From the Department of Biological Sciences,
University of Pittsburgh, Pittsburgh, Pennsylvania 15260 and the
¶ Department of Biology, University of Nevada,
Reno, Nevada 89557
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ABSTRACT |
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Polypeptide import into the yeast endoplasmic
reticulum (ER) requires two hsp70s, Ssa1p in the cytosol and BiP
(Kar2p) in the ER lumen. After import, aberrant polypeptides may be
exported to the cytoplasm for degradation by the proteasome, and
defects in the ER chaperone calnexin (Cne1p) compromise their
degradation. Both import and export require BiP and the Sec61p
translocation complex, suggesting that import and export may be
mechanistically related. We now show that the cne1 Results from recent studies have established the existence of an
intracellular protein degradation process that removes aberrant and
unassembled proteins from the endoplasmic reticulum
(ER)1 (reviewed in Refs.
1-3). ER-associated protein degradation (ERAD) operates as an
essential step in the quality control of newly synthesized proteins by
selectively degrading aberrant and unassembled secretory proteins that
accumulate within the ER. It is now clear that the degradation of many
soluble (4, 5) and integral membrane (6-17) proteins in the ER is
mediated by the cytoplasmic proteasome, indicating that protein export
from the ER to the cytosol must occur. The energy source required to
drive these proteins from the ER, however, remains unknown, but it has
been proposed to derive from the action of cytosolic molecular
chaperones (1).
Several studies suggest that the protein translocation channel,
composed of the Sec61p complex, mediates polypeptide extrusion from the
ER. First, CPY*, a mutated form of vacuole-targeted carboxypeptidase Y,
was stabilized in yeast strains containing a mutation in
SEC61 (18). Second, the degradation of an unglycosylated
form of the yeast mating pheromone, pro- A vital aspect of ERAD is its selectivity for specific soluble and
integral membrane proteins, whereas the majority of ER resident and
secreted proteins are stable (20, 21). Although the nature of this
substrate selectivity is not completely clear, structural motifs that
are buried when proteins are correctly folded and assembled may be
recognized by the ERAD machinery if protein folding is hindered
(22-24). Two ER lumenal molecular chaperones, BiP and calnexin, are
known to be involved in protein maturation and "quality control" in
the ER and are likely candidates to play a role in ERAD.
Calnexin is a lectin that binds transiently to monoglucosylated glycans
and retains unfolded polypeptides in the ER lumen (25, 26); therefore,
calnexin could target misfolded proteins for ERAD. Support for this
hypothesis derives from the observation that some mutant proteins in
the mammalian ER bind to calnexin prior to their degradation (12, 27,
28) and that partially inhibiting the interaction between calnexin and
ApoB attenuated the ER-associated degradation of ApoB ~2-fold (29).
In addition, we discovered that ER-derived microsomes prepared from a
yeast strain deleted for calnexin were compromised in their ability to
degrade p BiP, an hsp70 homologue, binds preferentially to hydrophobic arrays of
amino acids (31, 32), suggesting that BiP can recognize unfolded
proteins. BiP is also required for protein translocation into the ER
lumen in yeast, acting as a ratchet to prevent polypeptide egress from
the ER, as a motor to actively drive substrates into the ER, and/or in
conjunction with other proteins to regulate the translocation machinery
(reviewed in Ref. 33). Thus, BiP might promote polypeptide efflux
during ERAD.
Several investigators have uncovered a role for BiP during ERAD. First,
iodination of a misfolded proinsulin polypeptide in vivo
indicated co-localized signals for BiP binding and ERAD (24). Second,
the dissociation of BiP from unassembled Ig chains correlated with the
kinetics of Ig degradation (34). Third, Skowronek et al.
(35) recently determined that the accelerated degradation of
immunoglobulin light chain chimeras corresponded to enhanced BiP
binding. And fourth, Plemper et al. (18) observed the
inefficient degradation of CPY* in yeast containing a
temperature-sensitive mutation in the gene encoding BiP,
KAR2.
Because the kar2 mutant used by Plemper et al.
(18) to examine ERAD in yeast is also defective for protein
translocation into the ER (36, 37), BiP may have been performing the
same function during polypeptide export as in import. Therefore, to determine whether the function of BiP during polypeptide export and
degradation is more elaborate than its role during translocation, we
measured ERAD both in vitro and in vivo using
kar2 mutants that are translocation-proficient, as well as
the kar2 mutant used previously (18). Our data demonstrate
that each of these mutants is defective for ERAD, indicating for the
first time that BiP action during protein translocation into the ER and
polypeptide efflux from the ER differs.
Another hsp70 molecular chaperone, Ssa1p, is a cytosolic protein known
to function in polypeptide import into the yeast ER (38, 39). To
investigate whether Ssa1p was required to drive ERAD substrates from
the ER, we examined ERAD in vitro and in vivo
using strains either containing a temperature-sensitive mutation in
SSA1 (38) or in which the levels of Ssa1p can be regulated (39, 40); however, we observed that ERAD activity was unaffected. We
conclude from these combined results that BiP and Ssa1p play distinct
roles during protein import and export and that the molecular mechanisms of polypeptide import and export from the yeast ER must differ.
Yeast Strains and Growth Conditions--
RSY607
(MAT
To assay for a synthetic interaction between KAR2 and
CNE1, the calnexin-deleted strain (see above) (41) was mated
with MS111 and MS193, diploids were selected, and tetrad analysis was performed using standard methods (44). The genotype of the progeny was
assessed by replica-plating onto omission media.
