144 Resea ch A icle
In oduc ion
A e ansloca ion ac oss o inse ion in o he endoplasmic
e iculum (ER), memb ane p o eins ha e o old, a p ocess ha is
assis ed by a a ie y o ER esiden chape ones ( o a e iew see
Ellgaa d and Helenius, 2003). Folding is moni o ed by ER quali y
con ol mechanisms, and co ec ly olded p o eins a e allowed o
exi he ER by esicula budding (Ba lowe, 2003). Assis ance in
p o ein olding and quali y con ol a e o en o e lapping unc ions
o ER chape ones. A olding in e media e is ecognized by a
chape one h ough such uni e sal signa u es as exposed
hyd ophobic pa ches, unpai ed cys eines, and p oneness o
agg ega ion (F a e al., 1993; Hellman e al., 1999; Zhang e al.,
1997). I ini ial olding is slow o ails, hese ea u es will con inue
o be exposed and will ensu e immedia e e-binding o a chape one.
By his mechanism, slow- olding p o eins o p o eins ha mis old
will be e ained inside he ER o ex ended pe iods o ime. Some
ER e en ion mechanisms ha e addi ional complexi y, such as he
calnexin–cal e iculin cycle ound in mammalian cells, whe e se e al
chape ones and enzymes oge he moni o he olding o
glycop o eins (Hammond e al., 1994; Pa odi, 2000).
As well as aiding in p o ein olding and ER e en ion, some
uni e sal chape ones such as calnexin and BiP possess a hi d
unc ion in ha hey can a ge e minally mis olded p o eins o
deg ada ion (B odsky e al., 1999; Denic e al., 2006; McC acken
and B odsky, 1996; Plempe e al., 1997). Deg ada ion o abe an
ER p o eins occu s mos ly by a p ocess called ER-associa ed
p o ein deg ada ion (ERAD), whe eby p o eins a e
‘ e o ansloca ed’ in o he cy osol o be deg aded by he ubiqui in–
p o easome sys em ( o a e iew see Meusse e al., 2005). Co e
componen s o he esponsible machine ies o e o ansloca ion
a e he ER memb ane-embedded E3 ubiqui in ligases. In yeas ,
he e a e wo such ubiqui in ligases, Doa10p and H d1p, which a e
ound in dis inc memb ane p o ein complexes (Ca alho e al.,
2006; Denic e al., 2006; Swanson e al., 2001). Toge he hey
p omo e e o ansloca ion and deg ada ion o mos , i no all,
mis olded subs a es in he ER. Whe eas he Doa10p complex
a ge s memb ane p o eins wi h lesions in hei cy osolic po ions
o he p o easome, a ou e e med he ERAD-C pa hway, he H d1p
complex e o ansloca es memb ane p o eins wi h mis olded
ansmemb ane domains (ERAD-M pa hway) o memb ane and
soluble p o eins wi h de ec s in hei luminal po ion (ERAD-L
pa hway) (Ca alho e al., 2006; Vashis e al., 2001). All pa hways
con e ge a he cy osolic Cdc48p ATPase complex, which p obably
p o ides he ene gy o e o ansloca ion and o subs a e ans e
o he p o easome (Ca alho e al., 2006; Rabino ich e al., 2002;
Ye e al., 2001).
Whe eas he as majo i y o mis olded p o eins o he sec e o y
pa hway a e ecognized ea ly while s ill inside he ER and will
ul ima ely be a ge ed o ERAD, he e a e cases whe e a ac ion
o hese species exi s he ER no mally. This occu s i de ec i e
p o eins a e exp essed in la ge quan i ies o i expo signals a e
dominan enough ha hey compe e wi h e en ion mechanisms
(Caldwell e al., 2001; Haynes e al., 2002; Kincaid and Coope ,
2007; Vashis e al., 2001). In ei he case, i becomes mo e e iden
ha he cell possesses addi ional, albei less well-cha ac e ized,
quali y con ol mechanisms in pos -ER compa men s ha can
a ge abe an p o eins o deg ada ion (He ema e al., 2004;
Hong e al., 1996; Reggio i and Pelham, 2002; Wang and Ng,
2010).
One in e es ing bu poo ly unde s ood p o ein modi ica ion ha
occu s inside he ER is ha o p o ein O-mannosyla ion, which is
one o a a ie y o possible O-glycosyla ion e en s ha occu
h oughou he sec e o y pa hway ( o a e iew see Spi o, 2002).
