Department of Biochemistry and Cell Biology and the Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215
Received for publication, January 29, 2001, and in revised form, March 16, 2001
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ABSTRACT |
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In addition to a role in DNA repair events in
yeast, several lines of evidence indicate that the Rad23 protein
(Rad23p) may regulate the activity of the 26 S proteasome. We report
evidence that a de-N-glycosylating enzyme, Png1p, may be
involved in the proteasomal degradation pathway via its binding to
Rad23p. Interaction of Rad23p and Png1p was first detected by
two-hybrid screening, and this interaction in vivo was
confirmed by biochemical analyses. The Png1p-Rad23p complex was shown
to be distinct from the well established DNA repair complex,
Rad4p-Rad23p. We propose a model in which Rad23p functions as an escort
protein to link the 26 S proteasome with proteins such as Rad4p or
Png1p to regulate their cellular activities.
Proteins that transit through the secretory pathway are subjected
to a quality control system (1) in the endoplasmic reticulum (ER)1 that recognizes
aberrantly folded proteins/glycoproteins. It has been shown that in
some cases these misfolded and/or unfolded proteins are degraded by
ER-associated degradation mechanisms, which involves retrograde
transfer of proteins from the ER to the cytosol followed by degradation
by the proteasome (2-7). Previously, we described PNG1, a
gene encoding a cytoplasmic deglycosylating enzyme,
peptide:N-glycanase (PNGase), that is evolutionarily
conserved throughout eukaryotes (8). It has been suggested that this enzyme activity is linked to a proteasomal degradation pathway and has
a role in efficient degradation of glycoproteins by the proteasome
(8-13). This would be achieved by removing bulky N-linked glycans from misfolded glycoproteins that are translocated from the
lumen of the ER into the cytosol for degradation. However, a physical
link between the proteasome and this deglycosylating enzyme has not yet
been described.
Rad23p is known to have a pivotal role in nucleotide excision repair
(14-16). Yeast Rad23p stoichiometrically forms a complex with Rad4p to
form nucleotide excision repair factor 2 (NEF2). Unlike other NER
proteins, the biochemical functions of NEF2 still remain largely
unknown. However, NEF2 was recently shown to bind specifically to
damaged DNA in an ATP-independent manner (17-19). While the absence of
Rad4p (rad4 One structural feature of Rad23p is that it contains a ubiquitin-like
domain (UbL) at the N terminus that can bind to the 26 S proteasome
(24). This association appears to be important for DNA repair (24-26).
In addition, Rad23p was shown to have an overlapping function with
Rpn10p, a 19 S proteasome subunit that is known to be a multiubiquitin
chain-binding receptor for the proteasome (27). It has been suggested
that the Rad23p is a negative regulator of multiubiquitin chain
assembly (28). However, thus far, the link between DNA repair and
proteasome degradation with respect to Rad23p function remains elusive.
We report here a finding that the deglycosylation enzyme, Png1p, exists
as a high molecular weight complex with Rad23p. The Png1p-Rad23p
complex was found to be distinct from the well established DNA repair
complex, Rad4p-Rad23p (NEF2). In addition, we report that the
Png1p-Rad23p complex interacts with the 26 S proteasome. These findings
led us to hypothesize that Rad23p may function to link the 26 S
proteasome with other proteins, such as Rad4p or Png1p. This
"escort" property of Rad23p may explain the complex effect of
Rad23p-proteasome associations in a variety of cellular processes,
including deglycosylation of glycoproteins slated for degradation.
Yeast Strains and Media--
The yeast strains used in this
study were the following: BY4742 (MAT PNGase Activity Assay--
PNGase activity was assayed in yeast
lysates using fetuin-derived asialoglycopeptide I
([14C]CH3)2Leu-Asn(GlcNAc5Man3Gal3)-Asp-Ser-Arg)
as described previously (35, 36). Radioactivity was monitored on a
PhosphorImager (Molecular Dynamics, Inc.) and quantitated using
ImageQuant (version 1.2). One unit was defined as the amount of enzyme
that catalyzed hydrolysis of 1 µmol of fetuin-derived
asialoglycopeptide I/h.