Translocation and ERAD Assays--
The translocation of a form
of prepro- Purification of Ssa1p, Preparation of Cell Lysates, and
Immunoblotting--
Ssa1p was purified from strain MW141 as described
(45). Cell lysates were prepared from a total of 2.0 A600 units of exponentially growing cells that
were pelleted and washed once with 10 mM NaN3 before the cell pellet was frozen at Luciferase Assay--
The ability of an hsp110 chaperone to
retain heat-denatured firefly luciferase in a folding-competent
conformation was assayed as described (47) with minor modifications.
Briefly, firefly luciferase was incubated in the presence of the yeast
hsp110 chaperone, Sse1p, at a 1:5 molar ratio and the mixture was
incubated at 42 °C for 30 min. An ATP-regenerating system (37) and
yeast cytosol from the desired strain was added to a final
concentration of 4 mg/ml, and the reaction was incubated at 26 °C.
Aliquots at the indicated time points were assayed for luciferase
activity in an Analytical Luminescence Laboratory (Ann Arbor, MI)
Monolight luminometer using luciferin as the substrate. Although the
refolding of luciferase in this assay requires cytosol, purified
cytosolic hsp70 and a cognate DnaJ chaperone can substitute for the
cytosolic requirement (47), indicating that the chaperone activity of an hsp70 may be assayed using this procedure. Sse1p was purified from
yeast over-expressing a hexa-histidine tagged form of Sse1p using
nickel agarose affinity and ion exchange
chromatography,2 and yeast
cytosol was prepared as described previously (30).
The Genes Encoding BiP and Calnexin Interact--
We previously
observed that ERAD was inhibited by ~50% when assayed in yeast
microsomes prepared from a strain deleted for calnexin
(cne1) (30). Multichaperone complexes containing BiP and
calnexin have been noted in the mammalian ER (48), and BiP and calnexin
co-immunoprecipitate in extracts prepared from the yeast
Schizosaccharomyces
pombe.3 Therefore, we
sought genetic interactions between the cne1 mutation and
mutations in BiP (kar2-1 and kar2-133) that may
prevent the ability of BiP to chaperone protein folding in the ER (43,
49). To this end, we mated the cne1 deleted strain (41) to
kar2-1 and kar2-133 and dissected asci after
sporulation of the diploids. Tetratype tetrads with respect to
CNE1 and KAR2 were then identified. At least two
representative tetratypes from each cross were analyzed, replated, and
grown at 30, 35, or 37 °C. As shown in Fig.
1, A and B, we
observed that the kar2-1cne1 BiP Is Required for ERAD in Vitro and in Vivo--
To examine the
role of BiP in ERAD, we prepared ER-derived microsomes from an isogenic
wild type strain and three temperature-sensitive kar2
strains, kar2-1, kar2-113, and
kar2-133. The kar2-113 mutation converts a
phenylalanine at position 196 to a leucine in the ATPase domain of BiP
(42), obstructs the import of polypeptides at 37 °C by inhibiting
the association of translocating proteins with Sec61p (36), and
compromises the degradation of an ERAD substrate, CPY*, in
vivo (18). Interestingly, Kar2-113p exhibits wild type ATPase
activity but reduces the levels of both co- and posttranslational
protein translocation into ER-derived microsomes (37). These results,
and others, suggest that BiP may regulate the translocation machinery
(33), a function likely to hinder ERAD because degraded substrates must
be exported from the ER through the Sec61p complex (9, 18, 19). By
comparison, the kar2-1 and kar2-133 mutations
are in the peptide binding domain of BiP (kar2-1, P515L;
kar2-133, T473F) (42), and protein translocation is
unaffected, as determined by examining whether two ER-targeted substrates (pp
To test this hypothesis, we translocated radiolabeled
To determine whether the mutations in KAR2 prevented p
To observe whether these mutations also affected the degradation of an
ERAD substrate in vivo, the stability of A1PiZ was monitored
by pulse-chase protein radiolabeling in the kar2-1, kar2-113, and kar2-133 mutants. We had
previously shown that A1PiZ, a mutant form of the mammalian
One explanation for the recovery of ERAD activity after 90 min at
37 °C is that elevated levels of mutant BiP could compensate for its
compromised activity; for example, Scidmore et al. (51) determined that the presence of a kar2 mutation induces the
expression of its own message. To observe whether BiP was induced
during these in vivo ERAD reactions, we measured the
relative amounts of BiP in each strain cultured at both the permissive
(23 °C) and nonpermissive (37 °C) temperatures, and we detected a
time- and temperature-dependent increase in the wild type
and mutant forms of BiP (Fig. 5).
Although it is possible that other compensatory mechanisms restore ERAD
in the kar2 mutants (see under "Discussion"), the
simplest explanation is that elevated levels of Kar2-1p, Kar2-113p, and Kar2-133p alleviate their respective ERAD defects in
vivo.
