1 Biology Department, University of Nevada, Reno, NV 89557, USA
2 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA
15260, USA
* Author for correspondence (e-mail: mccracke{at}unr.edu)
Accepted 24 February 2003
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Summary |
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Key words: ERAD, Protein quality control, Degradation
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Introduction |
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Genetic screens for ERAD-defective Saccharomyces cerevisiae
mutants (Hampton et al., 1996;
Knop et al., 1996
;
McCracken et al., 1996
), the
use of mammalian cell-free systems
(Winitz et al., 1996
;
Wilson et al., 2000
;
Gusarova et al., 2001
;
Shamu et al., 1999
;
Shamu et al., 2001
;
Ye et al., 2001
) and a yeast
in vitro ERAD assay (McCracken and
Brodsky, 1996
; Werner et al.,
1996
; Pilon et al.,
1997
; Gillece et al.,
2000
) have facilitated the discovery of components required for
ERAD substrate specificity, protein export and delivery of substrates to the
proteasome; however, unanswered questions regarding the molecular details of
ERAD remain. Results from several laboratories reveal that both ubiquitination
and molecular chaperones aid in the targeting of ERAD substrates to the
proteasome, yet it appears that each substrate has a unique set of
requirements for its degradation (reviewed by
Fewell et al., 2001
). For
example, not all ERAD substrates are ubiquitinated
(Werner et al., 1996
).
Furthermore, integral ER membrane proteins appear to be degraded independently
of the ER molecular chaperone BiP (IgG heavy chain binding protein), whereas
soluble ERAD substrates require BiP to mediate their export to the cytoplasm
(Brodsky et al., 1999
;
Nishikawa et al., 2001
). By
contrast, the cytoplasmic heat-shock protein Hsp70 chaperone, Ssa1p,
facilitates the degradation of several integral membrane proteins but is
dispensable for the proteolysis of soluble substrates
(Hill and Cooper, 2000
;
Zhang et al., 2001
). Finally,
genes required for the degradation of one substrate may or may not be required
for the degradation of a related substrate (e.g.
Wilhovsky et al., 2000
).
Clearly, a greater number of substrates and factors required for ERAD must be
analyzed to better understand the molecular mechanisms of this pathway. By
analogy, the full spectrum of factors required for protein transport and a
more complete understanding of the secretory pathway emerged only after varied
genetic screening protocols and biochemical attacks were employed to examine
the transport of multiple diverse cargoes (e.g.
Schekman and Orci, 1996
).
One soluble ERAD substrate is an unglycosylated version of the yeast mating
pre-pheromone, pre-pro alpha factor (ppF). After signal sequence
cleavage, pp
F is converted to pro-alpha factor (p
F) and if
glycosylation is prevented, p
F is retro-translocated to the cytoplasm
and destroyed by the proteasome (Werner
et al., 1996
). An assay in which the degradation of p
F was
faithfully reconstituted indicated requirements for the ER-resident chaperones
calnexin (McCracken and Brodsky,
1996
), protein disulfide isomerase (PDI)
(Gillece et al., 1999
) and BiP
(Brodsky et al., 1999
). Ssa1p
was dispensable for p
F degradation, as was polyubiquitination
(Werner et al., 1996
;
Brodsky et al., 1999
). Indeed,
the 19S cap of the proteasome is sufficient for ATP-mediated p
F
retro-translocation, and the substrate can be degraded on addition of the 20S
particle (R. Lee and J.L.B., unpublished).
The mutant Z variant of 1-proteinase inhibitor (A1PiZ, also known as
1-antitrypsin-Z or AT-Z) is another soluble substrate for ERAD
(McCracken and Kruse, 1993
;
McCracken et al., 1996
;
Teckman and Perlmutter, 1996
).
In humans, secretion-incompetent A1Pi mutants may aggregate in the hepatic ER,
ultimately giving rise to liver disease and juvenile emphysema, although it is
unclear how the misfolded protein is converted from being an ERAD substrate
into an aggregation-prone polypeptide
(Lomas et al., 1992
;
Yu et al., 1995
).
To begin to dissect A1Pi maturation at the molecular level, both
A1PiZ-expressing human cell lines and yeast have been used. In yeast, optimal
A1PiZ degradation requires the ER-resident molecular chaperone BiP, yet is
degraded in the absence of functional Ssa1p
(Brodsky et al., 1999).
Ubiquitination does not appear to be required for A1Pi degradation in yeast,
whereas in mammalian cells both ubiquitin-dependent and ubiquitin-independent
A1PiZ degradation have been described
(Teckman et al., 2000
).
