-1 and kar2-133 mutations affect the structure of the
peptide-binding domain similarly.
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Yeast Expressing Kar2-1p and Kar2-133p Induced the Unfolded Protein
Response
The strain used for the screen in which kar2-1 was reisolated
contained kar2 on a single-copy plasmid. To ensure that experiments
were free of potential plasmid copy-number artifacts, subsequent in vivo
experiments were conducted with haploid yeast harboring a chromosomal copy of
kar2-1. The kar2-133 strain and wild-type yeast were
examined in parallel. UPR induction in the kar2-1 and
kar2-133 mutants was confirmed after introduction of a UPR reporter
plasmid (see MATERIALS AND METHODS): The UPR was enhanced 6–7-fold in
the mutant strains compared with wild-type yeast when cells were grown at
26°C and was activated further after a shift to 37°C for 1 h
(Table 1). In accordance with
these data, we observed that the levels of Kar2-1p and Kar2-133p were
2-fold higher than wild-type BiP in microsomes prepared from the
respective strains and that this difference became more pronounced after a
37°C shift.
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The Purified Kar2-1 and Kar2-133 Proteins Are Not Grossly
Mis-folded
To begin to elucidate the molecular basis of the kar2-1 and
kar2-133 ERAD defects we purified wild-type BiP, Kar2-1p, and
Kar2-133p by nickel affinity and ion exchange column chromatography (see
MATERIALS AND METHODS), and then we performed two assays to assess the global
conformation of the mutant proteins. First, CD spectroscopy was performed and
the spectral characteristics for the wild-type and mutant proteins were
identical (our unpublished data). Second, we examined the digestion patterns
of wild-type BiP and Kar2-1p and Kar2-133p after limited proteolysis in the
presence of ATP or ADP and observed that the mutant proteins, like wild-type
BiP, exhibited an ATP-dependent conformational change
(McClellan et al.,
1998; J. Endres and J.L.B., unpublished data); these results are
consistent with the fact that the single turnover and steady-state ATPase
activities of wild-type BiP and Kar2-1p and Kar2-133p are similar (see
MATERIALS AND METHODS; Figures
6 and
7; below).
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Kar2-1p and Kar2-133p Interact Functionally with the J Domains of
Sec63p and Jem1p
The interaction between BiP and the J-domain of Sec63p in the ER is
essential for protein translocation
(Brodsky and Schekman, 1993;
Brodsky et al., 1995;
Lyman and Schekman, 1995;
Corsi and Schekman, 1997;
Matlack et al., 1997;
McClellan et al.,
1998), and a mutation in SEC63 that abrogates this
interaction has been reported to modestly reduce the degradation of a soluble
ERAD substrate (Plemper et al.,
1997). Therefore, to determine if Kar2-1p and Kar2-133p associate
functionally with the J domain of Sec63p, the wild-type and mutant proteins
were incubated with increasing concentrations of a GST-Sec63p-J domain fusion
protein that was used previously to report on the interaction of BiP with
Sec63p (Corsi and Schekman,
1997; McClellan et
al., 1998). We found that the steady state ATPase activities
of wild-type BiP and Kar2-1p and Kar2-133p were enhanced identically in a
concentration-dependent manner by the Sec63p J domain
(Figure 6A). We also assessed
the ability of wild-type, Kar2-1p and Kar2-133p to associate with Sec63p after
purification of the BiP-Sec63p-Sec71p-Sec72p complex from
detergent-solubilized microsomes prepared from wild-type, kar2-1, and
kar2-133 mutant yeast (Brodsky and
Schekman, 1993). After ion exchange (DEAE) and gel filtration
(Super-soe-6) column chromatography, the complex eluted from hydroxylapatite
on a 0.2–0.5 M phosphate gradient and the ratio of BiP to Sec63p that
coeluted was comparable regardless of whether the microsomes derived from the
wild-type or kar2 mutant yeast (our unpublished data).
BiP also associates with two other J domain–containing proteins in
the ER, Scj1p and Jem1p. Deletion of both proteins, but neither one alone,
attenuates ERAD and increases ERAD substrate aggregation, suggesting that
Scj1p and Jem1p function redundantly to facilitate polypeptide
retrotranslocation (Nishikawa et
al., 2001). To measure the interaction of Jem1p with
wild-type and the mutant Kar2 proteins, we incubated increasing concentrations
of a GST-Jem1p-J domain fusion protein and measured steady state ATP
hydrolysis, as above. The results shown in
Figure 6B indicate that the J
domain of Jem1p also activated the ATPase activities of each protein
identically.