O-mannosyla ion is media ed by membe s o he p o ein O-
mannosyl ans e ase (PMT) amily (Lussie e al., 1995; S ahl-
Bolsinge e al., 1993). PMTs a e mul ispanning memb ane p o eins
Accep ed 9 No embe 2010
Jou nal o Cell Science 124, 144-153
©
2011. Published by The Company o Biologis s L d
doi:10.1242/jcs.072181
Summa y
In euka yo ic cells, p o eins en e he sec e o y pa hway a he endoplasmic e iculum (ER) as linea polypep ides and old a e
ansloca ion ac oss o inse ion in o he memb ane. I co ec olding ails, many p o eins a e O-mannosyla ed inside he ER by an
O-mannosyl ans e ase, he Pm 1p–Pm 2p complex. The consequences o his modi ica ion a e con o e sial and he cellula ole o
he Pm 1p–Pm 2p complex in his espec is unclea . He e, we ha e iden i ied he binding pa ne s o yeas Pm 1p and Pm 2p. These
include ER chape ones in ol ed in oxida i e p o ein olding; he H d1p complex, which is in ol ed in ER-associa ed p o ein
deg ada ion (ERAD); and he p24 p o ein complex in ol ed in ER expo . The esul s sugges ha he Pm 1p–Pm 2p complex
pa icipa es in hese p ocesses. We es ed his assump ion in a unc ional assay and ound ha whe eas he Pm 1p–Pm 2p complex
p omo es as ER expo o he GPI-ancho ed p o ein Gas1p, i e ains he mis olded e sion Gas1*p and a ge s i o he H d1p
complex o subsequen deg ada ion. Ou esul s e eal p e iously unknown cellula oles o he Pm 1p–Pm 2p complex in connec ion
wi h he ERAD machine y and show i s pa icipa ion in ER p o ein quali y con ol.
Key wo ds: Endoplasmic e iculum, ER p o ein quali y con ol, ER-associa ed p o ein deg ada ion, Pm complex
P o ein
O
-mannosyl ans e ases pa icipa e in ER
p o ein quali y con ol
Vei Gode * and Alejand o Mele o
Depa men o Gene ics, Uni e si y o Se ille, A e Reina Me cedes 6, 41012 Se ille, Spain
*Au ho o co espondence ([email p o ec ed])
Jou nal o Cell Science
wi h se en ansmemb ane domains and wo la ge luminal loops,
which oge he a e needed o he enzyma ic ac i i y (Gi bach e
al., 2000; S ahl-Bolsinge and Scheinos , 1999). They a e
conse ed om yeas o humans al hough hey appea o be missing
in plan s (Wille e al., 2003). Single mannose esidues a e a ached
o side chains o one o se e al se ine o h eonine esidues. I was
epo ed ha many p o eins will be O-mannosyla ed inside he ER
only in cases whe e hey mis old (Ha y e al., 2001; Vashis e al.,
2001). The a e o mis olded p o eins ha ha e been O-
mannosyla ed is con o e sial. Whe eas some o hem seem
inc easingly p o ec ed om deg ada ion, o he s a e epo ed o be
deg aded by he cy osolic p o easome o which hey a e a ge ed
by an unknown mechanism (Ha y e al., 2001; Hi ayama e al.,
2008). In e es ingly, like mos ER chape ones o membe s o he
ERAD machine ies, PMTs a e up egula ed du ing ER s ess by he
un olded p o ein esponse (UPR) (T a e s e al., 2000).
This s udy aimed a iden i ying he cellula ole o he PMTs
wi h espec o mis olded p o eins. We iden i ied he cellula
binding pa ne s o yeas Pm 1p and Pm 2p, which a e known o
o m an ac i e s ochiome ic complex (Pm 1p–Pm 2p) (Gi bach
and S ahl, 2003). Binding pa ne s include ER chape ones in ol ed
in oxida i e p o ein olding; he H d1p complex, which is in ol ed
in ERAD; and he p24 p o ein complex, which is in ol ed in
p o ein ER expo (Muniz e al., 2000; Schimmolle e al., 1995).