Oligosaccharyl trimming and the ER resident chaperone calnexin also play a
role in the ERAD of A1PiZ (Qu et al.,
1996
; Marcus and Perlmutter,
2000
; Cabral et al.,
2000
).
The proteolysis of a third soluble ERAD substrate, a mutated form of
carboxypeptidase Y (CPY*), also requires BiP in yeast
(Plemper et al., 1997).
Additional factors involved in CPY* degradation include an integral ER
membrane protein of unknown function, Der1p
(Knop et al., 1996
), two
proteins involved in transport between the ER and Golgi, Erv29p and Erv14p
(Caldwell et al., 2001
), and
Cdc48p, a cytosolic protein that may dislocate proteins from the ER and target
multi-ubiquitinated substrates to the proteasome
(Ye et al., 2001
;
Jarosch et al., 2002
;
Rabinovich et al., 2002
).
Unlike p
F, ubiquitination is necessary for the degradation of CPY*, and
several components of the ubiquitin-conjugating machinery have been implicated
in CPY* proteolysis: the ubiquitin-conjugating enzymes Ubc6p and Ubc7p
(Hiller et al., 1996
);
Der3p/Hrd1p, a membrane-anchored ubiquitin-protein ligase (E3)
(Bays et al., 2001
;
Deak and Wolf, 2001
); and
Cue1p, which recruits Ubc7p to the ER membrane
(Biederer et al., 1997
).
An integral membrane protein shown to be an ERAD substrate in both yeast
and mammalian cells is the cystic fibrosis transmembrane conductance regulator
(CFTR) (Yang et al., 1993;
Pind et al., 1994
;
Jensen et al., 1995
;
Ward et al., 1995
;
Zhang et al., 2001
).
Mutations in CFTR that prevent its maturation in the ER and subsequent
transport lead to cystic fibrosis. Like CPY*, CFTR degradation requires
ubiquitination (Ward et al.,
1995
; Jensen et al.,
1995
; Zhang et al.,
2001
), but unlike soluble substrates there is no requirement for
calnexin or BiP (Zhang et al.,
2001
). By contrast, Ssa1p facilitates CFTR degradation
(Zhang et al., 2001
).
In the event that ERAD is unable to rid the secretory pathway of aberrant
polypeptides the unfolded protein response (UPR) may be activated. The UPR is
present in all eukaryotic cells and increases the ability of the ER to
tolerate misfolded proteins (for a review, see
Kaufman, 1999;
Ng et al., 2000
). Because the
UPR and ERAD provide complementary facets of secretory protein `quality
control', it is not surprising that ERAD and the UPR are functionally
intertwined. For example, the scope of the UPR was examined by microarray
analysis on addition of the N-linked glycosylation inhibitor,
tunicamycin, and during accumulation of the mouse major histocompatibility
complex class I heavy chain (H-2Kb), a substrate for ERAD when
unassembled (Ploegh et al.,
1979
; Hughes et al.,
1997
; Casagrande et al.,
2000
). When H-2Kb was overexpressed in yeast, mRNAs
encoding several chaperones and known UPR targets were upregulated three- to
seven-fold, but many uncharacterized genes were also induced. Travers et al.
(Travers et al., 2000
)
determined the transcriptional scope of the yeast UPR using tunicamycin and
dithiothreitol (DTT), which prevents disulfide bond formation, and again,
known UPR-target genes and uncharacterized open reading frames (ORFs) were
induced. In both screens, UPR-target genes included those required for ERAD.
Moreover, yeast lacking nonessential components of both the ERAD and UPR
pathways exhibit synthetic growth defects, suggesting that the two pathways
function in concert (Travers et al.,
2000
; Ng et al.,
2000
; Friedlander et al.,
2000
).
Given the fact that many genes induced by the UPR are uncharacterized ORFs
and that a subset of known UPR targets encode ERAD-requiring proteins, we
selected 69 ORFs upregulated by the UPR. Mutants deleted for the corresponding
genes were then screened for A1PiZ degradation deficiencies (add).
From these analyses, six ADD gene products were identified.
Furthermore, analysis of CFTR, pF and CPY* degradation in the new
add mutants underscores the diverse requirements for the removal of
individual ERAD substrates and points to the complexity with which this
pathway functions.
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Materials and Methods |
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Construction of double mutants
Each newly identified add mutant strain was mated to
kar2-1 yeast (see above) using established methods
(Kaiser et al., 1994).
Sporulation in the selected diploids was induced by nitrogen starvation and
the spores were dissected and analyzed as described by Adams et al.
(Adams et al., 1997
) to
determine their genotypes.
