A plant peptide: N-glycanase orthologue facilitates glycoprotein ER-associated

degradation in yeast
Yuki Masahara-Negishi
a, 1
, Akira Hosomi
a, 1
, Massimiliano Della Mea
b
,
Donatella Serani-Fracassini
b
, Tadashi Suzuki
a,

a
Glycometabolome Team, RIKEN Advanced Science Institute, 21 Hirosawa, Wako, Saitama 3510198, Japan
b
Dipartimento di Biologia Evoluzionistica Sperimentale, Universit di Bologna, 40126 Bologna, Italy
a b s t r a c t a r t i c l e i n f o
Article history:
Received 24 February 2012
Received in revised form 14 May 2012
Accepted 21 May 2012
Available online 31 May 2012
Keywords:
Peptide:N-glycanase
ER-associated degradation
Yeast
Background: The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme involved in the ER-
associated degradation (ERAD) process, while ERAD-independent activities are also reported. Previous bio-
chemical analyses indicated that the cytoplasmic PNGase orthologue in Arabidopsis thaliana (AtPNG1) can
function as not only PNGase but also transglutaminase, while its in vivo function remained unclaried.
Methods: AtPNG1 was expressed in Saccharomyces cerevisiae and its in vivo role on PNGase-dependent ERAD
pathway was examined.
Results: AtPNG1 could facilitate the ERAD through its deglycosylation activity. Moreover, a catalytic mutant
of AtPNG1 (AtPNG1(C251A)) was found to signicantly impair the ERAD process. This result was found to
be N-glycan-dependent, as the AtPNG(C251A) did not affect the stability of the non-glycosylated RTA
(ricin A chain non-toxic mutant). Tight interaction between AtPNG1(C251A) and the RTA was conrmed
by co-immunoprecipitation analysis.
Conclusion: The plant PNGase facilitates ERAD through its deglycosylation activity, while the catalytic mutant
of AtPNG1 impair glycoprotein ERAD by binding to N-glycans on the ERAD substrates.
General signicance: Our studies underscore the functional importance of a plant PNGase orthologue as a
deglycosylating enzyme involved in the ERAD.
Crown Copyright 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction
Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that
cleaves the -aspartyl glycosylamine bond of N-linked glycoproteins,
releasing intact N-glycans fromproteins. PNGase activity was originally
discovered in almond [1] and subsequently conrmed in bacteria [2].
Since then, this enzyme has been widely used as a tool to analyse the
structure and functions of N-linked glycans on glycoproteins. The pres-
ence of PNGase activity in animals was rst reported in Medaka sh
(Oryzias latipes) [3], and later in various mammalian-derived cultured
cells [4,5]. While the sh enzyme is believed to be of lysosomal origin
[6], mammalian PNGase is localised in the cytosol (hence, cytoplasmic
PNGase) andits optimal activity is at neutral pH[5]. Agene encoding cy-
toplasmic PNGase (PNG1) was identied in Saccharomyces cerevisiae
[7]. The orthologous genes of PNG1 are widely distributed throughout
eukaryotes.
Endoplasmic reticulum (ER)-associated degradation (ERAD) is a
component of the quality control system for newly synthesised pro-
teins. In this system, proteins that fail to fold correctly are degraded,
while functional proteins are delivered to their intended destina-
tions through the secretory pathway [8,9]. ERAD involves the extrac-
tion of proteins from the ER to the cytosol, followed by proteasomal
degradation. Cytoplasmic PNGase is involved in the efcient degra-
dation of some ERAD substrates [1012]. The PNGase-mediated
deglycosylation during ERAD was also suggested to play an impor-
tant role in antigen presentation by class I major histocompatibility
complex in mammalian cells [1316]. However, some of the PNGase
orthologues were reported to be catalytically inactive, while its mu-
tant exhibited severe phenotypic consequences [17,18], raising
the possibility that, in addition to its enzyme activity, PNGase
orthologues might have signicant enzyme-independent roles.
From an evolutionary view, cytoplasmic PNGase is an interesting
protein with a diverse structural arrangement [19,20]. The core cat-
alytic domain of cytoplasmic PNGase is highly conserved throughout
eukaryotes and, because of its homology with transglutaminase
(TGase), cytoplasmic PNGase was categorised as a member of the
TGase superfamily [19,21]. The plant orthologue of cytoplasmic
PNGase was rst identied in Arabidopsis thaliana (AtPNG1),
based on the homology of a TGase domain [22], while the regions
Biochimica et Biophysica Acta 1820 (2012) 14571462
Abbreviations: PNGase, peptide:N-glycanase; ERAD, ER-associated degradation;
TG and TGase, transglutaminase; RTA, ricin A chain non-toxic mutant; RTL, RTA-
transmembrane-Leu2; SC, synthetic complete; endo H, endoglycosidase H
Corresponding author. Tel.: +81 48 467 9628; fax: +81 48 467 9626.