Construction of Plasmids--
DNA manipulations were performed
according to Sambrook et al. (37). Plasmids used in this
study are listed in Table I. pCS13 was a
kind gift from Dr. Kiran Madura. The yeast genes used in this study
were isolated from the genomic DNA of W303-1a by polymerase
chain reaction using Vent DNA polymerase (New England Biolabs). For
isolation of the PNG1 gene, the following primers were used:
5'-AAAAAGAATTC-ATGGGAGAGGTATACGAAAAAA-3' (5'-primer) and
5'-AAAAACTCGAG-CTATTTACCATCCTCCCCACGC-3' (3'-primer). The amplified
fragments were digested with EcoRI/XhoI and
cloned into EcoRI/SalI sites of pBTM116 (29) and
EcoRI/XhoI sites of pRD53 (38). The pBTM116-PNG1
was subsequently digested with MluI/PstI and
cloned into MluI/PstI sites of pBTM116-ADE2 (39)
to give rise to pBTM116-ADE2-PNG1. pRD53-PNG1His6 was
prepared by amplifying the PNG1 gene using another
3'-primer, 5'-AAAAACTCGAGTCAGTGGTGGTGGTGGTGGTGTTTACCATCCTCCCCACG-3', and the EcoRI/XhoI fragments were cloned
into pRD53. pRS314-GAL1PNG1His6 was prepared by
digestion of pRD53-PNG1His6 with
NotI/XhoI and cloning the PNG1-containing
fragment into NotI/XhoI sites of pRS314 (40).
pESC-TRP-RAD4(Myc) was constructed by co-transformation of
ApaI/SalI-digested pESC-TRP (Stratagene) and
polymerase chain reaction-amplified RAD4 gene using the
following primers:
5'-AGAAAAAACCCCGGATCCGTAATACGACTCACTATAGGGCGAATTCATGAATGAAGACCTGCCCAAGG-3' (5'-primer) and
5'-AAGCTTACTCGAGGTCTTCTTCGGAAATCAACTTCTGTTCGTCGACGTCTGATTCCTCTGACATCTC-3' (3'-primer). The Trp+ colonies were isolated, and the
transformants bearing plasmids with the correct insert were identified
by colony polymerase chain reaction. The expression of the Rad4Mycp was
further confirmed by Western blotting. The RAD23 gene was
isolated by polymerase chain reaction using the primers
5'-AAAAAGAATTCATGGTTAGCTTAACCTTTAAAAATTTC-3' (5'-primer) and
5'-AAAAAGTCGACTCAGTCGGCATGATCGCTGAATAG-3' (3'-primer), and the
amplified DNA was digested with EcoRI/SalI and
cloned into pGAD424 (41). Other truncated versions of RAD23
(amino acids 78-398, 186-398, 253-398, 1-185, 1-252, 1-317, and
1-354) were amplified in a similar manner using primers that contain EcoRI and SalI sites and cloned into pGAD424. The
sequences of the resulting constructs were confirmed.
Yeast Two-hybrid Library Screening--
The two-hybrid
experiments were carried out as described previously (39). Strain L4O
was transformed with pBTM116-ADE2-PNG1 (target plasmid) and a
two-hybrid genomic library (pGAD library (Ref. 42; kindly provided by
Dr. Phillip James). After screening 2.2 × 106
transformants in a yeast genomic library, two distinct GAD library plasmids that showed a reproducible His+
Gel Filtration Analysis--
Yeast cytosol was prepared using
glass beads as described previously (43) except that the extraction of
cytosol was carried out in the presence of a protease inhibitor mixture
(final concentrations: leupeptin, 1 µg/ml; antipain, 2 µg/ml;
benzamide, 10 µg/ml; chymostatin, 1 µg/ml; pepstatin, 1 µg/ml;
phenylmethanesulfonyl fluoride, 1 mM). Preparation of a
crude extract of bacterially expressed Png1p was reported elsewhere
(8). 0.5 ml (~10 mg of protein) of cytosol were loaded on a Sephacryl
S-300 column (Amersham Pharmacia Biotech; 1.5 × 50 cm)
equilibrated with elution buffer (20 mM Hepes buffer (pH
6.8), 5 mM magnesium acetate, 1 mM
dithiothreitol, 2 mM ATP, 150 mM NaCl,
and 0.4 M sorbitol with protease inhibitor mixture as
described above), and fractions of 0.9 ml were collected. Fractions were assayed for PNGase activity. For protein determination, 0.3 ml of
fractions were precipitated with 10% trichloroacetic acid, and the
tagged protein was visualized by Western blotting.
Western Blot Analysis--
Western blot analysis was carried out
as described (8) using 1:10 dilution of mouse anti-HA (tissue culture
supernatant; 12CA5) or 1:1000 dilution of rabbit or mouse anti-Myc
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) rabbit or mouse
anti-His6 (Santa Cruz Biotechnology), or mouse anti-FLAG
antibody (M2; Sigma) followed by a 1:2000 dilution with the anti-rabbit
or mouse IgG horseradish peroxidase-conjugated secondary antibody
(Roche Molecular Biochemicals). 10% SDS-PAGE gels were used, and gels
were visualized using chemiluminescence (KPL) after exposure to medical
x-ray film (Fuji).