Cloning and expression of genes
Genomic DNA was isolated from the BY4742 wild-type parent strain as
described by Hoffman and Winston (Hoffman
and Winston, 1987). WT ADD gene sequences were amplified
from genomic DNA by PCR using oligonucleotides (Life Technologies) specific
for each locus with alterations to introduce unique restriction endonuclease
cleavage sites immediately 5' of the ATG or
300 base pairs upstream
of the ATG, and unique restriction endonuclease cleavage sites 3' of the
stop codon. Primer sequences used are available on request. Wild-type
ADD genes lacking a promoter (i.e. those immediately 5' of the
ATG) were inserted into the p415.ADH vector, and each wild-type gene
containing a putative promoter sequence (i.e. those containing
300 base
pairs 5' of the ATG) was inserted into the p415.CYC1term vector. The
correct insertion of each gene was determined by automatic DNA sequence
analysis (following standard protocols) using primers specific for the
p415.ADH or p425.CYC1term vectors. The p415CYC1term vector was generated by
removing the CYC1 promoter from p415.CYC1 with a
SacI/BamHI digest, creation of blunt ends and ligation so
that the multiple cloning region (MCR) retained the BamHI through
XhoI restriction sites preceding the CYC1 termination
sequence.
Yeast and E. coli transformation
Yeast transformation was carried out by a standard lithium acetate
procedure (Gietz and Woods,
1994), and transformants were isolated after growth in selective
medium containing 2% dextrose. The Cell-Porator E. coli Pulser
(GibcoBRL, Series 1613, Rockville, MD) was used to electroporate HB101.
Plasmids were isolated from bacterial transformants using the Quantum Prep
Plasmid Miniprep kit (Bio-Rad, Hercules, CA).
Authentication of BY4742 mutants
Genomic DNA was isolated from BY4742 mutant strains: add06, add37,
add39, add66, add67 and add68, as described by Hoffman and
Winston (Hoffman and Winston,
1987). A kanamycin cassette for each corresponding ADD
locus was amplified from genomic DNA by PCR using oligonucleotides (Life
Technologies) as described by Research Genetic's published deletion module PCR
strategy (Wach et al., 1994
).
Upstream 45 bp and downstream 45 bp sequences and the kanamycin gene were
verified by automatic DNA sequence analysis (following standard protocols)
using primers specific for each locus.
Colony-blot immunoassay
The colony-blot immunoassay was a modification of a previously described
procedure (McCracken et al.,
1996). Three microliters of 0.001 OD/µl of overnight cell
cultures were spotted onto a nitrocellulose disc overlaid on medium containing
2% galactose to induce expression of A1Pi (M or Z) followed by incubation at
35°C for 36 hours. Cells were lysed and blots developed as described by
McCracken et al. (McCracken et al.,
1996
). The density of A1Pi at each colony spot was quantified
using Molecular Analyst.
Radiolabeling, immunoprecipitation and phosphorimaging
To assay A1PiZ degradation, yeast were grown at 30°C in selective
medium containing 2% galactose to induce A1Pi expression for 24 hours before
analysis. Using a protocol described by Brodsky et al.
(Brodsky et al., 1998), 13
OD600 cells were pulsed with 35S-Easy Tag (NEN) for 20
minutes and chased with 10x cold cysteine/methionine mix. Samples were
taken at the indicated time points. Cell lysis and immunoprecipitation were
performed as described (Brodsky et al.,
1998
). Proteins were resolved by 10% SDS-PAGE and visualized using
a BioRad PhosphorImager (Hercules, CA). Quantification was performed using
Molecular Analyst. CPY* degradation was measured similarly by pulse-chase
radiolabeling and immunoprecipitation as previously published
(Zhang et al., 2001
),
starting with HA-tagged CPY* expression vector (see above) transformed cells
and using anti-HA antibody.
CFTR degradation assay
Yeast expressing CFTR were grown to an OD600 of 0.5 at 30°C
before cycloheximide was added to make a final concentration of 100 µg/ml.
The cells were incubated at 30°C with shaking and 0.5 OD units were
harvested at the indicated time points. Total protein in the lysates (10 µl
of 100 µl final volume) (Zhang et al.,
2001) were resolved by SDS-PAGE, followed by immunoblot analysis
using monoclonal mouse anti-HA (Roche Molecular Biochemicals), sheep
anti-mouse IgG horseradish peroxidase-conjugated antibody (Amersham Pharmacia
Biotech), SuperSignal® West Pico Chemiluminescent Substrate
(Pierce, Rockford, IL), and visualizing using a BioRad PhosphorImager.
Quantification was performed using Molecular Analyst.