To determine whether the recovery of ERAD could be recapitulated
in vitro, the degradation of p Ssa1p Is Not Required for ERAD in Vitro or in Vivo--
If soluble
ERAD substrates are exported to the cytoplasm for degradation, there
must be a driving force to expel these polypeptides. Because BiP has
been proposed to drive polypeptides into the ER lumen by acting as a
ratchet or a motor (reviewed in Ref. 33), one might imagine that a BiP
homologue in the cytosol may similarly pull or ratchet polypeptides
from the ER (1). In fact, one such homologue is Ssa1p, the hsp70 that
is required for polypeptide import into the ER (38, 39). Cytosolic
hsp70s may also be required for ERAD by retaining a polypeptide in an
unfolded conformation or by actively unfolding the polypeptide (52) and
ushering it into the proteasome: some polypeptides can be degraded in
the absence of ubiquitination (1, 5, 15) and thus must be unfolded
and/or targeted into the proteasome by an alternate mechanism. In
addition, the degradation of polypeptides in bacteria requires specific
chaperones that catalyze protein unfolding (53). By analogy, a
chaperone activity in eucaryotes may be necessary for degradation
because only unfolded proteins can interact with the catalytic
core of the proteasome (54), some of which may not be ubiquitinated (1,
5, 15).
To determine whether Ssa1/2p was required for ERAD, we used a wild type
SSA1 strain in which the homologous ssa2,
ssa3, and ssa4 genes contained insertion
mutations and an ssa1 temperature-sensitive strain (known as
ssa1-45) in which the ssa2, ssa3, and
ssa4 genes were similarly inactivated (38). Craig and
co-workers (38) had previously shown that the translocation of pp
By performing quantitative immunoblotting, we determined that Ssa1p
comprises ~3% of the total protein in our cytosol preparations (data
not shown). Because Ssa1p has been observed to associate with pp
One concern was that the ssa1-45 cytosol used for these
studies may not exhibit chaperone-dependent defects
in vitro. To examine whether this was the case, we utilized
an established assay in which the ability of cytosolic chaperones to
refold heat-denatured firefly luciferase is measured (47). Because
Ssa1p has been shown to directly facilitate the folding of firefly
luciferase in vitro (56, 57), we chose to examine whether
the ssa1-45 cytosols and the wild type cytosol could
support heat-denatured luciferase refolding. In these studies, the
denatured material must be "held" in a folding competent
conformation until the cytosol is added (47). Because mammalian Hsp110
has previously been shown to fill this role, we used purified Sse1p, a
yeast hsp110 homologue.2 As shown in Fig.
7, we discovered that the
ssa1-45 cytosols were largely unable to refold luciferase,
whereas the refolding activity in wild type cytosol was intact.
Interestingly, the ssa1-45 cytosols were defective for this
activity regardless of whether they had been prepared from cells
shifted to 37 °C; similar results in which in vitro
defects of temperature-sensitive mutants are more severe than those
observed in vivo have been observed previously (37).
Nevertheless, these results indicate that the ssa1-45 mutant cytosols are defective for a known chaperone activity, and
combined with the data presented above, they indicate that this
activity is not required for ERAD.
To confirm that ERAD was Ssa1p-independent, Ssa1p-depleted cytosol was
prepared from the MW141 strain. MW141 lacks the SSA1, SSA2, and SSA4 genes encoding the three major
cytosolic hsp70s but carries the SSA1 gene on a
galactose-regulated promoter (39, 40). Whereas growth of this strain
for 8-10 h on 2% glucose leads to a 2-fold decrease in the amount of
Ssa1p and a strong defect in posttranslational protein translocation
(39), growth of the strain on a 2% galactose/0.2% glucose mixture
yields cytosol containing wild type levels of Ssa1p as determined by
quantitative immunoblotting.4 Thus, we prepared cytosol
from MW141 grown under each condition to use in an in vitro
ERAD assay. When we measured p
It was possible in these in vitro studies that wild type
Ssa1/2p may have contaminated the microsomes used to assay the
ssa1-45 cytosols, a supposition we confirmed by
immunoblotting the microsomes with an Ssa1p-specific antibody (Fig.
8). If Ssa1/2p acts catalytically during
ERAD, even low concentrations of membrane-bound wild type chaperone may
have masked an ssa1 mutant effect on ERAD. Thus, we prepared
microsomes from the ssa1 temperature-sensitive strain grown
exclusively at the permissive temperature or shifted to 37 °C for 45 min. Because the translocation of yeast lysate-synthesized pp
As shown above, mutations in KAR2 prevent export of p
Finally, to confirm further that ERAD was Ssa1p-independent and to
determine whether Ssa1p was required for proficient ERAD in
vivo, we measured the degradation of A1PiZ in the wild type and
ssa1-45 strain at both the permissive temperature and
during a 37 °C incubation. As shown in Fig.
9, we noted that the rate of A1PiZ
degradation over a 90-min chase was similar in both strains when
monitored at 23 or 37 °C.
The experiments reported here demonstrate that mutations in the
gene encoding BiP inhibit the degradation of p We also show that mutations in KAR2 prevent the efficient
degradation of A1PiZ in vivo and that the defect in A1PiZ
degradation at the nonpermissive temperature can be rescued over time
(Fig. 4). We suggest that this rescue may arise from the induction of BiP during the incubation (Fig. 5). We do not discount the alternative explanation that increased expression of other chaperones may rescue
this defect, as incubating yeast at higher temperatures or expressing a
misfolded secretory protein (such as A1PiZ) may increase the synthesis
of some ER chaperones by the heat shock and unfolded protein stress
responses, respectively. This response is not limited to yeast:
hepatocytes from some individuals homozygous for A1PiZ and with chronic
liver disease cannot degrade A1PiZ and induce the synthesis of BiP, the
stress protein SP90, and ubiquitin (59, 60). In contrast, these
proteins are not induced in heterozygous individuals or in those
homozygous for A1PiZ but who exhibit normal liver function (59). This
result indicates that when ERAD efficiently removes A1PiZ, no
stress response is triggered.