Co-immunoprecipitation--
Immunoprecipitation experiments were
carried out as previously described (27). Briefly, cell extracts were
prepared in lysis buffer (20 mM Hepes-KOH, pH 7.5, 100 mM potassium acetate, 5 mM EDTA, 10% glycerol)
including various protease inhibitors as described above, and equal
amounts of extract (2 mg of total protein) were incubated with 20 µl
of protein G-agarose with and without respective anti-Tag antibodies
(rabbit anti-Myc (Santa Cruz Biotechnology); rabbit anti-HA (Santa Cruz
Biotechnology); rabbit anti-His6 (Santa Cruz
Biotechnology), 1:50 in dilution; or mouse anti-FLAG antibody (Sigma),
1:250 in dilution) and incubated overnight at 4 °C. The immunoprecipitates were washed twice with buffer A (50 mM
Hepes-NaOH, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1%
Triton X-100), dissolved in 20 µl of sample buffer, and analyzed by
10% SDS-PAGE. Subsequently, Western blotting analysis was carried out
as described above.
UV Sensitivity Analysis--
For qualitative UV sensitivity
analysis, cells were grown in synthetic dextrose (SD)
For quantitative assay for UV treatment, cells were grown in the
respective medium (SD Png1p Forms a High Molecular Weight Complex in Yeast
Cytosol--
Our initial assumption that Png1p might be a part of a
multiprotein complex in yeast was based on the results of gel
filtration analysis of a cell-free yeast extract. As shown in Fig.
1, PNGase activity measurement of the gel
filtration fractions showed distinct differences between the elution
position of Png1p in the yeast cytosol and that of Png1p expressed in
Escherichia coli. While the bacterially expressed protein
showed the expected molecular mass of a monomer form (~45 kDa), the
yeast cytosol protein had a much higher molecular mass (estimated to be
~200 kDa). This result suggested that in the yeast cytosol Png1p may
bind to other proteins to form a high molecular weight protein
complex.
Rad23p Specifically Binds to Png1p--
The observation described
above led us to carry out two-hybrid library screening with
PNG1 as a bait to search for possible Png1p-binding
proteins. Upon screening 2.2 × 106 transformants with
a yeast genomic library, we recovered two distinct plasmids that showed
a reproducible His+ Rad23p Binds to Png1p in Yeast Cytosol--
To confirm the
interaction of Png1p with Rad23p in vivo, gel filtration
analysis was carried out to examine the migration of these two
proteins. First, we determined the elution position of Png1p in
rad23
To provide direct evidence for interaction of Rad23p and Png1p,
FLAG-tagged Rad23p (FLAG-Rad23p; Ref. 24) was expressed under an
inducible CUP1 promoter. This protein construct was used to
show interaction of Rad23p with the 26 S proteasome as well as with
Rad4p (24, 27). Png1p is known to be present in extremely low abundance
in cells under normal experimental conditions (8), while Rad23p is
relatively abundant (17). Therefore, only a minute fraction of Rad23p
was expected to be bound to Png1p; this would make it difficult to
observe co-migration of Png1p and Rad23p. For this reason, Png1p was
overexpressed using a inducible GAL1 promoter in a
png1 The C Terminus of Rad23p Is Critical for Its Binding to
Png1p--
Having biochemical evidence that Png1p binds to Rad23p in
yeast cytosol, we determined which domain of Rad23p was involved in
interaction with Png1p using the two-hybrid assay. Deletion constructs
of GAL4 activation domain-RAD23 were tested for
binding against lexA-PNG1. As shown in Table
II, deletion of N-terminal portions of
Rad23p did not have a significant effect on its binding to
Png1p. In sharp contrast, when the C-terminal region of Rad23p was
truncated, no interaction with Png1p was observed. This result most
likely suggested that the C-terminal ubiquitin-associated domain
(UBA) of Rad23p (see Fig. 2) was important for its binding to Png1p.
Png1p Interacts with the 26 S Proteasome Subunit in a
Rad23p-dependent Manner--
Since Rad23p has been shown
to require the N-terminal UbL for its interaction with the 26 S
proteasome (24), we assumed that Png1p might associate with the 26 S
proteasome through Rad23p. To test this hypothesis,
co-immunoprecipitation analysis with His6-tagged Png1p
(Png1-His6p) and the GFP/HA-tagged 26 S proteasome subunit,
Rpt1p, was carried out. The His6-tagged Png1p was expressed under the GAL1 promoter. The His6-tagged Png1p
was shown to express enzyme activity (data not shown). The
GFP/HA-tagged Rpt1p was previously shown to be integrated into a
functional 26 S proteasome complex (31). Cell lysates were prepared
from cells expressing Png1-His6p with or without
FLAG-Rad23p in png1 Png1p Associates with Rad23p in a Complex Distinct from the
Rad23p-Rad4p Complex (NEF2)--
The C-terminal domain of Rad23p,
identified above as a critical binding region to Png1p, was previously
shown to be critical for the Rad23p-Rad4p interactions (44). Therefore,
it was possible that Png1p and Rad4p might compete for the same binding
site on Rad23p. If so, one might expect Png1p to associate with Rad23p as a complex distinct from the Rad4p-Rad23p (NEF2 complex). To test
this hypothesis, co-immunoprecipitation experiments were carried out
using three different tags: His6 tag on Png1p, FLAG tag on
Rad23p, and Myc tag on Rad4p. As shown in Fig.