E-mail address: tsuzuki_gm@riken.jp (T. Suzuki).
1
Both authors contributed equally to this work.
0304-4165/$ see front matter. Crown Copyright 2012 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2012.05.009
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
j our nal homepage: www. el sevi er . com/ l ocat e/ bbagen
outside the TGase domain are quite unique and show no apparent
homology with the primary structures of animal/fungal
orthologues (Fig. 1A). This indicates that the plant cytoplasmic
PNGase may have followed a distinct evolutional route from others
to acquire its unique function. Interestingly, AtPNG1 was reported
to possess TGase activity [23], besides PNGase activity [24], in vitro.
However, the functional role of this plant protein in vivo remains
poorly understood.
In this study, we utilized the assay system for PNGase-dependent
ERAD in yeast, using RTA (ricin A chain non-toxic mutant; [10]) or
RTL (RTA-transmembrane-Leu2; [12]) as substrates to examine if
AtPNG1 can function as PNGase in vivo. Our results clearly indicated
that AtPNG1, when expressed in yeast, can act as a deglycosylating
enzyme and facilitate the degradation of RTA/RTL. Interestingly,
its catalytic mutant, AtPNG1(C251A), was found to signicantly
stabilise the RTA. Most importantly, these results were found to
be N-glycan specic, since non-glycosylated RTA did not require
AtPNG1 for efcient degradation. Consistent with this nding,
stable binding of AtPNG1(C251A) to RTA was conrmed by co-
immunoprecipitation experiments. Taken all results together, we
demonstrated that AtPNG1 can function as a PNGase in vivo and facil-
itate the degradation of ERAD substrate in an N-glycan-dependent
fashion.
2. Material and methods
2.1. Yeast strains and media
We used the following yeast strains: cim5-1 png1 cells (MATa
cim5-1 png1:: URA3 ura3-52 leu21 his3200 FOA
R
; [25]) and png1
cells (MATa his31 leu20 met150 ura30 png1::kanMX4; [7]).
Standard yeast media and genetic techniques were used [26,27].
2.2. Plasmid construction
Mutation of the catalytic Cys residue 251 to Ala was introduced to the
pET28b-AtPNG1 plasmid [23] using QuikChange (Stratagene) to establish
pET28b-AtPNG1(C251A). To construct pRS423
GPD
-AtPNG1-FLAG and
pRS423
GPD
-AtPNG1(CA mutant)-FLAG, cDNA of A. thaliana AtPNG1 and
AtPNG1(C251A) were amplied from pET28b-AtPNG1 and pET28b-
AtPNG1(C251A), respectively, using the following primers, 5-CACCA-
TGGGAGAGGTATACGAA-3 and 5- ATGCGGCCGCCTACTTATCGTCGTC-
ATCCTTGTAATCCTGGTGACTTCTGTACAGAT-3, in which the second
primer was designed to add a C-terminal FLAG tag, and were cloned
into pENTR/D-TOPO(Invitrogen). The DNA sequences of the constructs
were conrmed using BigDye ver. 3.1 and an ABI DNA sequencer
(3730xl). The AtPNG1 and AtPNG1(C251A) genes in the pENTR vectors
A
B
C
Fig. 1. Heterologous expression of AtPNG1 and deglycosylation of RTA. (A) Schematic representation of AtPNG1 and ScPng1 (S. cerevisiae Png1). The transglutaminase (TG) do-
main (catalytic PNGase domain) is indicated. (B) Western blotting analysis of RTA. RTA was co-expressed with an empty vector (control; V), ScPng1 (Sc) or AtPNG1 (At) in cim5-
1 png1 cells. Cell extracts were resolved by SDS-PAGE and RTA was visualised by immunoblotting using an anti-ricin antibody. The arrows indicate RTA modied with one (g1)
or no (g0) glycan. The immunoblot was also probed with anti-PGK antibody as a loading control. At, Arabidopsis thaliana; Sc, Saccharomyces cerevisiae. (C) RTL assay for png1 cells
expressing AtPNG1 or its catalytic mutant, AtPNG1(C251A). AtPNG1 or AtPNG1(C251A) were co-expressed with RTL in png1 cells. Cells were plated on SC-galactose medium with
or without leucine. The plates were incubated for 3 days at 30 C.