Kar2-1p and Kar2-133p Exhibit a Reduced Peptide Affinity and
Compromised Peptide-stimulated ATPase Activity
BiP binds polypeptides in the lumen of the yeast ER to drive protein
translocation (Sanders et al.,
1992; Matlack et al.,
1999), to engineer protein folding
(Te Heesen and Aebi, 1994;
Simons et al., 1995),
and to facilitate ERAD (Gillece et
al., 1999; Nishikawa
et al., 2001). We therefore wished to define whether the
affinities of the wild-type and mutant proteins for a peptide substrate
differed. Using a fluorescein-labeled peptide (F-APPY), increasing
concentrations of the proteins, and measurements of fluorescence anisotropy to
detect F-APPY-BiP complexes (Montgomery
et al., 1999), we found that the peptide affinities for
wild-type BiP (5.1 ± 0.21 µM), Kar2-1p (13.9 ± 0.60 µM),
and Kar2-133p (15.4 ± 0.41 µM) differed by 2.7–3.0-fold
(Figure 7A). Because saturation
was not achieved for the mutant proteins (yeast BiP aggregates in vitro at
concentrations required to achieve saturation; our unpublished data), the
calculated KDs for Kar2-1p and Kar2-133p are likely an
under-estimate of the true KDs. Therefore, the peptide
affinities for the ERAD-defective Kar2 mutants are significantly reduced
relative to wild-type BiP. Furthermore, the peptide affinity for wild-type BiP
is similar to that reported for the cytoplasmic yeast Hsp70, Ssa1p (5
µM; Pfund et al.,
2001), which is 63% identical to Kar2p, supporting the efficacy of
using this experimental method.
Hsp70-peptide interactions are controlled by interdomain coupling between
the ATPase and peptide-binding domains (see for example,
Ha et al., 1997;
Davis et al., 1999).
If the coupling between these domains is compromised, then the regulation of
peptide binding and/or release may be altered and the delivery of an ERAD
substrate to the cytoplasm would be attenuated. To assess interdomain coupling
of wild-type BiP and the Kar2-1 and Kar2-133 proteins, we first needed to
identify a more soluble, but related peptide substrate for yeast BiP. The
peptide ultimately chosen, known as p5, is a shorter derivative of APPY
(Pierpaoli et al.,
1998) and when labeled with the fluorescent reporter, AEDANS bound
to the wild-type and mutant proteins, as measured by fluorescence anisotropy
(our unpublished data). Next, we added saturating concentrations of unlabeled
p5 (700 µM peptide to 0.7 µM BiP) and measured steady state ATP
hydrolysis overtime. Although the endogenous ATPase activities of wild-type
and the mutant Kar2 proteins were similar (Figures
6 and
7B), we observed an
twofold reduced p5-mediated activation of ATP hydrolysis for the mutant
proteins compared with wild-type BiP
(Figure 7B). This second result
suggests that the interdomain coupling between peptide interaction and ATP
hydrolysis is altered to some degree in the Kar2 mutants.
Wild-type BiP Increases the Solubility of an ERAD Substrate
in kar2-1 Microsomes But Exacerbates the ERAD Defect
BiP retains ERAD substrates in an extended conformation as they
retro-translocate from the ER, and defects in BiP function can lead to
substrate aggregation (Nishikawa et
al., 2001). To examine whether the solubility of an ERAD
substrate was reduced in the ER of the kar2 mutants, we measured the
oligomeric state of pF. Microsomes containing radiolabeled pF
were treated with detergent, the extract was centrifuged on a continuous
sucrose gradient, and the amount of pF in each fraction was measured
after SDS-PAGE and phosphorimager analysis
(Nishikawa et al.,
2001). When the sedimentation of pF derived from wild-type
microsomes was examined, we found that 5% of the total pF resided
at the bottom of the gradient, whereas 40% of the pF resided at
the bottom of the gradients when kar2-1– and
kar2-133–derived microsomes were used, respectively
(Figure 8). These data suggest
that the propensity of pF to aggregate in the ER of kar2-1
yeast is enhanced, an effect that would hinder retro-translocation. We then
examined pF solubility in extracts prepared from kar2-1 mutant
yeast that had been transformed with a wild-type KAR2 expression
vector (pMR109) and observed that pF was now largely absent from the
bottom of the gradient and that the percentage of soluble pF (fractions
2–5) increased.
|
To explore whether increasing pF solubility enhanced its
degradation, we performed the in vitro ERAD assay using microsomes from the
wild-type and kar2-1 strains containing either pMR109 or a vector
control. The concentration of BiP was greater in microsomes prepared from the
wild-type and kar2-1 strains containing pMR109 compared with yeast
harboring the vector control: BiP levels were elevated by 50% when pMR109
was present in the wild-type strain and by 90% when the same plasmid was
present in the kar2-1 strain as assessed by quantitative immunoblot
analysis (our unpublished data). We then observed that microsomes from the
kar2-1 strain degraded pF less efficiently than microsomes
from the isogenic wild-type strain, as shown previously
(Brodsky et al., 1999;
also see Figure 4B).