6, Rad23p could be detected when either
Png1p His6 or Rad4p (Myc) was precipitated (lanes 1 and 3). This interaction was
also confirmed by reciprocal precipitation (lanes
5 and 9). However, no interaction (above background level) was observed between Png1p and Rad4p (compare lane 7 with lane 8, and
compare lane 11 with lane
12), supporting the idea that the Png1p-Rad23p complex is
distinct from the Rad4p-Rad23p complex (NEF2).
Overexpression of Png1p Causes Moderate UV Sensitivity--
Having
immunochemical evidence for the existence of two distinct complexes,
Rad4p-Rad23p and Png1p-Rad23p, we carried out genetic experiments to
confirm the formation of two complexes. The assumption made was that
overexpression of Png1p would prevent the formation of NEF2
(Rad4p-Rad23p complex), thereby causing UV sensitivity of yeast cells
even in the presence of Rad23p. For this experiment, we used
png1
Using a more quantitative UV sensitivity assay, we confirmed that
overexpression of Png1p under the experimental conditions described
above caused moderate sensitivity (10-fold decrease in survival) (Fig.
7B). This relatively moderate effect may be caused by a
residual amount of NEF2 (Rad4p-Rad23p) formation. As expected when
Png1p alone (without Rad23p) was induced, the sensitivity was very
great (1,000-fold decrease in survival).
The de-N-glycosylation process catalyzed by PNGase has
been proposed to be involved in proteasomal degradation of misfolded glycoproteins following their transfer from the ER to the cytosol (9-13). Earlier we reported that S. cerevisiae has a
soluble PNGase activity that is very similar to the soluble PNGase
found in higher eukaryotes (36). Subsequently, we isolated the gene
encoding this enzyme, PNG1, by isolating a mutant
that is defective in PNGase activity and then mapping of the locus
responsible for the loss of this activity (8). Comparison of the
protein sequence of yeast Png1p with a number of sequences in other
eukaryotic data bases revealed that this enzyme is highly conserved,
suggesting that it was functionally important in all eukaryotes.
Following the finding that the apparent molecular mass of PNGase
detected in the cytosol by an enzyme assay was much greater than its
calculated mass of 42.5 kDa, we carried out a two-hybrid analysis to
identify yeast proteins that interact with Png1p. By this screening,
two distinct candidates were isolated, both of which encoded a part of
Rad23p. In a rad23 In this experiment, using Png1p-overexpressed cells, the finding of two
distinct peaks of PNGase activity may be due to the fact that a
fraction of the large amount of Png1p produced under these conditions
was bound to Rad23p. The calculated apparent molecular mass of this
complex is ~200 kDa, which is approximately twice as large as the
expected molecular mass of a 1:1 stoichiometric Rad23p-Png1p complex.
This large molecular mass could be due to the presence of more
than one molecule of each of the subunits or a change in conformation
of the proteins in a complex. Alternatively, other proteins may also be
present in the complex; currently, we are investigating this possibility.
Interaction studies of Png1p using deletion constructs of Rad23p
provided two important conclusions. First, the N-terminal UbL, which is
important for Rad23p to interact with the proteasome (24), was found
not to be required for Rad23p binding to Png1p. This finding led us to
hypothesize that a physical interaction of Png1p with the 26 S
proteasome might be mediated by Rad23p. This hypothesis was confirmed
by a co-immunoprecipitation analysis that utilized an antibody to an
epitope-tagged subunit of the 26 S proteasome (Rpt1p) and
His6-tagged Png1p. This interaction between Png1p with the
26 S proteasome was Rad23p-dependent, which is consistent
with the idea of Rad23p being an "escort protein" to connect Png1p
and the 26 S proteasome. It has been reported that there is a so-called
"membrane-associated form" of PNGase, which can be precipitated by
ultracentrifugation and has enzymatic characteristics similar to those
of the soluble enzyme, in both animal cells and yeast (11, 36, 45). The
possibility that this so-called membrane-bound form is actually a
proteasome-associated form should be evaluated.