We previously suggested that a cytosolic molecular chaperone may drive
ERAD substrates from the ER by acting as a ratchet or molecular motor
(reviewed in Ref. 1), and a likely candidate for this factor was
Ssa1/2p because: 1) BiP performs this role during translocation into
the ER (reviewed in Ref. 33) and BiP and Ssa1p are >60% identical;
and 2) Ssa1p is required to chaperone proteins into the ER (38, 39, 45,
55) and may unfold native proteins (52), and ERAD substrates may
require this activity when delivered to the proteasome. In addition,
cytosolic hsc70s in mammals are required for ERAD (see below). To our
surprise, however, we discovered that ERAD is Ssa1p-independent. Thus,
it is uncertain what protein drives substrates such as p We suggest four scenarios by which ERAD substrates may be driven from
the ER. First, unidentified cytosolic chaperones may associate with
polypeptides as they emerge from the translocation channel and export
them from the ER, acting either as a ratchet, or In contrast to the results presented here, a requirement for cytosolic
hsp70s in the proteasome-mediated degradation of some mammalian
proteins has been obtained (14, 63). In one of these cases, the hsp70
cognate protein, hsc70, was required for the conjugation of ubiquitin
onto protein substrates (63). Ssa1p may not be required in our system
because we failed to note ubiquitination of p Finally, the action of BiP and calnexin during ERAD remains to be fully
elucidated. Both calnexin (30) and BiP (this report and Ref. 18) are
required for maximal ERAD. Molecular chaperone complexes exist in the
ER of mammalian cells (48) and S. pombe,3 and
Helenius and colleagues (64) have recently suggested that BiP and
calnexin may act together to ensure that proteins fold properly in the
S. cerevisiae ER. To directly test this hypothesis, they
attempted to create a haploid yeast strain containing both the
kar2-127 mutation and a CNE1 deletion but were
unsuccessful, suggesting that the combination of the two mutations was
lethal and that they might function in the same pathway. Our
observation that a strain with the kar2 and cne1
mutations show a synthetic interaction at 35 °C (Fig. 1) supports
their hypothesis. One possibility is that misfolded proteins in the
Sec61p translocation channel must be released from ER chaperone
complexes prior to retro-translocation. In this case, the
kar2 defects that compromise ERAD may derive from the
inability of the mutant chaperone to 1) release the misfolded protein,
2) maintain the polypeptide in a transport-competent conformation, 3)
interact with calnexin, or 4) gate the Sec61p channel. Of note, we
failed to detect a more pronounced ERAD defect in microsomes prepared
from the cne1 and
two kar2 mutant alleles exhibit a synthetic interaction and
that the export and degradation of pro-
factor is defective in
kar2 mutant microsomes. Pulse-chase analysis indicates that
A1PiZ, another substrate for degradation, is stabilized in the
kar2 strains at the restrictive temperature. Because two of
the kar2 mutants examined are proficient for polypeptide import, the roles of BiP during ER protein export and import differ, indicating that these processes must be mechanistically distinct. To
examine whether Ssa1p drives polypeptides from the ER and is also
required for degradation, we assembled reactions using strains either
containing a mutation in SSA1 or in which the level of Ssa1p could be regulated. We found that pro-
factor and A1PiZ were
degraded normally, indicating further that import and export are
distinct and that other cytosolic factors may pull polypeptides from
the ER.
INTRODUCTION
Top
Abstract
Introduction
References
factor (p
F), was
inhibited in microsomes prepared from sec61 mutant strains
(19). And third, ERAD substrates were co-immunoprecipitated with the
mammalian Sec61p homologue en route to degradation (9,
17).
F in vitro (30).
EXPERIMENTAL PROCEDURES
leu2-3, 112 ura3-52
PEP4::URA3); W303-calnexin delete (MATa
ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3-1
cne1::LEU2) (41); MS10 (MATa ade2-101
leu2-3, 112 ura3-52), MS543 (MAT
ade2-101
trp1-
1 ura3-52 kar2-113), MS111 (MATa
ade2-101 leu2-3, 112 ura3-52 kar2-1), and MS193 (MATa ade2-101 leu2-3, 112 ura3-52 kar2-133)
(36, 37, 42, 43); MW141 (MAT
his3-11, 15 leu2-3,
112 trp1-
1 ura3-52 ssa1::HIS3 ssa2::LEU2
ssa4::URA3 pGAL-SSA1-TRP1) (39); JN516 (MAT
his3-11, 15 leu2-3, 112 lys2 ura3-52 trp1-
1
SSA1 ssa2-1 ssa3-1 ssa4-2), and a1-45
U (MAT
his3-11, 15 leu2-3, 112 lys2 ura3-52 trp1-
1 ssa1-45BKD ssa2-1
ssa3-1 ssa4-2) (38); cells containing ssa1-45BKD are
temperature-sensitive for growth and protein translocation (38).