Surprisingly, ERAD was inhibited by 40–50% in microsomes prepared from
either strain containing the wild-type BiP expression vector compared with the
vector control (Figure 9). This
result suggests that the kar2-1 mutation may be dominant with respect
to ERAD because the ERAD defect was not rescued by expression of wild-type
protein; however, it is formally possible that wild-type BiP failed to rescue
the ERAD defect in kar2-1–derived microsomes because the amount
of BiP may be too high in this system. Regardless, these data show that
increasing the amount of BiP augments polypeptide solubility in a
kar2 mutant but is insufficient to restore ERAD to wild-type levels.
Furthermore, increasing the amount of BiP in microsomes prepared from
wild-type cells inhibited ERAD. A model for these observations is presented
below.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The position of the mutated residue in Kar2-1p suggested
that another previously isolated kar2 mutant, known as
kar2-133, would exhibit similar properties. Indeed, kar2-1
and kar2-133 yeast are defective for the ERAD of three soluble
substrates (CPY*: Zhang et al.,
2001; pF and AiPiZ:
Brodsky et al., 1999),
and we report here that expression of Kar2-1p or Kar2-133p induces the UPR and
leads to the aggregation of one of these substrates. Furthermore, the Kar2-1
and Kar2-133 proteins exhibit reduced peptide affinities compared with
wild-type BiP, and the addition of peptide fails to enhance ATP hydrolysis by
the mutant proteins to the same degree as for wild-type BiP. This effect,
although partial, most likely results from interdomain communication defects
in the mutant proteins because an equal degree of J domain–stimulated
ATPase activity was observed for Kar2-1/133p and wild-type BiP. Although we
cannot formally exclude the possibility that the reduced peptide affinity of
the mutants was the cause of lower peptide-stimulated ATPase activity, we note
that the concentration of peptide used in this latter experiment was 1000-fold
greater than the concentration of BiP, and—if the affinities of BiP for
APPY and p5 are comparable—was >40-fold higher than the predicted
KD.
In light of these data, why are Kar2-1p– and
Kar2-133p–expressing cells and microsomes derived from these strains
translocation-proficient? The ability of BiP to bind a translocating
polypeptide is essential to drive protein import, whether through its action
as a "motor" or as a "ratchet"
(Sanders et al.,
1992; Matlack et al.,
1999). However, it was previously noted by Rapoport and colleagues
that 6–7 BiPs bind ppF, and a BiP mutant lacking the peptide
binding domain "lid"—and that exhibits a faster off-rate for
peptide when ADP-bound (Misselwitz et
al., 1998)—supported ratcheting, but higher
concentrations of the protein were required
(Matlack et al.,
1999). Thus, a BiP mutant with a lower peptide affinity should
catalyze protein translocation as long as greater amounts of the protein are
present. In fact, this may be the case in kar2-1 and
kar2-133 yeast and in Kar2-1/133p-containing microsomes
(Table 1). We further note that
the positions of the kar2-1 and kar2-133 mutations are
likely to weaken interactions between the lid and the loops in the
peptide-binding domain, resulting in proteins with similar properties as the
"lid-less" BiP mutants used by Matlack et al.
(1999); as expected, Hsc70
mutants with predicted weakened interactions between the loops and the lid
exhibit an increase in peptide off-rates
(Hu et al., 2002; see
below). In contrast, a BiP mutant in which peptide binding is completely
abolished should be lethal and would not be isolated in our screen: Mutations
in DnaK that significantly reduce the chaperone's peptide affinity cannot
complement the dnaK phenotype (for example, see
Burkholder et al.,
1996). Therefore, we propose that kar2-1 and
kar2-133 yeast are viable and translocation-proficient because they
can balance the reduced affinity between BiP and peptides by synthesizing
greater amounts of BiP via the UPR.