The second important observation regarding domain interaction of Rad23p
was that the C-terminal ubiquitin-associated domain of Rad23p was
critical for its binding to Png1p. This result is reminiscent of the
previously reported domain interaction of Rad4p-Rad23p (NEF2) (44). Our
results, together with the previous observation, suggest that Png1p and
Rad4p might both independently bind to the same domain of Rad23p. In
fact, the formation of a Png1p-Rad23p complex that is distinct from
NEF2 (Fig. 8) was confirmed by
co-immunoprecipitation analysis and further supported by genetic
analysis. We have also confirmed that the mouse homologue of Png1p can
bind to mouse Rad23p
homologues.2 In this
connection, it should be noted that Rad4p is the only structurally
related protein among Saccharomyces cerevisiae
proteins that was found to contain a domain similar to one in Png1p
(based on BLAST analysis; E value = 0.023; Png1p amino
acids 220-321 versus Rad4p amino acids 316-434;
identities, 30/119 (25%); positives, 53/119 (44%); gaps, 17/119
(14%)). This fact may mean that this region has in common the same
folding structure, which could be important for binding to the C
terminus region of Rad23p.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) causes extreme sensitivity to UV light in
yeast, rad23
mutants exhibited only moderate UV sensitivity, indicating that Rad23p may affect the efficiency of the
excision repair process rather than directly mediating the repair of
damaged DNA (20-23).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
his3
1 leu2
0 lys2
0
ura3
0; Ref. 8); Research Genetic strain 10278 (BY4742 rad23
::KanMX4); L4O
(MATa ade2 his3 leu2 trp1 LYS2::lexAop-HIS3
URA3::lexAop-lacZ; Ref. 29); AMR70 (MAT
ade2 leu2 his3 trp1 URA3::lexAop-lacZ; Ref. 29);
W303-1a (MATa ade2-101 his3-11, 15 leu2-3, 112 trp1-1
ura3-1 can1-100); TSY190 (W303-1a
rad23
::URA3
png1
::his5+(pombe)
FOAR); and TSY195 (TSY190
RPT1-GFP-HA::URA3::HIS3).
TSY190 was prepared by crossing TSY146 (W303-1a
png1
::his5+(pombe);
Ref. 8) and MGSC101 (W303-1b (W303-1a MAT
)
rad23
::URA3; Ref. 30; kindly
provided by Dr. Jaap Brouwer, Leiden) followed by isolating haploid
segregants of the appropriate genotype and selection of 5-fluoroorotic
acid (FOA)-resistant cells on FOA plates. TSY195 was prepared by
transforming TSY190 with XhoI/NotI digests of
pBS-CIM5-GFPHA-HU (Ref. 31; kindly provided by Dr. Cordula
Enenkel, Humboldt Universität, Berlin) and isolating Ura+His+ transformants. Correct integration of
the transformant was confirmed by colony PCR as well as the expression
of Rpt1-GFP-HAp by Western blotting using mouse anti-HA antibody
(12CA5). Standard yeast media and genetic techniques were used
(32-34).
Plasmids used in this study
-galactosidase+ phenotype in PNG1-specific
manner were recovered. These candidate GAD plasmids were then
transformed into strain L4O and mated with AMR70 cells with each of
several plasmids encoding various lexA-plasmids. These
candidate plasmids did not exhibit an interaction with the lexA-lamin or lexA-SIR4 C terminus,
both of which have been used to test for potential false positives in
two-hybrid library screening (39). However, isolated plasmids showed
the His+
-galactosidase+ phenotype with the
original target plasmids.
Leu
Ura
+galactose or
Leu
Ura +glucose medium to saturation. Cells were
further cultured in the presence of 0.1 mM
CuSO4 for another 2 h, and the cell density was
normalized to A600 = 1.0 (~5.0 × 106 cells/ml). Cells were taken from the aliquot with
Q-tips and streaked as a single line onto the respective plate (
Leu
Ura +galactose +0.1 mM CuSO4 or
Leu
Ura
+glucose +0.1 mM CuSO4). After these plates
were covered with a glass plate, they were placed 20 cm distant from
the germicidal UV light (254 nm; Sylvania). Then the glass plate was
slid parallel to the line of cells so that the cells were exposed to UV
for different times from 0 to 8 s. The plate was then incubated at
25 °C in the dark for 4 days. If a cell line had UV sensitivity,
cells would not be expected to grow all the way across the line when
exposed to UV. In contrast, wild-type cells would be expected to
exhibit growth across the entire line even when they were exposed to
the maximum dose of UV.
Leu
Ura +glucose or SD
Leu
Ura +galactose) overnight, and after 0.1 mM CuSO4
was added, they were cultured for 2 h, plated on YPAD at
appropriate dilutions, and then exposed to 254-nm UV light using a UV
cross-linker (UV Statalinker model 1800; Stratagene) at given doses.
Cells were plated in triplicate and incubated at 25 °C in the dark
for 3 days, and the number of surviving colonies were counted.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Gel filtration profile of PNGase activity in
yeast cytosol and an E. coli extract expressing
Png1p. Yeast cytosol or E. coli extracts (0.5 ml) were
applied to a Sephacryl S-300 gel filtration column (1.5 × 50 cm),
and fractions of 0.9 ml were collected and assayed for PNGase activity.