Strains were grown in YPD (1% yeast extract, 2% peptone, 2%
dextrose) at 25-30 °C unless a temperature-sensitive mutant
phenotype was desired, in which case half of each log phase culture
(A600 = 0.5-1.0) was incubated with shaking at
37 °C for 45 min. MW141 was grown in YP-2% galactose/0.2% glucose
(wild type) or the log phase culture was harvested, washed once with water, and resuspended in YP-2% glucose to deplete cellular Ssa1p (Ssa1p-depleted), as described (39).
factor (pp
F) in which the three core glycosylation
consensus sites have been mutated (
Gpp
F) has been described (30).
In brief, ER-derived microsomes are incubated in the presence of an
ATP-regenerating system and gel-purified, in
vitro-translated 35S-labeled pp
F (37). The
translocation of pp
F is apparent by the generation of a signal
sequence-cleaved product, p
F, that is protected from exogenous
protease. Translocation is ATP-, time-, and
temperature-dependent and is linear for up to 40 min.
In vitro ERAD reactions to measure the degradation of p
F
were then assembled as described (5, 30). Microsomes containing p
F
were pelleted, washed once, and resuspended in a "chase" reaction
containing an ATP-regenerating system and cytosol from the desired
yeast strain. At the indicated time points, aliquots were removed, the proteins were precipitated with trichloroacetic acid, and the products
were resolved by 18% SDS-polyacrylamide gel electrophoresis/urea gels
(30). The degradation of p
F in these reactions is also ATP-, time-,
and temperature-dependent, but it requires the additional presence of yeast cytosol. The export of p
F from microsomes was detected by trypsin-treatment of ERAD reactions after incubating microsomes in the presence of cytosol and ATP, as described (5). Pulse-chase analysis to measure the in vivo degradation of
A1PiZ was performed as previously published (5).
20 °C and thawed on ice. A
20-µl aliquot of sample buffer (80 mM Tris, pH 6.8, 2%
SDS, 0.1% bromphenol blue, 100 mM dithiothreitol, and 10%
glycerol) was then added, and the samples were incubated at 95 °C
for 2 min. A total of 0.12 g of glass beads (Sigma G-9268) was
added, the cells were disrupted by agitation on a vortex mixer for 1 min, and 80µl of sample buffer was added before aliquots were reheated and applied to SDS-polyacrylamide gels. After the transfer of
the electrophoresed proteins to nitrocellulose filters, antibodies against yeast BiP (45, 46) and Ssa1p (45) were used to probe the cell
lysates and horseradish peroxidase-conjugated anti-rabbit IgG or
125I-protein A (Amersham) was used to detect the primary
antibodies. The resulting blots were washed, and then exposed and
processed either by phosphorimager analysis (when
125I-protein A was used) or according to the
manufacturer's specifications (when enhanced chemiluminescence
detection was desired).
RESULTS
and
kar2-133cne1
strains grew less well than the
corresponding kar2-1 and kar2-133 strains at
the semipermissive temperature of 35 °C, indicating a mild synthetic
interaction between the kar2 and the cne1
mutations. To confirm this result, we performed growth curves at
35 °C of each strain obtained from the kar2 × cne1
crosses (Fig. 1, C and D). We
noted again that the kar2 mutants grew somewhat better than
the kar2cne1
double mutant strains at this temperature. These results suggest that BiP and calnexin may act in a common pathway
in S. cerevisiae.
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Fig. 1.
KAR2 and CNE1
synthetically interact. Colonies derived from tetratype
spores in crosses between the indicated kar2 mutants and the
cne1 deleted strain were obtained, re-streaked, and then
incubated at the indicated temperatures on YPD medium for 2-3 days.
A, tetratype progeny from a cross between kar2-1
and cne1 ; B, tetratype progeny from a cross
between kar2-133 and cne1
. Growth curves from
single colonies for each cross in A and B are
shown in C and D, respectively. Saturated
cultures were diluted into YPD medium and grown with shaking at
35 °C. Open circles, wild type; closed
circles, cne1
; open triangles,
kar2; closed triangles,
kar2cne1
.
F and preinvertase) accumulate in vivo at
the nonpermissive temperature (42) and by examining whether the
posttranslational translocation of an ER-targeted substrate (pp
F)
(37) was compromised in vitro (see
below);4 however, these
mutations may affect the folding of specific proteins in the yeast ER
(43, 49). We anticipated that defects in the ability of BiP to
chaperone protein folding may also obstruct ERAD.
Gpp
F into
microsomes prepared from each mutant strain and an isogenic wild type
strain.
Gpp
F is a form of the yeast mating pheromone precursor,
pp
F, that cannot become glycosylated after signal sequence cleavage.
The resulting product, p
F, is a substrate for ERAD in
vitro when a posttranslocation chase is performed in the presence
of wild type cytosol and hydrolyzable ATP (5, 19, 30). We found that
the extent of
Gpp
F translocated into kar2-1 and
kar2-133 mutant microsomes and wild type microsomes was
similar, whereas translocation in the kar2-113 microsomes was ~42% as efficient as in wild type microsomes (data not shown), in accordance with previous data (36, 37, 42). In a subsequent ERAD
assay, we observed that the degradation of p
F was slower in each of
the mutants at both permissive (23 °C) and nonpermissive (37 °C)
temperatures and that the defect was more pronounced at the
nonpermissive temperature (Fig. 2). At
23 °C, ~64% of p
F was degraded in wild type microsomes during
the 40-min chase incubation, whereas only 45, 34, and 45% was degraded
in microsomes derived from kar2-1, kar2-113,
and kar2-133, respectively, at the 40-min time point. At
37 °C, whereas 64% of p
F was degraded in wild type microsomes,
only 36, 22, and 41% was degraded in microsomes derived from
kar2-1, kar2-113, and kar2-133,
respectively. These results suggest that Kar2p may be a component of
the recognition and/or export machinery required for the degradation of
p
F, as was suggested previously for CPY* (18).