1458 Y. Masahara-Negishi et al. / Biochimica et Biophysica Acta 1820 (2012) 14571462
were then transferred to the destination vector (pRS423
GPD
) [17] by LR
Clonase II reactions (Invitrogen) to generate pRS423
GPD
-AtPNG1-FLAG
and pRS423
GPD
-AtPNG1(C251A)-FLAG. To generate pRS316-
GPD
RTA,
pRS315
GPD
-RTA [11] was cut by SalI/NotI digestion, and the insert was
cloned into the equivalent site of pRS316. pRS316-
GPD
HA-RTAwas gen-
erated by site-directed mutagenesis (Quikchange).
2.3. Cycloheximide (CHX) decay assay
Strains harbouring the RTA expression plasmid were grown and
CHX was added to the cultures (nal concentration, 250 g/ml). The cul-
tures were collected at the indicated times, and the cells were subjected
to western blotting analysis.
2.4. Preparation of yeast cell extracts and western blotting
Preparation of yeast cell extracts and western blotting were carried
out as previously described [12]. Antibodies were used at the following
dilutions: 1:10,000 for anti-PGK (22C5) (Invitrogen) and anti-
DYKDDDDK (Wako Chemicals Co.), and 1:500 for anti-HA (sc-805)
(Santa Cruz).
2.5. Spotting assay
RTL spotting assay was carried out as previously described [12]. In
brief, strains harbouring the RTL expression plasmid were spotted
onto SC-uracil-histidine mediumor SC-uracil-histidine-leucine medium
containing galactose, and cells were incubated at 30 C for 3 days. Pho-
tographs of the gel were taken using FUJIFILM LAS-3000 mini (Fujilm
Co., Tokyo, Japan).
2.6. Immunoprecipitation
Immunoprecipitation was carried out as previously described [12].
Fig. 2. AtPNG1 acts as a PNGase and facilitates degradation of RTA, while AtPNG1(C251A) stabilises RTA. (A) Cycloheximide (CHX) decay assay for RTA in png1 cells expressing
ScPng1, AtPNG1 or AtPNG1(C251A). ScPng1, AtPNG1 and AtPNG1(C251A) were co-expressed with RTA in png1 cells. The CHX decay assays were performed in each transformant
cell line. CHX was added at t =0 min. Samples were collected at the indicated times and subjected to SDS-PAGE, followed by immunoblotting using an anti-ricin antibody. The immuno-
blot was also probed with anti-PGK antibody as a loading control. (B) Quantitation of the CHX decay assay shown in A. The log scale of the remaining protein (%) was plotted versus time.
Linear regression lines for each experiment are shown.
1459 Y. Masahara-Negishi et al. / Biochimica et Biophysica Acta 1820 (2012) 14571462
3. Results
3.1. AtPNG1 acts as a PNGase in yeast and facilitates ERAD processes
It has been reported that cytoplasmic PNGase possesses a core TGase
domain, which contains a catalytic triad of Cys, His and Asp, and is es-
sential for enzyme activity [21]. Previous studies showed that AtPNG1
has both TGase [23] and PNGase activity [24]. However, the role of
AtPNG1 in living cells is still unclear, as its enzymatic activities were
only detected by in vitro assays. In fact, PNGase activity was not detected
in a crude extract of Arabidopsis leaves [24] and we could not detect
PNGase activity in AtPNG1 expressed in E. coli either (data not shown).