, PNGase activity in yeast cytosol;
, PNGase activity in E. coli extract. V0, void volume. The
arrows denote positions of the marker proteins: 440 kDa,
ferritin; 69 kDa, bovine serum albumin; 45 kDa, ovalbumin.
-galactosidase+
phenotype in a target plasmid (lexA-PNG1)-specific manner.
Sequencing of inserts recovered from these two plasmids showed that
they consisted of two different Rad23p fragments (one containing amino acid residues 218-398 and the other containing residues 229-398) fused in frame to the GAL4 activation domain sequence (Fig.
2). These plasmids did not exhibit an
interaction with either lexA-lamin or lexA-SIR4 C
terminus, which have been used to test for potential false positives in
two-hybrid library screening (39), suggesting that the interaction
observed was specific (data not shown).
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Fig. 2.
Schematic representation of Rad23p. UbL,
ubiquitin-like domain; UBA, ubiquitin-associated domains,
which are found in various ubiquitin-related proteins (55). The
bars below indicate the domains of Rad23p that
were isolated as Png1p-binding proteins by two-hybrid screening.
cells. As shown in Fig.
3, a drastic shift in elution position of
this enzyme to a lower apparent mass was observed in
rad23
cells, and the elution position was similar to that
for bacterially expressed Png1p. This result suggested that the high
molecular complex containing Png1p might also contain Rad23p.
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Fig. 3.
Gel filtration profile of PNGase activity in
yeast cytosol prepared from wild-type and rad23D
cells. Yeast cytosol (0.5 ml) was applied to a Sephacryl
S-300 gel filtration column (1.5 × 50 cm) and fractions of 0.9 ml
were collected and monitored for PNGase activity. , PNGase activity
in yeast cytosol prepared from wild-type yeast (BY4742);
, PNGase activity in yeast cytosol prepared from
rad23
cells (BY4742
rad23
::KanMX4; purchased from
Research Genetics).
rad23
strain for this experiment
(TSY190; for details of genotypes of strains, see "Experimental
Procedures"). When both proteins were expressed, Png1p exhibited two
peaks of activity, one corresponding to the higher molecular mass peak and one similar to bacterially expressed Png1p (Fig.
4A). FLAG-Rad23p was shown to
co-migrate with the first peak and not with the second peak (Fig. 4,
A and B). The proteasome was shown to be present in fractions 34-40 by activity assay as well as Western blotting using
anti-Rpt1p antibody, showing that the first PNGase activity peak did
not contain the proteasome complex (data not shown). When only Png1p
was overexpressed in the absence of Rad23p (rad23
), the
higher molecular weight peak was not detectable (data not shown; see
also Fig. 3). When only Rad23p was overexpressed in the absence of
Png1p (png1
), the elution position of Rad23p was shifted
to a lower molecular weight (Fig. 4C), suggesting that when
Png1p was expressed, all of Rad23p detected co-migrated with Png1p.
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Fig. 4.
Co-migration of Png1p and Rad23p.
A and B, yeast cytosol was prepared from
TSY190 (W303-1a rad23 ::URA3
png1
::his5+(pombe)
FOAR) bearing pCS13
(PCUP1::FLAG-RAD23) and pRD53-PNG1
(PGAL1::PNG1) grown overnight in SD
Ura
Leu +galactose medium containing 0.1 mM
CuSO4. Gel filtration was performed on a Sephacryl S-300
gel filtration column (1.5 × 50 cm), and fractions of 0.9 ml were
collected. A, profile of PNGase activity after gel
filtration. B, Western blotting using mouse anti-FLAG
antibody to detect FLAG-Rad23p. FLAG-Rad23p was detected in fractions
corresponding to the first peak (fractions 44-50) of PNGase activity.
C, yeast cytosol was prepared from TSY190 bearing pCS13 and
pRD53 as a png1
control. Culture conditions were same as
above for PNG1-overexpressed cells (Fig. 4A).
FLAG-Rad23p was detected in fractions 54-62 using mouse anti-FLAG
antibody.
Mapping of interaction domains of the Rad23p to Png1p assessed by yeast
two-hybrid assay
rad23
cells, and
co-immunoprecipitation was carried out using anti-His6 or anti-HA antibody. As shown in Fig. 5,
Png1p and Rpt1p co-immunoprecipitated considerably more than background
level, which might represent nonspecific interaction of proteins with
resin (compare lanes 1 and 3 with
lanes 2 and 4). This interaction was
observed only in the presence of Rad23p (Fig. 5; lanes
5 and 7). The fraction of Png1p-Rad23p bound to
the 26 S proteasome under these experimental conditions was estimated
to be 2-5% based on a comparison of intensity of a band detected by
Western blotting of whole extract with the intensity of
immunoprecipitate pulled down by Rpt1-GFP-HAp (data not shown).
View larger version (27K):
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Fig. 5.