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Fig. 2.
Defects in Kar2p inhibit the in
vitro degradation of unglycosylated
p F. Radiolabeled
Gpp
F was
translocated into ER-derived microsomes prepared from
kar2-1, kar-113, kar2-133, and a
wild type strain grown at the permissive temperature. Posttranslocation
chase reactions were performed as described under "Experimental
Procedures" in the presence of wild type cytosol at either 23 °C
(squares) or 37 °C (triangles), and the
products of the chase samples, collected at 0, 10, 20, and 40 min, were
precipitated with trichloroacetic acid and resolved by SDS-18%/4
M urea polyacrylamide gel electrophoresis. Relative amounts
of p
F were measured using the Bio-Rad Phosphor Analyses program.
Reactions using wild type microsomes (circles) exhibited
identical levels of ERAD at 23 and 37 °C.
F
export from the ER-derived microsomes, a protease protection analysis was performed (5). In this assay, exported p
F is degraded by trypsin
while p
F sequestered inside the microsomes remains shielded. When
trypsin accessibility was examined in ERAD reactions at 23 and 37 °C
using wild type microsomes, we found that the amount of p
F decreased
from 92 to 54% over a 40-min chase, in agreement with previous results
and indicating export of 46% of the p
F to the cytosol (5) (Fig.
3). On the contrary, the amount of
trypsin-sensitive p
F was low and remained constant in the kar2 microsomes; only 5-7% of p
F was exported to the
cytosol (Fig. 3). We concluded that p
F remained sequestered inside
the microsomes and that the mutations in KAR2 abrogate ERAD
by preventing efficient p
F export to the cytosol.
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Fig. 3.
Defects in BiP/Kar2p prevent
p F export from ER-derived microsomes.
Radiolabeled
Gpp
F was translocated into kar2-1,
kar2-113, kar2-133, and wild type microsomes,
and posttranslocation chase incubations were performed in the presence
of wild type cytosol at either 23 °C (squares) or
37 °C (triangles). Samples were collected at 0, 10, 20, and 40 min, and the reactions were treated with trypsin and processed
as described in the legend to Fig. 2. Reactions performed with wild
type microsomes exhibited identical amounts of protected p
F at 23 and 37 °C (circles).
-1
protease inhibitor, could be imported into the yeast ER but was
subsequently degraded by the proteasome (5, 50). The degradation of
A1PiZ is also proteasome mediated in mammalian cell lines (12). After a
30-min chase at 37 °C, we found that A1PiZ was stabilized in each of
the kar2 mutant strains (Fig.
4); 8, 16, and 16% of A1PiZ was degraded in kar2-1, kar2-113, and kar2-133,
respectively, compared with 61% in the isogenic wild type strain.
However, at the end of the 90-min chase, 69, 84, and 97%, was degraded
in kar2-1, kar2-133, and kar2-113,
respectively, which was quite similar to the 87.5% degraded in the
wild type strain (Fig. 4). These results demonstrate that
KAR2 mutations initially inhibit A1PiZ proteolysis, but ERAD activity can be recovered at later time points.
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Fig. 4.
Defects in Kar2p initially inhibit the
in vivo degradation of A1PiZ. Pulse-chase protein
radiolabeling experiments were performed in kar2-1,
kar2-113, kar2-133 (closed symbols),
and a wild type strain (open symbols) expressing A1PiZ as
described under "Experimental Procedures." Chase reactions were
performed at either 23 °C (circles) or 37 °C
(triangles and squares). A1PiZ immunoprecipitated
from the chase reactions at 0, 30, and 90 min was resolved by 10%
SDS-polyacrylamide gel electrophoresis, and the relative amounts of
A1PiZ were determined using the Bio-Rad Phosphor Analyses
program.
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Fig. 5.
Kar2p levels increase upon expression of
A1PiZ at 37 °C. kar2-1 (triangles),
kar2-113 (squares), kar2-133
(diamonds), and wild type cells (circles)
expressing A1PiZ were incubated for the indicated times at either
23 °C (closed symbols) or 37 °C (open
symbols) before cell lysates were prepared and immunoblots using
anti-BiP antibody were performed. Relative amounts of BiP were obtained
from scanned images using the Bio-Rad Phosphor Analyses program. Values
represent the means of at least three independent experiments. Note
that at 90 min, a precursor form of Kar2-113p was observed, consistent
with the expected translocation defect in this strain (see text for
details).
F was monitored using
microsomes prepared from wild type and kar2-1 cells
cultured under growth conditions used for the in vivo
experiments, conditions that led to elevated levels of Kar2-1p (Fig.
5). We found that 49% of p
F remained following a 10-min chase in
kar2-1 microsomes, and 44% of p
F remained in the wild
type microsomes. These results indicate that the ERAD defect was
alleviated in both microsomes and cells cultured under conditions in
which the level of Kar2-1p was induced.