These features make the functional assessment of AtPNG1 as a PNGase
difcult. Therefore, we aimed to utilize an in vivo assay using a yeast sys-
tem to examine whether AtPNG1 can function as a deglycosylating en-
zyme in the ERAD processes. To achieve this aim, we used a model
ERAD substrate, RTA, a non-toxic mutant of ricin A-chain [10]. This sub-
strate is knownto be degradedby ERADina PNGase (ScPng1)-dependent
fashion [1012], and provides an excellent system to measure in vivo
PNGase activity. To determine whether AtPNG1 can facilitate the degra-
dation of RTA, C-terminal FLAG-tagged ScPng1 or AtPNG1 were co-
expressed with RTA in the cim5-1 png1 cells, which bear a mutation
in a proteasome subunit and deletion of the PNG1 gene [25]. Three inde-
pendent clones were picked up from ScPng1-expressing, AtPNG1-
expressing and vector-bearing (control) cells, and the accumulation of
RTA in each clone was examined by western blotting. As shown in
Fig. 1B, the g0 form of RTA (i.e., un- or deglycosylated RTA) was
detected in all cells expressing ScPng1 or AtPNG1 (lanes 2, 3, 5, 6, 8 and
9), whereas the g1 formof RTA(i.e., glycosylated RTA) was almost ex-
clusively detected in control cells (lanes 1, 4 and 7). These results
suggested that AtPNG1 can act as a PNGase and deglycosylates RTA in
vivo during ERAD. It should also be noted that the amount of RTA was
lower in AtPNG1-expressing cells compared with control cells (compare
lanes 1, 4, 7 and 3, 6, 9), indicating that AtPNG1 can, to some extent, sub-
stitute ScPng1 in facilitating degradation of RTA in yeast.
RTL (RTA-transmembrane-Leu2 protein) is another Png1-
dependent ERAD substrate [12]. RTL is a membrane protein that con-
sists of a luminal RTA, transmembrane domain of Pdr5, and a cytoplas-
mic Leu2 protein involved in biosynthesis of leucine. RTL assay is a
method for detection of defective ERAD by yeast cell growth. The
logic behind this assay is that strains with leu2 mutation (bearing
RTL-expressing plasmid) are usually unable to grow in media lacking
leucine, whereas yeast mutants with defective ERAD pathway can
support cell growth under such conditions because they are defective
in degrading Leu2 proteins on RTL. It has been shown that RTL, as was
the case with RTA, was degraded in yeast in ScPng1-dependent man-
ner [12]. RTL spotting assay therefore is a genetic device to character-
ize the Png1-dependent ERAD pathway in yeast. To conrm whether
AtPNG1 is also required for RTL degradation, RTL spotting assay was
performed. As shown in Fig. 1C, AtPNG1-expressing cells resulted in
impaired growth on media lacking leucine (Fig. 1C, middle row), indi-
cating the efcient degradation of RTL. On the other hand, cells ex-
pressing AtPNG1(C251A), where a catalytic Cys [28] is converted to
Ala, exhibited defect in degrading RTL, and therefore could grow on
media lacking leucine to the level of vector control (Fig. 1C, bottom
row). This result clearly indicated that PNGase activity of AtPNG1 is
also required for efcient RTL degradation.
3.2. A catalytic mutant of AtPNG1 stabilises glycosylated ERAD substrates
To further examine whether the degradation of RTA is indeed facili-
tatedby AtPNG1-mediateddeglycosylation, we performedcycloheximide
(CHX)-decay experiments. As showninFig. 2A, the g0 formwas detected,
as was efcient degradation, in ScPng1- (top-right panel) and AtPNG1-
expressing cells (bottom-left panel). By comparison, RTA remained in
the g1 form during the chase period in control cells (top-left panel) and
in cells expressing a catalytic mutant AtPNG1(C251A) (bottom-right
panel) (Fig. 2A). By quantifying the amount of the g1 form of RTA, it
seemed that facilitated degradation was occurring in ScPng1- and
AtPNG1-expressing cells (Fig. 2B). Surprisingly, expressionof the catalytic
mutant AtPNG1(C251A) resulted in signicant stabilization of RTA, as
demonstrated by the accumulation of RTA in the steady state (Fig. 2A)
and in the CHX-chase experiment (Fig. 2B; compare AtPNG1(C251A)
with vector control). Taken together, these results indicate that AtPNG1
facilitates the degradation of RTA, while its catalytic mutant stabilises
RTA.
3.3. Non-glycosylated RTA was degraded in an AtPNG1-independent
fashion
Previously it was shown that the cytoplasmic PNGase is only required
for degradation of RTA, and its non-glycosylated mutant (RTA-
(N10QN236Q)), in which Asn in potential N-glycosylation sites are mu-
tated into Gln, was not degraded in an ScPng1-dependent manner [12].
To nd out that it is also the case with AtPNG1, the effect of AtPNG1 on
degradation of non-glycosylated version of RTA, RTA(N10QN236Q)
[12], was examined. As shown in Fig. 3, the nonglycosyled RTA was
found to be degraded in the presence of AtPNG1 as efcient as the vector
control (compare the left panel and middle panel). As expected, expres-
sion of the catalytic mutant, AtPNG1(C251A), also did not alter the ef-
ciency of the degradation (right panel). This result contrasted with the
case with glycosylated RTA (Fig. 2A and B), strongly indicating that
the stabilization effect of AtPNG1(C251A) was specic to N-glycosylated
ERAD substrates.