Assays for ERAD and UPR induction
Yeast ER-derived microsomes and cytosol were prepared and the in vitro ERAD
assay was performed as described by McCracken and Brodsky
(McCracken and Brodsky, 1996).
Quantification of the resulting phosphorimaged gels was performed using
Molecular Analyst. UPR induction was measured after growth of each transformed
strain in selective medium to log phase (OD600=
1). Cell
extracts were prepared by agitation of washed cells with glass beads, and
ß-galactosidase activity was measured using published protocols
(Adams et al., 1997
).
Database searches
NCBI BLASTN 2.2.3 (Altschul et al.,
1990; Karlin and Altschul,
1990
; Karlin and Altschul,
1993
; Tatusova and Madden,
1999
)
(http://www.ncbi.nlm.nih.gov/blast/Blast.cgi),
the PSORT WWW Server (Nakai,
1991
; Nakai, 2000
)
(http://psort.nibb.ac.jp/)
and reports on the random and systematic epitope-tagging of ORFs in the yeast
genome (Ross-Macdonald et al.,
1999
)
(http://ygac.med.yale.edu/ygac-cgi/front_page_OE.html)
were used to identify and predict cellular locations of the Add proteins.
BLASTP 2.2.3 generated multiple protein sequence alignments and sequence
relatedness data. The Add06p inosine-uridine hydrolase family signature was
identified using The Swiss Institute of Bioinformatics ScanProsite
(http://www.expasy.ch/tools/scanprosite/).
Signal peptide cleavage sites were identified by Center for Biological
Sequence Analysis SignalP V1.1 (Nielsen et
al., 1997
)
(http://www.cbs.dtu.dk/services/SignalP/).
PSORT WWW Server identified N- and C-terminal ER retention signals. Structural
homology predictions were made using the 3D-PSSM Web Server V.2.6.0
(Fischer et al., 1999
;
Kelley et al., 1999
;
Kelley et al., 2000
)
(http://www.sbg.bio.ic.ac.uk/~3dpssm/).
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Results |
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A1PiZ accumulation in mutants deleted for UPR-target genes
To determine whether yeast lacking the UPR-target genes were
ERAD-defective, 73 deletion mutants were transformed with a
galactose-inducible A1PiZ expression vector and tested for A1PiZ degradation
deficiencies (add mutant phenotype)
(McCracken et al., 1996) using
a colony-blot immunoassay (see Materials and Methods). On the basis of the
knowledge that A1PiZ accumulates in ERAD-defective cells
(McCracken et al., 1996
;
Werner et al., 1996
), strains
exhibiting an add mutant phenotype should contain an increased amount
of immunoreactive A1PiZ under steady-state conditions and thus display a
`spot' of greater intensity on the colony-blot compared with wild-type (WT)
cells (Fig. 1). Controls for
this assay were WT cells carrying the expression vector lacking an A1Pi gene,
which resulted in background intensity, WT cells expressing but degrading
A1PiZ, which produced a low-intensity spot, and WT cells expressing A1PiM, a
stable protein that accumulates in the yeast ER and thus displays a
high-intensity spot (McCracken et al.,
1996
). Of the uncharacterized yeast mutants examined in this
screen, six (add06, add37, add39, add66, add67 and add68)
were putative add mutants after initial screening and re-screening
(Table 2, Fig. 1). The amount of A1PiZ
accumulated reproducibly in these mutants was >125% of that in the WT
parent strain. Two genes upregulated by the UPR and known to be involved in
ERAD (#1DER1, and #72ERV29)
(Plemper et al., 1997
;
Caldwell et al., 2001
) were
included in our analyses, along with known ERAD genes that are not UPR-induced
(#38CUE1, #41EPS1, and
#43HRD1/DER3, #61-HRD3, #73-SSA1)
(Lenk and Sommer, 2000
;
Wang and Chang, 1999
;
Hampton and Bhakta,1997
;
Plemper et al., 1999
;
Zhang et al., 2001
).
Interestingly, with the exception of ERV29, these deletion mutants
displayed a WT ADD phenotype
(Table 1, Fig. 1), indicating that the
proteins encoded by the genes are not essential for the degradation of
A1PiZ.
|
|
To authenticate the identity of the deleted genes in each mutant that we
obtained commercially, the polymerase chain reaction (PCR) was used with
primers corresponding to the insertion site of the deletion modules
(Wach et al., 1994), and the
products were sequenced (Materials and Methods). We thus confirmed the
identity of the deleted genes (data not shown).