F
factor into the ER of ssa1-45 was inhibited after 2 min at
37 °C and found that the translocation block was complete. Thus, we
grew both of these strains at a permissive temperature of 20 °C, and
then one-half of each culture was incubated at the nonpermissive
temperature of 37 °C for 45 min. Cytosols were prepared from the
cultures and used to measure the degradation of microsome-occluded
p
F in an in vitro ERAD reaction identical to that used in
Fig. 2 and described previously (5, 30). As shown in Table
I, we observed that cytosol prepared from
the ssa1 temperature-sensitive strain was proficient for
ERAD when present at a final concentration of 5 mg/ml, regardless of
whether the culture used to prepare the cytosol had been grown at a
nonpermissive temperature and regardless of the assay temperature.
However, one explanation for this lack of an effect on ERAD is that
saturating amounts of cytosol may have been used and that subtle
defects in p
F degradation could have been obscured. For example, if
Ssa1p acts with other components to facilitate ERAD,
Ssa1p-dependent ERAD may be evident only if limiting
amounts of cytosol are used. Therefore, we measured p
F degradation
at varying concentrations of SSA1 and ssa1-45 cytosols from cells grown exclusively at the permissive temperature or
that had been shifted to 37 °C for 45 min, and from an unrelated wild type yeast strain, RSY607. As shown in Fig.
6, we found that the degree of p
F
degradation was similar at each concentration regardless of which
cytosol preparation was used. These results suggest that the presence
of wild type Ssa1p was not required for proficient ERAD in
vitro.
Cytosol from an ssa1 mutant strain is proficient for ERAD in vitro
F in the presence of
an ATP regenerating system. The degradation of p
F was measured, as
described under "Experimental Procedures" and in the legend to Fig.
2, after a 5-min reaction at the indicated temperatures. Values
represent the mean percentages of p
F remaining of at least three
independent experiments ± S.D.
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Fig. 6.
ERAD dependence on cytosol
concentration: ssa1 mutant cytosol is proficient
for ERAD. ERAD reactions were assembled as in Table I except that
varying amounts of wild type (crosses), ssa1
(20 °C) (closed circles), and ssa1 (37 °C)
(open circles) cytosol were used. Reactions were conducted
at 20 °C for 5 min. Values represent the means of at least three
independent experiments.
F
(55), it was also possible that excess Ssa1p may inhibit p
F
degradation, perhaps by shielding the substrate from the proteasome. Thus, we performed an in vitro ERAD reaction using
nonsaturating concentrations of cytosol (1.5 mg/ml) with the different
cytosol preparations either in the absence or presence of a ~3-fold
excess of Ssa1p (10% of total protein). However, as displayed in Table I, we discovered that ERAD was unaffected.
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Fig. 7.
ssa1-45 mutant cytosol is
defective for refolding heat-denatured firefly luciferase.
Luciferase was incubated in the presence of the Sse1p chaperone at a
1:5 molar ratio for 30 min at 42 °C and then supplemented with
either wild type cytosol (filled triangles),
ssa1-45 cytosol from cells grown at the permissive
temperature (filled circles), or ssa1-45 cells
grown at the permissive temperature and then shifted to 37 °C for 45 min (open circles). Refolding reactions were conducted at
26 °C, and the final concentration of each cytosol was 4 mg/ml.
Luciferase activity (defined by relative light units) was detected as
described under "Experimental Procedures" at the indicated time
points.
F degradation using the Ssa1p-depleted
cytosol or cytosol containing wild type levels of Ssa1p at a range of
concentrations, we found that ERAD activities were identical (data not
shown). We also noted that the amount of p
F remaining at saturating
cytosol concentrations was higher than in previous experiments
(~40%, as compared with ~10%; see Fig. 6). This may reflect the
altered cellular growth conditions used prior to the cytosol
preparation or the absence of Ssa2-4p (see under "Discussion").
These results further support our conclusion that Ssa1p is not required
for p
F degradation in vitro.
F does
not require the addition of exogenous Ssa1p (37, 39, 45), efficient
import of our ERAD substrate occurred. Each of these microsome
preparations were then combined with cytosol prepared from the
ssa1 temperature-sensitive strain grown exclusively at 26 °C or shifted to 37 °C for 45 min before cytosol was prepared. When ERAD was assayed for 5 min at 30 °C, using the cytosols at a
final concentration of 3 mg/ml, we found that the amounts of p
F
remaining were statistically identical (range, 51-65%), regardless of
whether the microsomes and/or cytosols derived from the
ssa1-45 mutant grown at the permissive or nonpermissive
temperature. We concluded that residual amounts of microsome-bound
Ssa1p had not altered our results presented above.
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Fig. 8.
Ssa1p is associated with ER-derived
microsomes. Cytosol and microsomes were immunoblotted for BiP and
Ssa1p as described under "Experimental Procedures." Whereas BiP was
enriched about 20-fold in microsomes compared with cytosol, Ssa1p
levels were similar in the two preparations. Lane 1, 1 µg
of total protein; lane 2, 10 µg of total protein;
lane 3, 25 µg of total protein.
F to
the cytosol for degradation; thus, we asked whether or not Ssa1p had a
role in p
F export. A defect in export due to loss of Ssa1p activity
may not have been observed in the experiments presented above if
proteolysis is the rate-determining step in the in vitro ERAD assay. To this end, we monitored the pool of exported p
F using
the trypsin protection assay and cytosol prepared from a wild type
yeast strain and from ssa1-45 cells following incubation at
either the permissive or the nonpermissive temperature. We found that
the trypsin-sensitive pool of p
F in 15-min chase samples containing
ssa1-45 cytosol was not significantly different than that
seen with wild type cytosol (~75% of p
F protected in all cases).