Co-immunoprecipitation of Png1p with 26 S proteasome. TSY195 (W303-1a
rad23 ::URA3
png1
::his5+(pombe)
FOAR
RPT1-GFP-HA::URA3::HIS3)
bearing pRS314-GAL1PNG1His6
(PGAL1::PNG1-His6) and either
pCS13 (PCUP1::FLAG-RAD23) or YEp351
(control) was cultured in SD
Trp
Leu +galactose containing 0.1 mM CuSO4 overnight. Yeast extract was prepared,
and equal amounts of protein were incubated with antibody
(Ab) against His6 (lanes 1 and 5) or HA (lanes 3 and
7). Then protein G-agarose was added for immunoprecipitation
(IP). Following SDS-PAGE, the immunoprecipitated proteins
were detected by immunoblotting using anti-HA (lanes
1, 2, 5, and 6) or
anti-His6 (lanes 3, 4,
7, and 8). For controls, incubation of protein
G-agarose without antibody was also carried out (lanes
2, 4, 6, and 8). In some
experiments, a background level of proteins was detected in these
control lanes, which is most likely due to antibody-independent
precipitation of proteins. For comparison of protein amounts between
cells bearing Rad23p (pCS13; lane 9) and control
(YEp351; lane 10), one-tenth of the extract used
for immunoprecipitation was analyzed by SDS-PAGE followed by Western
blotting using anti-His6 as a probe to detect
Png1-His6p.
View larger version (25K):
[in a new window]
Fig. 6.
Png1p forms a complex with Rad23p that is
distinct from nucleotide excision repair factor 2 (Rad23p-Rad4p
complex). TSY190 (W303-1a
rad23 ::URA3
png1
::his5+(pombe)
FOAR) bearing pESC-TRP-RAD4(Myc)
(PGAL1::RAD4-Myc), pCS13
(PCUP1::FLAG-RAD23), or
pRD53-PNG1His6)
(PGAL1::PNG1-His6) was
cultured in SD
Ura
Trp
Leu +galactose medium containing 0.1 mM CuSO4 overnight. After the cell extract was
prepared, immunoprecipitation (IP) was carried out using
antibody (Ab) against FLAG (lanes 5 and 9), Myc (lanes 3 and 7)
and His6 (lanes 1 and 11).
The immunoprecipitate was analyzed by 10% SDS-PAGE followed by Western
blotting. For controls, incubation of protein G-agarose without
antibody was also carried out (lane 2,
4, 6, 8, 10, and
12).
rad23
cells. Png1p was expressed overnight using an inducible GAL1 promoter, and then
FLAG-Rad23p was transiently expressed for 2 h using the
CUP1 promoter. First, a qualitative test was performed to
check the UV sensitivity of these cells. As shown in Fig.
7A, under the experimental
conditions described above, cells overexpressing Png1p exhibited
moderate UV sensitivity (compare sample 2 with sample 1). This level of sensitivity was not seen when the same experiment was carried out
without induction of Png1p (compare sample 2C and sample 1C), indicating that this effect was Png1p-dependent. That a
comparable amount of Rad23p was expressed in samples 1 and 2 was
confirmed by Western blotting, excluding the possibility that the
observed UV sensitivity was due to the amount of Rad23p expressed in
cells (data not shown). In contrast to the above experiment, when we expressed FLAG-Rad23p overnight, we observed no apparent UV sensitivity in PNG1-overexpressing cells, suggesting that the observed
UV sensitivity is a transient effect and cells can make enough
Rad4p-Rad23p complex at stationary stage even in the presence of large
amount of Png1p (data not shown). With this assay, png1
cells exhibited no apparent UV sensitivity, implying that
PNG1 itself does not have a role in nucleotide excision
repair (data not shown).
View larger version (45K):
[in a new window]
Fig. 7.
Sequential overexpression of Png1p followed
by Rad23p causes moderate UV sensitivity. A, cells used
were as follows. 1, TSY190 (W303-1a
rad23 ::URA3
png1
::his5+(pombe)
FOAR) bearing pCS13
(PCUP1::FLAG-RAD23) and pRD53 (no
Png1p control); 2, TSY190 bearing pCS13 and pRD53-PNG1
(PGAL1::PNG1); 3, TSY190
bearing YEp351 (no Rad23p control) and pRD53-PNG1. The absence of cell
growth at the right end of the streak is
indicative of UV sensitivity of these cells. For control, the same
experiment was carried out using glucose medium so that expression of
Png1p could not occur (samples 1C, 2C, and 3C). B,
quantitative analysis of UV sensitivity was carried out. Yeast cells
were exposed to 254-nm UV light, and the survival was determined.