These indings sugges ha he Pm 1p–Pm 2p complex migh
pa icipa e in all hese cellula p ocesses. In a subsequen unc ional
assay, we es ed his hypo hesis and showed ha he Pm 1p–Pm 2p
complex is equi ed o as ER expo o he GPI-ancho ed p o ein
Gas1p, whe eas i e ains he mis olded e sion Gas1*p and a ge s
i o he H d1p complex o subsequen deg ada ion. Ou esul s
pu he Pm 1p–Pm 2p complex in a ca ego y wi h ER chape ones
ha unc ion in ER p o ein quali y con ol. We p o ide a model o
how he machine ies o p o ein O-mannosyla ion, ER expo and
ERAD a e connec ed on a molecula le el. Finally, ou esul s
uni y p e iously con adic ing da a o he ole o he Pm 1p–
Pm 2p complex in ERAD.
Resul s
In e ac ion pa ne s o Pm 1p and Pm 2p
To e eal he cellula unc ion o he Pm 1p–Pm 2p complex, we
i s asked which componen s i is associa ed wi h. We sepa a ely
agged Pm 1p and Pm 2p ch omosomally wi h a usion ag ha
con ained a P o ein A moie y and a calmodulin binding pep ide
(CA ag) and exp essed he p o eins om hei endogenous
p omo e s. These agged p o eins we e ully unc ional
(supplemen a y ma e ial Fig. S1). Yeas cells we e g own in
olumes o 3 l. A e cell lysis, memb ane ac ions we e isola ed
and solubilized wi h 1% digi onin. Tagged p o eins we e a ini y
pu i ied oge he wi h hei binding pa ne s using IgG-coupled
magne ic beads. The elua e was subjec ed o SDS-PAGE ollowed
by Coomassie Blue s aining (Fig. 1A). The isualized bands we e
cu ou and hei iden i y de e mined by andem mass spec ome y
(Table 1 and supplemen a y ma e ial Table S1). Al e na i ely, we
p ecipi a ed he en i e elua e wi h ichlo oace ic acid (TCA) and
iden i ied he pool o bound p o eins by andem mass spec ome y
(Table 1 and supplemen a y ma e ial Table S1). A wild- ype s ain
wi hou agged p o eins was used as a con ol in all expe imen s.
As shown in Fig. 1A, Pm 1p and Pm 2p we e isola ed oge he
bu no abundan addi ional binding pa ne s we e isible (Fig. 1A,
lanes 2 and 3, bands 1 and 2; Table 1). Howe e , a ain smea was
seen con aining p o eins o he p24 p o ein complex (Fig. 1A,
145
Pm 1p–Pm 2p complex in ER quali y con ol
lanes 2 and 3, band 5; Table 1). In Fig. 1A, bands 3 and 4 con ained
esidual IgG hea y and ligh chain om incomple e coupling o
magne ic beads. Using TCA p ecipi a ion, we iden i ied addi ional
and less abundan binding pa ne s o he Pm 1p–Pm 2p complex.
Along wi h p o eins o he p24 p o ein complex, we ound Cdc48p,
H d1p, Usa1p and Yos9p (componen s o he ERAD-media ing
H d1p complex), E o1p and Pdi1p (p o eins in ol ed in oxida i e
p o ein olding), Ub 1p and Cue4p (p o eins in ol ed in p o ein
ubiqui yla ion), D m1p (a dis inc co- ac o o he Cdc48p ATPase)
and Ted1p (a p o ein linked o GPI-ancho emodeling) (see Table
1).
In summa y, he Pm 1p–Pm 2p complex associa es weakly and
hus p obably ansien ly, wi h ER componen s ha ha e es ablished
oles in p o ein olding and ER expo as well as in ERAD. We
Fig. 1. The Pm 1p–Pm 2p complex associa es wi h ER machine ies
in ol ed in ER p o ein expo and in ERAD. (A)

In e ac ion pa ne s o
Pm 1p and Pm 2p. Wild- ype yeas cells (con) o cells exp essing ei he Pm 1-
CA o Pm 2-CA we e lysed and memb ane ac ions solubilized wi h 1%
digi onin. The ex ac was incuba ed wi h IgG-coupled magne ic beads and
bound ma e ial analyzed by SDS-PAGE and Coomassie Blue s aining.
Visualized bands (lanes 2 and 3) and egions om he con ol eac ion (lane 1)
we e numbe ed, cu ou and hei p o ein con en de e mined by mass
spec ome y (Table 1; supplemen a y ma e ial Table S1). (B)

Yeas cells
exp essing Emp24-3H wi h o wi hou exp essing Pm 2-CA we e lysed.