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Fig. 9.
A temperature-sensitive defect in Ssa1p does
not affect the in vivo degradation of A1PiZ.
Pulse-chase protein radiolabeling experiments were performed in the
ssa1 temperature-sensitive mutant (triangles) and
an isogenic wild type strain (circles) at either 23 °C
(closed symbols) or 37 °C (open symbols).
A1PiZ immunoadsorbed from the chase samples at 0, 30, 60, and 90 min
was resolved by 10% SDS-polyacrylamide gel electrophoresis. Relative
amounts of A1PiZ were determined using the Bio-Rad Phosphor Analyses
program. First order decay curves were generated with average values
from at least three independent experiments to determine the half-life
of A1PiZ.
DISCUSSION
F in vitro and A1PiZ in vivo. We also discovered that defects in or
depletion of the homologous cytoplasmic hsp70, Ssa1p, have no effect on ERAD, even though these conditions prevent protein import into the ER
both in vivo (as determined by pulse-chase analyses) and in vitro (as shown using wheat germ-translated pp
F) (38,
39, 45). We show further that BiP is required to export p
F from the
ER and thus is involved in the recognition of ERAD substrates and/or
directly in the export process. In support of this first hypothesis,
other reports indicated that mammalian BiP recognizes aberrantly folded
or unassembled polypeptides in the ER and targets them for degradation
(24, 34, 35). A direct role for BiP in the export process is more
difficult to imagine unless it regulates the translocation channel to
permit access of ERAD substrates to the cytoplasm. In accordance with
this view, the kar2-113 mutant that was used both by
Plemper et al. (18) and in this report prevents an early
event during protein translocation (36). Early events may include the
transfer of the signal sequence-bearing nascent polypeptide to the
translocation pore, Sec61p (reviewed in Ref. 33), or the gating of the
pore. Defects in Sec61p gating are expected to compromise p
F or CPY*
degradation as the channel must transport ERAD substrates to the
cytosol. Indeed, Johnson and co-workers (58) have recently shown that
BiP regulates the opening of the mammalian translocation channel during
protein translocation into the ER. Whether yeast BiP similarly gates
the translocation pore and whether Kar2-113p is unable to exhibit this
activity are currently unknown.
F and A1PiZ from the ER before degradation may ensue and whether other cytosolic chaperones are required to usher substrates to the proteasome. In this
regard, we did note that ERAD was less efficient when measured in
vivo in the SSA1 strain deleted for ssa2-4
(Fig. 9) than in a bona fide wild type strain (that contains
SSA2-4) used in Fig. 4, and when cytosol that was depleted
for Ssa1p, Ssa2p, and Ssa4p was used to support ERAD in
vitro. These differences may arise from either the unique strain
backgrounds used in the experiments or because Ssa2p and/or Ssa4p play
supporting but nonessential roles during ERAD in vivo.
Regardless, it is clear that the essential role of Ssa1p in protein
translocation into the ER is not required for ERAD.
if harnessed to the
membrane
as a motor. Second, ubiquitination may provide the favorable
energy needed to dislocate ERAD substrates from the ER (10, 17, 61).
Third, the chaperone-like 19S cap (PA700) on the proteasome (54) may
bind emerging polypeptides and pull the substrate from the lumen;
indeed, a recent report indicates that the 26s proteasome may exhibit
this activity (62). Fourth, the BiP-Sec63p pair that is required for
posttranslational translocation into the ER (reviewed in Ref. 33) may
function in reverse to engineer polypeptide export.
F en route
to its degradation (5). Therefore, it will be interesting to determine
whether the proteolysis of CPY*, to which ubiquitin is attached before
degradation (4, 10), requires Ssa1p.
kar2-133 strain compared with microsomes
prepared from either the cne1
or kar2-133
strains (data not shown), a result that may arise from redundant
functions of BiP and calnexin during ERAD in vitro.
Regardless, future work will address each of these hypotheses.
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ACKNOWLEDGEMENTS |
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We thank Drs. Randy Hampton and Beth Jones for critical reading of the manuscript, Dr. E. A. Craig for generously providing strains and reagents, and Stephanie Stewart and Anna Young for technical assistance.
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FOOTNOTES |
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* This work was supported by Grants MCB-9506002 and MCB-9722889 from the National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Junior Faculty Achievement Award from the American Cancer Society. To whom correspondence should be addressed: Dept. of Biological Sciences, Univ. of Pittsburgh, 267 Crawford Hall, Pittsburgh, PA 15260. Tel.: 412-624-4831; Fax: 412-624-4759; E-mail: jbrodsky+{at}pitt.edu.
Supported by a Research Experience for Undergraduates Award
from the National Science Foundation.
The abbreviations used are:
ER, endoplasmic
reticulum; ERAD, ER-associated protein degradation; pF, pro-
factor; pp
F, prepro-
factor.
2 J. L. Goeckeler and J. L. Brodsky, manuscript in preparation.
3 Jannatipour, M., Callejo, M. Parodi, A. J., Armstrong, J., and Rokeach, L. A. (1998) Biochemistry 37, 17253-17261
4 J. L. Brodsky, unpublished observations.
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