The rate of A1PiZ degradation is decreased in ADD deletion
mutants
To verify the mutant phenotype in add06, add37, add39, add66,
add67 and add68, we performed pulse-chase analyses to monitor
A1PiZ degradation. As anticipated, A1PiZ was stabilized in each add
deletion mutant; 40-50% of A1PiZ was degraded in the deletion mutants at
60 minutes compared with 60% degraded in the WT parent strain
(Fig. 2). This level of A1PiZ
stabilization was similar to that seen in temperature-sensitive kar2
strains (Brodsky et al., 1999
)
and previously described ADD mutant strains
(McCracken et al., 1996
).
|
To complement the ERAD defects observed in the ADD deletion
strains, WT copies of ADD06, ADD37, ADD39, ADD66, ADD67 and
ADD68 were cloned into the p415.ADH constitutive expression vector
and were transformed into the corresponding strains. However, only three
strains (add66, add67 and add68) displayed complementation
of the add phenotype. Next, the ADD genes were cloned along
with their native promoters into the p425.CYC1term vector. For this analysis,
the putative promoters were assumed to be located within 300 base pairs
upstream from the ATG translation start site. We then assayed the ADD
deletion strains expressing the appropriate WT gene from their native
promoters and found that all strains, except add67, showed
complementation of the add phenotype
(Fig. 3). The add67
strain showed complementation only when the ADD67 gene was expressed
from the p415.ADH vector (Fig.
3). We note that the various promoters used for these
complementation analyses direct expression at different levels; however, it is
also possible that positional effects might influence expression from native
promoters when inserted into the corresponding plasmids. Because
complementation of the mutant phenotype is seen when WT ADD genes are
expressed from specific promoters and not others, we speculate that the level
of expression of these ADD genes may be crucial for their function in
ERAD. In support of this hypothesis, Lenk et al.
(Lenk et al., 2002) reported
that the level of expression of the yeast ubiquitin-conjugating enzyme Ubc6p
influenced ERAD activity.
|
The degradation of other ERAD substrates is compromised in the add
mutants
Because of the diverse requirements for the degradation of ERAD substrates
(see Introduction), we examined pF, CPY* and CFTR degradation in each
add mutant strain and in a WT parent. We discovered that none of the
new add mutants strongly affected the rate of CPY* degradation as
determined by pulse-chase analysis (data not shown), whereas CPY* degradation
was attenuated in kar2-1 mutant yeast that express an ERAD-defective
form of BiP (Brodsky et al.,
1999
; Zhang et al.,
2001
). In accordance with these data, Caldwell et al.
(Caldwell et al., 2001
)
previously analyzed yeast deleted for YKL206c and YMR184w, which encode Add66p
and Add37p, respectively, and reported that the mutant strains degraded CPY*
with WT efficiency.
To examine whether the degradation of an ER membrane protein is affected in these add mutants, we next measured CFTR stability using a cycloheximide chase protocol (Materials and Methods). CFTR was stabilized to varying extents when compared with the WT parent strain (Fig. 4). The add06 and add39 deletion mutants displayed the greatest degradation defects; 10% and 17% of CFTR, respectively, was degraded at 20 minutes, compared with 35% CFTR degradation observed in the WT parent.
|
Finally, we assayed pF degradation in vitro using cytosol and
ER-derived microsome fractions prepared from deletion mutants and WT cells. In
principle, this assay should permit us to define the compartment(s) in which
the corresponding proteins normally function. However, it is possible that
cytosolic factors can contaminate membrane fractions, that the requirement for
gene products can be bypassed in vitro and/or that the action of regulators
may be obviated in defined, in vitro systems. Nevertheless, we found that both
cytosol and microsomes from add37, add39, add66 and add67
supported the degradation of p
F, suggesting that the gene products were
not required for p
F degradation in vitro. However, reactions with
add06 or add68 microsomes and cytosol displayed a noticeable
degradation defect; 46% of p
F was proteolyzed at 40 minutes compared
with 68% proteolysis in WT reactions (Fig.
5A). Although the stabilization of p
F with both
add06 and add68 cytosol was significantly greater than that
seen with WT cytosol, the difference in the rate of degradation between the WT
and the mutant strain was not as great as that observed with other strains
that stabilize p
F. For example, WT KAR2 microsomes and cytosol
proteolyzed 64% of p
F in 40 minutes, whereas the reaction with
kar2-1 cytosol and microsomes showed only 35% p
F degradation
(Brodsky et al., 1999
).