3.4. The catalytic mutant of AtPNG1 stabilises RTA by binding to N-
glycans on the substrate
Since AtPNG1(C251A) stabilised RTA in an N-glycan dependent
manner, we assumed that the catalytic mutant may form a stable com-
plex withRTAby binding to N-glycans on RTA. To investigate this hy-
pothesis, we performed co-immunoprecipitation experiments using
AtPNG1 or AtPNG1(C251A). For this purpose, HA-tagged RTA was
constructed and AtPNG1(C251A) was immunoprecipitated with the
anti-FLAG antibody. As shown in Fig. 4A, co-precipitation was observed
for HA-tagged RTAand AtPNG1(C251A) (lane 2, top panel). The inter-
action was specic because no band was observed in the absence of
AtPNG1(C251A) (lane 1, top panel). Moreover, wild-type AtPNG1
could not be immunoprecipitated with RTA under the same experi-
mental conditions (data not shown), suggesting that mutation in the
catalytic residue render the AtPNG1 to form the stable complex with
RTA.
It should be noted that larger species of RTA tended to be con-
centrated in the precipitated fraction (compare top panel and middle
panel of lane 2). The upper band on precipitates was conrmed to be
Fig. 3. Effects of AtPNG1 or AtPNG1(C251A) on degradation of non-glycosylated RTA, RTA(N10QN236Q). AtPNG1 or AtPNG1(C251A) were co-expressed with the non-
glycosylated RTA, RTA(N10QN236Q), in png1 cells. The cycloheximide decay assays were performed as shown in Fig. 2A.
1460 Y. Masahara-Negishi et al. / Biochimica et Biophysica Acta 1820 (2012) 14571462
di-N-glycosylated RTA, because endo H treatment resulted in com-
plete deglycosylation of RTA around the same position of the g0
form (compare lanes 3 and 4 in the top panel). It has been shown
that, of two potential N-glycosylation sites, only Asn10 was efciently
N-glycosylated, while the other site (Asn236) was scarcely used for
N-glycosylation [12]. The fact that di-N-glycosylated RTA was signif-
icantly concentrated in the immunoprecipitates further suggests that
AtPNG1 preferentially binds to N-glycans. Considering all of these re-
sults together, we concluded that the catalytic mutant of AtPNG1 sta-
bilises RTA by binding to the substrate in an N-glycan-dependent
fashion. Consistent with this observation, the amino acids critical for
carbohydrate binding are all conserved in AtPNG1 [29] (Fig. 4B).
4. Discussion
While recent studies have unequivocally demonstrated that cyto-
plasmic PNGase is involved in ERAD, the biological signicance of this
enzyme has not been fully established in any system. For example,
png1 cells derived fromS. cerevisiae showno apparent growth defects
under various experimental conditions [7], but they do exhibit delayed
degradation of some ERAD substrates [7,10,12]. More recently, using C.
elegans, it was shown that a defect in a gene orthologue (png-1) caused
excessive axon branching in specic neurons [30], and this phenotype
appeared to be associated with the deglycosylation activity [31,32].
On the other hand, a mutation of the PNGase orthologue in N. crassa
caused temperature-sensitive growth/hyphae malformation pheno-
types [33], despite the fact that PNGase is inactive because of the
amino acid substitutions at the catalytic amino acids [17]. It was also
reported that, while the catalytic triad was conserved, an orthologue
in fruit y (Pngl) exhibited no detectable PNGase activity possibly be-
cause of the absence of a CXXC motif critical for enzyme activity, and
the mutants exhibited severe developmental delay [18]. Considering
the remarkable structural diversity among species, researchers must
take care when evaluating the functional importance of this protein, be-
cause we need to establish whether the observed properties are com-
mon for all orthologues or unique for a certain subset of organisms.
In previous studies, it was reported that AtPNG1 retains PNGase ac-
tivity [24] as well as TGase activity [23] in vitro. While multiple roles of
plant transglutaminases have beensuggested, it remainedto be demon-
strated which activity can be attributed to AtPNG1 in vivo [34]. In this
study, we examined whether AtPNG1 has PNGase activity in vivo,
using an ERAD assay for cytoplasmic PNGase-dependent substrates
[10,12]. Our results indicate that AtPNG1 can function as a cytoplasmic
PNGase and facilitates the degradation of RTA/RTL in similar manner
to the yeast orthologue ScPng1. These results are consistent withanear-
lier observation that ricin protein undergoes deglycosylation during
ERAD in tobacco cells [35,36].