To explain the fact that kar2-1 and kar2-133 cells and
microsomes obtained from these strains are ERAD-defective, we note that
pF aggregates in kar2-1– and
kar2-133–derived microsomes
(Figure 8), a phenomenon that
would preclude retro-translocation through the pore formed by the Sec61p
complex in the ER membrane. We suggest further that nucleotide and/or
cochaperone-mediated pF release from Kar2-1/133p is altered. For
example, if pF is released prematurely, then the polypeptide may
aggregate before it can be retro-translocated. Several lines of evidence
support this view. First, expression of wild-type BiP restores pF
solubility (Figure 8),
suggesting that it binds Kar2-1/133p–released polypeptides. Second,
mutations in bovine Hsc70 in which interdomain coupling is altered exhibit an
increase in peptide off-rate (Ha et
al., 1997). Third, conversion of glycine-468 to aspartic acid
in DnaK was proposed to perturb the conformation of loop 5,6 in the
peptide-binding domain, and when combined with a second mutation (G455D), the
peptide off-rate increased (Buchberger
et al., 1999). Based on their positions
(Figure 5), the kar2-1
and kar2-133 gene products may exhibit a similar kinetic defect;
however, the contribution of single mutations, ATP, and/or Hsc70/DnaK
cochaperones on peptide release was not examined in these other studies. Thus,
it will be vital in future work to assess the peptide affinities and on- and
off-rates for wild-type BiP and Kar2-1/133p in the presence and absence of
each of the growing number of known BiP cochaperones, which include activators
of ATP hydrolysis (e.g., Sec63p, Scj1p, and Jem1p) and nucleotide exchange
factors (reviewed in Fewell et
al., 2001); for example, the Sls1p nucleotide exchange factor
catalyzes nucleotide release from yeast BiP, and the introduction of the
kar2-1 and kar2-133 alleles into sls1 yeast
results in synthetic effects on cell growth
(Kabani et al.,
2000b).
Another, nonmutually exclusive scenario is that the Kar2-1p– and
Kar2-133p–mediated ERAD defects arise at least in part from defective
"gating" of the translocation channel during retro-translocation.
Experiments from Johnson and colleagues
(Hamman et al., 1998;
Haigh and Johnson, 2002)
indicate that BiP helps seal the translocation channel in the mammalian ER
during translocation, but it is not clear how BiP facilitates channel
reopening during retro-translocation
(Johnson and Haigh, 2000).
Therefore, it is formally possible that the Kar2p mutants are able to gate the
translocon during protein import, but cannot do so during ERAD; this
hypothesis is currently being examined. BiP-dependent gating might also
require the participation of cochaperones, and the differential interaction
between Kar2-1p and translocation/retro-translocation-specific factors might
also explain why kar2-1 yeast are selectively defective for ERAD.
Finally, as noted above, we have shown that increasing the amount of BiP in
the ER does not facilitate ERAD in wild-type yeast but instead slows
retro-translocation and degradation. In the kar2-1 background, the
introduction of wild-type BiP helps resolubilize pF but ERAD efficiency
is also reduced. These data may be explained by the fact that prevention of
polypeptide aggregation requires only BiP binding, whereas ERAD requires both
polypeptide binding and release, which might be regulated by cochaperones.
PF might have become bound to and trapped in the ER by exaggerated
amounts of BiP (Figure 9), but
the concentrations and/or activities of cochaperones required for release
might not have risen in parallel. Consistent with our work, increasing the
level of BiP slows the ERAD of mutant ribophorin in mammalian cells
(deVirgilio et al.,
1999) and the concept that ERAD can proceed only after BiP release
is well established (Knittler et
al., 1995; Beggah et
al., 1996; Skowronek
et al., 1998;
Chillaron and Haas, 2000). It
is possible that under conditions of ER stress, when the concentration of BiP
rises via the UPR, ERAD efficiency may decline, but the solubility of putative
ERAD substrates is maintained. In turn, if ER stress is removed and/or the
level of BiP can return to that in the unstressed state, BiP-bound,
aggregation-prone substrates may be given a "second-chance" to
fold or may then be targeted for degradation. To test this hypothesis, it will
be important in the future to correlate how different levels of ER stress
affect the concentration of BiP, ERAD efficiency, and the solubility of ERAD
substrates.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Present address: Laboratoire de Physiogenomique, Service de Biochemie et de
Genetique Moleculaire, CEA/Saclay, Gif-sur-Yvette, France. 
¶ Corresponding author. E-mail address: jbrodsky{at}pitt.edu.
|
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), Kar2-1p
(
), and Kar2-133p (
) was measured using 5 µg of BiP and either
the (A) GST-Sec63p-J or (B) GST-Jem1p-J fusion proteins. ATPase activity is
plotted as nmol of ATP hydrolyzed/min/mg BiP vs. the molar ratio of the J
domain–containing fusion proteins to BiP.
), and Kar2-133p (
) cells, and kar2-1 yeast
transformed with a KAR2 overexpression plasmid (pMR109; 