Additionally, when studying the role of calnexin in ERAD we observed 80%
degradation of p
F using materials prepared from the CNE1
parent, whereas 55% proteolysis was apparent when reagents from the
cne1 delete were used (McCracken
and Brodsky, 1996
). Thus, the WT parents of the various mutant
strains show variations in the efficiency of p
F degradation in vitro,
indicating that strain variation and perhaps proteasome activity in the
cytosol preparations influence the extent of p
F proteolysis in this
assay.
|
Next, pF degradation was assayed using microsomes and cytosol from
WT and add06 (Fig. 5B)
or add68 (Fig. 5C)
strains in all combinations. Only reactions containing mutant cytosol with
either WT or mutant microsomes exhibited a decreased rate of p
F
degradation similar to that seen in Fig.
5A. These results suggest that both Add06p and Add68p function in
the cytosol to facilitate p
F degradation. In accordance with this
hypothesis, the Add68 and Add06 proteins lack putative signal sequences, and
Add06p tagged at the C-terminus was shown to reside in the yeast
cytoplasm/nucleus (Ross-Macdonald et al.,
1999
).
The unfolded protein response is modestly enhanced in add66 and add67
yeast
ERAD defects can lead to induction of the UPR because aberrant proteins
accumulate in the ER (reviewed by Fewell
et al., 2001). By contrast, more subtle or substrate-specific ERAD
defects might not induce the UPR. To examine UPR levels in the add
mutants, a UPR reporter plasmid containing four repeats of the UPR element
(UPRE) 5' to the ß-galactosidase-encoding gene (pJC104) (see
Cox et al., 1993
) was
transformed into each strain. As a control, isogenic wild type and
kar2-1 mutant yeast were also transformed with pJC104. Cells were
grown at 30°C to log phase, cell extracts were prepared and
ß-galactosidase activity was measured. We found that the kar2-1
strain exhibited an
11-fold increase in the UPR compared with the wild
type; this is consistent with the fact that all soluble ERAD substrates
examined in this mutant are stabilized and that protein folding is compromised
in this strain (Fig. 6)
(Simons et al., 1995
;
Brodsky et al., 1999
;
Zhang et al., 2001
). We also
noted that UPR induction in the add strains varied considerably
relative to the ADD parent. For example, add06, 37, 39, 68
displayed either no increase or a modest decrease in the UPR, whereas
add66 and add67 exhibited a 3-4-fold induction. These data
suggest that the Add66 and Add67 proteins may be required more generally for
ERAD and/or that the lack of these proteins affects ER physiology. By
contrast, cells might be able to compensate for loss of the proteins encoded
by the other add mutants, or the absence of these gene products might
lead to a defect in the degradation of only select proteins.
|
The ADD68 deletion strain is hypersensitive to cadmium
Addition of cadmium to living cells causes oxidative damage to cellular
components, catalyzes protein unfolding, induces the expression of heat-shock
proteins in yeast and is toxic at elevated concentrations
(Jungmann et al., 1993).
Furthermore, compromising the cellular ubiquitin-proteasome pathway by
mutation or expression of dominant-negative mutants leads to hypersensitivity
to cadmium (Tsirigotis et al.,
2001
). Thus, hypersensitivity to cadmium may be indicative of
defects in proteasome-mediated degradation of aberrant proteins.
To determine whether the ADD mutants were sensitive to cadmium,
exponentially growing cells were spotted in tenfold serial dilutions on plates
containing 15 µM or 30 µM cadmium chloride (CdCl2) and
incubated at either 30°C or 37°C for 3 days. All strains tested showed
mild sensitivity to cadmium, as indicated by slowed growth, and one strain,
add68, was hypersensitive to cadmium
(Fig. 7). The effect of 30
µM CdCl2 was marginally greater than that of 15 µM
CdCl2, and a slight increase in sensitivity was seen at 37°C
(data not shown). One control strain, add72, deleted for the gene
ERV29 that is required for the HIP pathway of ERAD
(Haynes et al., 2002),
displayed sensitivity to cadmium. Interestingly, a strain deleted for the
HRD1/DER3 gene (add43) that encodes an ubiquitin-protein
ligase (Bays et al., 2001
) was
not hypersensitive to CdCl2, indicating that defective ERAD does
not necessitate cadmium hypersensitivity.
|
Yeast exhibit a growth defect when ADD37 deletion is combined with
the kar2-1 allele
To determine the growth phenotypes of strains lacking the newly identified
ADD genes and a known ERAD-requiring gene, add mutant strains were mated to
kar2-1 yeast, an ERAD-specific mutant allele of BiP. Previous results
indicated that yeast containing this allele and deleted for the ER lumenal
chaperone, calnexin (CNE1), exhibited poorer growth than cells either
lacking calnexin or containing kar2-1 alone
(Brodsky et al., 1999). We
first found that add66 and add68 mutant cells exhibit severe
sporulation defects; thus, only kar2-1 progeny combined with
add06, add37, add39 and add67 could be obtained by this
method. Next, we noted that only add37 yeast exhibited a synthetic
growth defect at 37°C when combined with the kar2-1 allele
(Fig. 8).
|
Bioinformatic analysis of the ADD gene products
To gain insight into the protein product of each ADD gene and
their possible roles in ERAD, sequence homology and structural analysis
searches were performed. This information is summarized in
Table 2, and we present models
for how the corresponding gene products might facilitate ERAD in the
Discussion, below.