What surprised us, however, was the inhibitory effect of
AtPNG1(C251A), a catalytic mutant, on RTA degradation. This result
clearly suggested that the catalytic mutant was not only functional,
but can also impair substrate degradation. This activity was specic
to N-glycosylated substrates, because the degradation of a non-
glycosylated RTA mutant was not affected by the expression of
AtPNG1(C251A). Furthermore, co-immunoprecipitation analysis re-
vealed that the catalytic mutant can forma stable complex with glyco-
sylated RTA. Consistent with this result, cytoplasmic PNGase is
known to bind carbohydrates with a preference for N-glycans [29,37].
Although AtPNG1(C251A) did not result in enhanced stability for
RTL, it should be noted that RTL assay depends on stability of the cy-
toplasmic Leu2 proteins, and while most of the components required
for RTL and RTA are shared, there may be a difference, and such dis-
crepancy has been observed for degradation of CPY* (a mutant form
of carboxypeptidase Y), a well-studied ERAD substrate, and CTL*, a
Leu2-bearing, transmembrane version of CPY* similar to RTL [38,39].
Fig. 4. AtPNG1(C251A) can bind to RTA. (A) Immunoprecipitation of the AtPNG1 catalytic mutant with RTA. AtPNG1(C251A) was coexpressed with HA-tagged RTA in png1
cells. (B) Sequence alignment of the catalytic domains important for carbohydrate binding. The catalytic residues (*) and residues important for carbohydrate binding (#) [29] are
indicated.
1461 Y. Masahara-Negishi et al. / Biochimica et Biophysica Acta 1820 (2012) 14571462
It should be noted that, although our data clearly indicate that
AtPNG1 functions as a PNGase in vivo, it does not necessarily exclude
the possibility that AtPNG1 may also function as a TGase in certain
cellular processes. On-going research should be directed at dissecting
the physiological function of AtPNG1 and whether defects in this gene
have any phenotypic consequences. If so, one must clarify whether
the effects are due to enzyme (PNGase or other activities)-dependent
or enzyme-independent activities, as is the case with N. crassa [17].
Acknowledgements
We thank the members of the Glycometabolome Team (RIKEN),
particularly Dr. Hiroto Hirayama, for helpful discussion. We also
thank Dr. Yoshiki Yamaguchi (RIKEN) for valuable comments on
this manuscript. The work was supported in part by a Grant-in-Aid
for Scientic Research from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (to T.S. and A.H.) and RFO 2009 Uni-
versity of Bologna (Italy) to D.S.F. We also thank COE (Center of Excel-
lence) Program, Osaka University for their support to M.D. to carry
out experiments in Japan.
References
[1] N. Takahashi, Demonstration of a new amidase acting on glycopeptides, Biochem.
Biophys. Res. Commun. 76 (1977) 11941201.
[2] T.H. Plummer Jr., J.H. Elder, S. Alexander, A.W. Phelan, A.L. Tarentino, Demonstra-
tion of peptide:N-glycosidase F activity in endo-beta-N-acetylglucosaminidase F
preparations, J. Biol. Chem. 259 (1984) 1070010704.
[3] A. Seko, K. Kitajima, Y. Inoue, S. Inoue, Peptide:N-glycosidase activity found in the
early embryos of Oryzias latipes (Medaka sh). The rst demonstration of the oc-
currence of peptide:N-glycosidase in animal cells and its implication for the pres-
ence of a de-N-glycosylation system in living organisms, J. Biol. Chem. 266 (1991)
2211022114.
[4] T. Suzuki, A. Seko, K. Kitajima, Y. Inoue, S. Inoue, Identication of peptide:
N-glycanase activity in mammalian-derived cultured cells, Biochem. Biophys.
Res. Commun. 194 (1993) 11241130.
[5] T. Suzuki, A. Seko, K. Kitajima, Y. Inoue, S. Inoue, Purication and enzymatic prop-
erties of peptide:N-glycanase from C3H mouse-derived L-929 broblast cells.
Possible widespread occurrence of post-translational remodication of proteins
by N-deglycosylation, J. Biol. Chem. 269 (1994) 1761117618.