Sample 2 (
; +Rad23p, +Png1p) displayed intermediate sensitivity
between sample 1 (
; +Rad23p,
Png1p) and sample 3 (×;
Rad23p,
+Png1p).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain, we observed a dramatic
decrease in elution position of the yeast Png1p to a position similar
to that of bacterially expressed Png1p. These results supported the
idea that the formation of a higher molecular weight complex of Png1p
involved Rad23p. This was further confirmed by showing that
epitope-tagged Rad23p co-migrated with Png1p during gel filtration
analysis of the yeast cytosol.
View larger version (25K):
[in a new window]
Fig. 8.
Diagram of postulated two distinct Rad23p
complexes. The Rad4p-Rad23p (left) is a well
characterized complex required for DNA repair (17-19) that is known to
associate with the proteasome (24). This interaction has been
shown to be important for the DNA repair functions (24, 25) in a
proteolytic activity-independent manner (26). A postulated second
complex contains Png1p and Rad23p (right). This complex is
also associated with the 26 S proteasome and may have a function as a
specialized protein degradation complex used for degradation of
misfolded glycoproteins (see "Discussion").
The biological significance of the interaction of the 26 S proteasome with Rad23p still remains to be understood. Now it is evident that there are at least two distinct complexes involving the proteasome and Rad23p (Fig. 8). This finding may to some extent explain the complex role of Rad23p-proteasome interactions. We hope that in the future it will be possible to dissect the specific functions of proteasome or Rad23p in each distinct complex. In this regard, it is of interest that a number of domain-specific Rad23p-binding proteins, other than the nucleotide repair proteins, have been identified in mammalian cells (46-48). These studies, combined with our results, lead us to speculate that such domain-specific interactions may allow Rad23p to connect a proteasome protein through its N terminus to other proteins at its C terminus. This link might be necessary to modulate functions of various binding proteins. Most intriguing is the finding that a human homologue of Dsk2p, a Rad23p-like protein, has recently been shown to provide a link between the proteasome and ubiquitin ligase (49). Thus, a general feature of UbL-containing proteins would be to link proteins to the proteasome in order to control or modulate the function of these proteins.
While the precise biological function of Png1p is still unclear, it is
important to note that this enzyme has been proposed to be involved in
proteasome degradation of misfolded glycoproteins in mammalian cells
(9, 10). We have previously shown that glycopeptides exported from the
ER to the cytosol in yeast are subsequently deglycosylated by the
activity of Png1p (36). However, because of the inefficient uptake of
proteasome inhibitors (50), it has not been possible to observe an
accumulation of de-N-glycosylated protein intermediates in
yeast. Interestingly, biochemically purified PNGase from mammalian
cells, as well as the purified bacterially expressed yeast Png1p, do
not act on intact glycoprotein substrates in vitro (8, 35).
As discussed earlier (8), these observations may be relevant to the
fact that the 20 S catalytic proteasome subcomplex can only act
efficiently on small peptide substrates in vitro, and it has
been proposed that the function of the 19 S ATPases is to unwind
protein substrates prior to their degradation by the 20 S proteolytic
complex (51, 52). Such "protein-unfolding" molecular chaperone
activities of 19 S subunits have been recently reported (53, 54).
Therefore, the association of Png1p with the 26 S proteasome would
produce a complex in which de-N-glycosylation and
proteolysis of unwound glycoprotein substrates could be accomplished in
an efficient manner. Our findings indicate that the highly conserved
enzyme, Png1p, is a part of the proteasomal degradation machinery.
Further studies should reveal its precise role in the degradation of
misfolded glycoproteins by this pathway.
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ACKNOWLEDGEMENTS |
---|
T. S. gratefully acknowledges the continuous encouragement of Drs. Yasuo and Sadako Inoue (Academia Sinica, Taiwan). We thank Dr. Jaap Brouwer (Leiden University, Leiden, Netherlands), Dr. Cordula Enenkel (Humboldt Universität, Berlin, Germany), Dr. Kiran Madura (UMDNJ, Piscataway, New Jersey), and Dr. Phillip James (University of Wisconsin, Madison) for providing various materials. We also thank our associates, Drs. JoAnne Engebrecht, Xiao-Dong Gao, Robert Haltiwanger, Noritaka Hirohashi, Nancy Hollingsworth, Janet Leatherwood, Aaron Neiman, Rolf Sternglanz, and Ann Sutton, for various suggestions and technical comments on this study. We thank members of the Lennarz laboratory for useful discussions and Lorraine Conroy for manuscript preparation.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM33184 (to W. J. L.).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.
To whom correspondence should be addressed. Tel.: 631-632-8560;
Fax: 631-632-8575; E-mail: wlennarz@notes.cc.sunysb.edu.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M100826200
2 H. Park, T. Suzuki, and W. J. Lennarz, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ER, endoplasmic reticulum; PNGase, peptide:N-glycanase; UbL, ubiquitin-like domain; FOA, 5-fluoroorotic acid; PAGE, polyacrylamide gel electrophoresis; GFP, green fluorescent protein; HA, hemagglutinin.
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