![]() |
Discussion |
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The concept that ERAD may be comprised of multiple pathways is supported by
the diverse requirements for the degradation of ERAD substrates
(Table 3) and by their
diversity in structures and post-translational modifications. For example,
most, but not all, ERAD substrates are ubiquitinated before their degradation,
and most, but not all, are glycosylated (reviewed by
Fewell et al., 2001). These
features will play a profound role in dictating which partners a given ERAD
substrate will encounter en route to its degradation. Also, ERAD substrates
are either integral membrane or soluble proteins in the ER, and differences in
the chaperone requirements for the degradation of these two classes of
substrates have led us and others to propose distinct mechanisms for their
removal (see below). Finally, we suspect that the degree to which a protein
misfolds to become an ERAD substrate may be quite varied. Some ERAD substrates
probably attain little, if any, secondary structure in the ER, whereas others
at least partially fold and become glycosylated and disulfide-bonded before
their retrotranslocation and degradation. For A1PiZ, this issue is
particularly pertinent as it is highly aggregation prone
(Lomas et al., 1992
). Thus,
gene products that are required for the degradation of A1PiZ may be
specialized, catalyzing the degradation and maintaining the solubility of such
aggregation-prone ERAD substrates. In the future, it will be vital to
understand whether the add mutants such as those identified
in this study affect the solubility of A1PiZ in the ER and/or whether
they are directly involved in its degradation.
|
Our results also serve as an important starting point to develop hypotheses
regarding the functions of these ADD gene products during ERAD.
Although striking sequence identities were not apparent between the
ADD genes and other characterized genes, each ADD gene
contains motifs that suggest specific functions. Such suggested homologies
will drive our future research efforts. For example, the ADD06 gene
(YDR400w) encodes a 378 amino acid residue uridine nucleoside
N-ribohydrolase (Urh1p) (Table
2). ScanProsite analysis of the amino acid sequence indicates a
nucleoside hydrolase family signature at residues 49-59 and previous studies
have demonstrated its role in hydrolyzing nucleosides
(Magni et al., 1975;
Kurtz et al., 1999
). More
relevant, however, may be the observation that Add06p interacts with Yih1p
(Uetz et al., 2000
), a protein
involved in protein synthesis regulation during stress
(Kubota et al., 2000
). These
data suggest that defects in stress-induced regulation might impact ERAD.
Consistent with this notion, cell stress was recently shown to regulate ERAD
(VanSlyke and Musil,
2002
).
YMR184w, the gene deleted in add37, encodes a cytosolic protein of
unknown function. BLAST2 sequence alignments show that the region of Add37p
spanning amino acids 105 and 186 is 25% identical and 45% similar to the
mitochondrial J-type chaperone Jac1p, which is involved in the assembly of
iron sulfur clusters (Lutz et al.,
2001; Voisine et al.,
2001
). However, these sequence homologies occur outside of the J
domain of Jac1p. Potentially more relevant, the region between amino acids 99
and 180 displays 23% identity and 54% similarity with Gos1p, a v-SNARE protein
(McNew et al., 1998
).
Moreover, the region of Add37p spanning amino acids 95 and 170 is 27%
identical and 44% similar to Uso1p, a protein necessary for ER to Golgi
protein transport (Nakajima et al.,
1991
), and the amino acid region 62 to 78 displays 52% identity
and 63% similarity with Sec13p, a component of the COPII vesicle coat
(Salama et al., 1997
).
Intriguingly, 3D-PSSM predicted the Add37p to structurally resemble the
ubiquitin-conjugating enzyme, Ubc4p. Although it is hard to reconcile these
data into a working model for Add37p function in ERAD, it is tempting to
speculate that Add37 is required for protein secretion and that A1PiZ
degradation, like three other soluble ERAD substrates, requires a functional
ER to Golgi pathway (Caldwell et al.,
2001
; Vashist et al.,
2001
). In accordance with this model, we found that deletion of
the gene encoding Erv29p, which is a soluble cargo receptor in the ER
(Belden and Barlowe, 2001
),
inhibits AiPiZ degradation (Fig.