[6] A. Seko, K. Kitajima, T. Iwamatsu, Y. Inoue, S. Inoue, Identication of two discrete
peptide: N-glycanases in Oryzias latipes during embryogenesis, Glycobiology 9
(1999) 887895.
[7] T. Suzuki, H. Park, N.M. Hollingsworth, R. Sternglanz, W.J. Lennarz, PNG1, a yeast gene
encoding a highly conserved peptide:N-glycanase, J. Cell Biol. 149 (2000) 10391052.
[8] W. Xie, D.T. Ng, ERAD substrate recognition in budding yeast, Semin. Cell Dev.
Biol. 21 (2010) 533539.
[9] R. Bernasconi, M. Molinari, ERAD and ERAD tuning: disposal of cargo and of ERAD
regulators from the mammalian ER, Curr. Opin. Cell Biol. 23 (2011) 176183.
[10] I. Kim, J. Ahn, C. Liu, K. Tanabe, J. Apodaca, T. Suzuki, H. Rao, The Png1-Rad23 com-
plex regulates glycoprotein turnover, J. Cell Biol. 172 (2006) 211219.
[11] K. Tanabe, W.J. Lennarz, T. Suzuki, A cytoplasmic peptide:N-glycanase, Methods
Enzymol. 415 (2006) 4655.
[12] A. Hosomi, K. Tanabe, H. Hirayama, I. Kim, H. Rao, T. Suzuki, Identicationof anHtm1
(EDEM)-dependent, Mns1-independent Endoplasmic Reticulum-associated Degra-
dation (ERAD) pathway in Saccharomyces cerevisiae: application of a novel assay
for glycoprotein ERAD, J. Biol. Chem. 285 (2010) 2432424334.
[13] M.L. Altrich-VanLith, M. Ostankovitch, J.M. Polefrone, C.A. Mosse, J. Shabanowitz, D.F.
Hunt, V.H. Engelhard, Processing of a class I-restricted epitope from tyrosinase re-
quires peptide N-glycanase and the cooperative action of endoplasmic reticulum
aminopeptidase 1 and cytosolic proteases, J. Immunol. 177 (2006) 54405450.
[14] E. Kario, B. Tirosh, H.L. Ploegh, A. Navon, N-linked glycosylation does not impair
proteasomal degradation but affects class I major histocompatibility complex
presentation, J. Biol. Chem. 283 (2008) 244254.
[15] M. Ostankovitch, M. Altrich-Vanlith, V. Robila, V.H. Engelhard, N-glycosylation
enhances presentation of a MHC class I-restricted epitope from tyrosinase,
J. Immunol. 182 (2009) 48304835.
[16] A. Dalet, P.F. Robbins, V. Stroobant, N. Vigneron, Y.F. Li, M. El-Gamil, K. Hanada, J.C.
Yang, S.A. Rosenberg, B.J. Van den Eynde, An antigenic peptide produced by re-
verse splicing and double asparagine deamidation, Proc. Natl. Acad. Sci. U. S. A.
108 (2011) E323E331.
[17] S. Maerz, Y. Funakoshi, Y. Negishi, T. Suzuki, S. Seiler, The Neurospora peptide:
N-glycanase ortholog PNG1 is essential for cell polarity despite its lack of enzy-
matic activity, J. Biol. Chem. 285 (2010) 23262332.
[18] Y. Funakoshi, Y. Negishi, J.P. Gergen, J. Seino, K. Ishii, W.J. Lennarz, I. Matsuo, Y. Ito,
N. Taniguchi, T. Suzuki, Evidence for an essential deglycosylation-independent
activity of PNGase in Drosophila melanogaster, PLoS One 5 (2010) e10545.
[19] T. Suzuki, H. Park, W.J. Lennarz, Cytoplasmic peptide:N-glycanase (PNGase) in eu-
karyotic cells: occurrence, primary structure, and potential functions, FASEB J. 16
(2002) 635641.
[20] T. Suzuki, Cytoplasmic peptide:N-glycanase and catabolic pathway for free
N-glycans in the cytosol, Semin. Cell Dev. Biol. 18 (2007) 762769.
[21] K.S. Makarova, L. Aravind, E.V. Koonin, A superfamily of archaeal, bacterial, and
eukaryotic proteins homologous to animal transglutaminases, Protein Sci.
8 (1999) 17141719.
[22] T. Suzuki, H. Park, E.A. Till, W.J. Lennarz, The PUB domain: a putative
protein-protein interaction domain implicated in the ubiquitin-proteasome path-
way, Biochem. Biophys. Res. Commun. 287 (2001) 10831087.
[23] M. Della Mea, D. Caparros-Ruiz, I. Claparols, D. Serani-Fracassini, J. Rigau,
AtPng1p. The rst plant transglutaminase, Plant Physiol. 135 (2004) 20462054.
[24] A. Diepold, G. Li, W.J. Lennarz, T. Nurnberger, F. Brunner, The Arabidopsis AtPNG1
gene encodes a peptide:N-glycanase, Plant J. 52 (2007) 94104.
[25] T. Suzuki, W.J. Lennarz, Glycopeptide export from the endoplasmic reticulum into
cytosol is mediated by a mechanism distinct from that for export of misfolded
glycoprotein, Glycobiology 12 (2002) 803811.
[26] M.D. Rose, F. Winston, P. Hieter, Methods in Yeast Genetics, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY, 1990.
[27] F. Sherman, Getting started with yeast, Methods Enzymol. 194 (1991) 321.
[28] S. Katiyar, T. Suzuki, B.J. Balgobin, W.J. Lennarz, Site-directed mutagenesis study
of yeast peptide:N-glycanase. Insight into the reaction mechanism of
deglycosylation, J. Biol. Chem. 277 (2002) 1295312959.
[29] G. Zhao, G. Li, X. Zhou, I. Matsuo, Y. Ito, T. Suzuki, W.J. Lennarz, H. Schindelin,
Structural and mutational studies on the importance of oligosaccharide binding
for the activity of yeast PNGase, Glycobiology 19 (2009) 118125.
[30] N. Habibi-Babadi, A. Su, C.E. de Carvalho, A. Colavita, The N-glycanase png-1 acts
to limit axon branching during organ formation in Caenorhabditis elegans, J. Neu-
rosci. 30 (2010) 17661776.
[31] T. Suzuki, K. Tanabe, I. Hara, N. Taniguchi, A. Colavita, Dual enzymatic properties
of the cytoplasmic peptide: N-glycanase in C. elegans, Biochem. Biophys. Res.
Commun. 358 (2007) 837841.
[32] T. Kato, A. Kawahara, H. Ashida, K. Yamamoto, Unique peptide:N-glycanase of
Caenorhabditis elegans has activity of protein disulphide reductase as well as of
deglycosylation, J. Biochem. 142 (2007) 175181.
[33] S. Seiler, M. Plamann, The genetic basis of cellular morphogenesis in the lamen-
tous fungus Neurospora crassa, Mol. Biol. Cell 14 (2003) 43524364.
[34] D. Serani-Fracassini, M. Della Mea, G. Tasco, R. Casadio, S. Del Duca, Plant and an-
imal transglutaminases: do similar functions imply similar structures? Amino
Acids 36 (2009) 643657.
[35] A. Di Cola, L. Frigerio, J.M. Lord, A. Ceriotti, L.M. Roberts, Ricin A chain without its
partner B chain is degraded after retrotranslocation from the endoplasmic reticu-
lum to the cytosol in plant cells, Proc. Natl. Acad. Sci. U. S. A. 98 (2001)
1472614731.
[36] A. Di Cola, L. Frigerio, J.M. Lord, L.M. Roberts, A. Ceriotti, Endoplasmic
reticulum-associated degradation of ricin A chain has unique and plant-specic
features, Plant Physiol. 137 (2005) 287296.
[37] T. Suzuki, I. Hara, M. Nakano, G. Zhao, W.J. Lennarz, H. Schindelin, N. Taniguchi, K.
Totani, I. Matsuo, Y. Ito, Site-specic labeling of cytoplasmic peptide:N-glycanase
by N, N'-diacetylchitobiose-related compounds, J. Biol. Chem. 281 (2006)
2215222160.
[38] B. Medicherla, Z. Kostova, A. Schaefer, D.H. Wolf, A genomic screen identies
Dsk2p and Rad23p as essential components of ER-associated degradation,
EMBO Rep. 5 (2004) 692697.
[39] S. Kohlmann, A. Schafer, D.H. Wolf, Ubiquitin ligase Hul5 is required for
fragment-specic substrate degradation in endoplasmic reticulum-associated
degradation, J. Biol. Chem. 283 (2008) 1637416383.
1462 Y. Masahara-Negishi et al. / Biochimica et Biophysica Acta 1820 (2012) 14571462