1).
The ADD67 gene (YLR380w) is an endocytic
membrane/vesicle-associated phosphatidylinositol transferase known as
Csr1p/Sfh2p (Li et al., 2000;
Cockcrost and De Matteis, 2001). The region spanning amino acid 109 and 316 of
Csr1p/Sfh2p displays 29% identity and 44% similarity with Sec14p and like
Sec14p appears to function in ER-to-Golgi vesicle transport
(Li et al., 2000
), suggesting
another possible link between the ADD gene products and the secretory
pathway. An alternative scenario is that lipid composition is altered in the
csr1 mutant, leading to an ERAD-defect. Pertinent to this view is the
recent finding that lipid rafts may be important to sort proteins that have a
potential to become ERAD substrates if mutated
(Bagnat et al., 2001
).
The ADD66 gene (YKL206c) encodes a cytosolic protein that has been
found to be associated with an ADP-ribosylation factor-like protein known as
Arl3p, and with Pre1p, a proteasome subunit
(Ho et al., 2002). Two models
can be envisioned for Add66p function. First, the protein may associate with
Pre1p and be involved in proteasome assembly or function. Second, because
arl3 mutants exhibit defects in secretion
(Huang et al., 1999
), the
add66 mutation may similarly be compromised for ERAD because of
defects in ER-to-Golgi trafficking (see above). Because of these strong
connections to components/pathways that impact upon ERAD, it may not be
surprising that deletion of the ADD66 gene induces the UPR
(Fig. 7).
YHR043c encodes a 2-deoxyglucose-6-phosphate phosphatase (Add39p/Dog2p),
cytoplasmic protein involved in carbohydrate metabolism. The entire
Dog2p sequence displays 88% identity and 92% similarity to Dog1p, the region
spanning amino acids 6 and 231 of Dog2p is 35% identical and 51% similar to
Rhr2p, and amino acids 6 to 219 of Dog2p display 35% identity and 50%
similarity to Hor2p, all of which are phosphatases. Yeast two-hybrid assays
have shown interactions between Dog2p and Apg17p, a protein involved in
autophagy (Kamada et al.,
2000
; Ito et al.,
2001
). Perlmutter and colleagues have found that mutant A1Pi
accumulation leads to the propagation of autophagocytic vesicles in mammalian
cells (Teckman and Perlmutter,
2000
), suggesting that this ERAD substrate may be degraded by the
vacuole/lysosome when the ERAD machinery is overwhelmed. Thus, add39
yeast might be compromised for autophagocytosis, leading to an increased
demand on the ERAD pathway.
The ADD68 gene (YFL049w) is predicted to encode a nuclear membrane
protein. The region spanning amino acids 50 and 220 displays 29% identity and
44% similarity with Npl6p, a protein isolated originally in a screen for
nuclear protein localization mutants
(Nelson et al., 1993).
Intriguingly, another gene isolated from that screen, Npl4p, associates with
Cdc48p and with a third protein, Ufd1, that together are required for the ERAD
of several substrates (Bays and Hampton,
2002
; Tsai et al.,
2002
). An additional link between Add68p and ERAD comes from
structural predictions that Add68p resembles an N-terminal nucleophile
aminohydrolase, such as the proteasome
subunit protein. Like the
proteasome (Enenkel et al.,
1998
), Add68p can be found associated with the ER and in the
cytosol (Ross-Macdonald et al.,
1999
) and appears to function in the cytosol
(Fig. 5). Moreover, the
ADD68 deletion mutant is sensitive to cadmium, a phenotypic
characteristic of cells with defective proteasome activity. Overall, as with
the other new ADD gene products, further work is clearly needed to
elucidate, at the molecular level, how these factors facilitate ERAD.
In summary, the results of our screen and the timeliness of available bioinformatic/genome-wide analyses in yeast have provided us with many testable hypotheses about the functional roles for these ADD gene products in ERAD. Our results provide further insight into the complexity of ERAD, and underscore the importance of analyzing additional ERAD substrates and in defining the genetic requirements for their degradation. A biochemical characterization of the ADD gene products will also lead to a better, more complete understanding of the complexity of the substrate specific requirements for ERAD. However, we note that our search was limited to those genes that are not essential for cell viability. Thus, microarray analysis of differential gene expression using cells over-expressing A1PiZ should identify essential and other nonessential genes required for the degradation of A1PiZ and will further help elucidate the molecular mechanism of the degradation of this important ERAD substrate. Finally, and more generally, our screening approach may be applicable to the analysis of any ERAD substrate in yeast for which antiserum is available.
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