Samples we e analyzed di ec ly (inpu ; 5% o o al ma e ial) o a e
immunop ecipi a ion using IgG-coupled magne ic beads (IP:IgG; 95% o o al
ma e ial). All samples we e sepa a ed by SDS-PAGE (4-20% g adien gel) and
analyzed by immunoblo ing (IB) wi h he indica ed an ibodies. The
a owhead indica es he co-immunop ecipi a ed ac ion. The s a indica es
Pm 2-CA ha was also ecognized by he seconda y an ibody. (C)

As o B,
bu using cells exp essing Pm 1-3HA wi h o wi hou exp essing H d1-CA.
The SDS-PAGE was pe o med using a 7% s anda d gel. The a owhead
indica es he co-immunop ecipi a ed ac ion. The s a indica es H d1-CA ha
was also ecognized by he seconda y an ibody.
Jou nal o Cell Science
hus hypo hesized ha he Pm 1p–Pm 2p complex i sel ac i ely
pa icipa es in hese cellula p ocesses.
Be o e es ing ou hypo hesis expe imen ally we wan ed o con i m
and isualize he in e ac ions o he Pm 1p–Pm 2p complex wi h he
p edominan binding pa ne s ha we iden i ied: he H d1p complex
and he p24 p o ein complex. To his end, we cons uc ed s ains in
which wo p o eins we e di e en ially agged. As can be seen in Fig.
1B, a ac ion o HA- agged Emp24p (Emp24-3HA, a membe o
he p24 p o ein complex) co-p ecipi a es wi h CA- agged Pm 2p
(Pm 2-CA). A con ol s ain wi hou Pm 2-CA did no b ing down
agged Emp24p (Fig. 1B, compa e lanes 5 and 6). As can be seen in
Fig. 1C, we could co-isola e a ac ion o HA- agged Pm 1p (Pm 1-
3HA) wi h CA- agged H d1p (H d1-CA, he E3-ligase o he H d1p
complex). Only ain backg ound s aining was isible i H d1p was
un agged (Fig. 1C, compa e lanes 5 and 6).
To assess whe he deg ada ion o he agged Pm 1p–Pm 2p
complex by he H d1p complex leads o hei obse ed associa ion,
we pe o med a cycloheximide (CHX)-chase analysis o CA- agged
Pm 2p. Su p isingly, Pm 2-CA was ela i ely uns able and was
deg aded wi h an app oxima e hal li e o 1.5 hou s (supplemen a y
ma e ial Fig. S2). Howe e , he u no e was independen o H d1p
and hus he physical in e ac ion was no due o deg ada ion by he
H d1p complex (supplemen a y ma e ial Fig. S2).
Gas1*p is a subs a e o he Pm 1p–Pm 2p complex and
is la gely deg aded by he ERAD-L pa hway
To add ess ou hypo hesis ha he Pm 1p–Pm 2p complex
pa icipa es in p o ein olding, ER expo and ERAD, we nex
looked o a sui able model subs a e. Ideally, such a subs a e
should no mally be expo ed om he ER in a p24 p o ein complex-
dependen manne , whe eas a mu an should be deg aded ia H d1p
complex-media ed ERAD. The GPI-ancho ed p o ein Gas1p comes
closes o hese c i e ia. In i s wild- ype o m, he p o ein lea es
146 Jou nal o Cell Science 124 (1)
he ER depending on he p24 p o ein complex and is a ge ed o
he plasma memb ane (Muniz e al., 2001; Muniz e al., 2000;
Schimmolle e al., 1995). Impo an ly, a mu an e sion o Gas1p,
Gas1*p, is uns able and deg aded by an uniden i ied p o easome-
dependen pa hway (Fuji a e al., 2006). In e es ingly, whe eas
Gas1p is O-glycosyla ed by Pm 4p and Pm 6p alone, Gas1*p is
u he O-glycosyla ed by Pm 1p and Pm 2p (Hi ayama e al.,
2008). Thus, he wild- ype p o ein Gas1p and i s mu an e sion
Gas1*p appea well sui ed o ou analysis.
We i s con i med ha Gas1*p is O-mannosyla ed by he
Pm 1p–Pm 2p complex as epo ed p e iously (Hi ayama e al.,
2008). To his end, we exp essed ch omosomally HA- agged species
o wild- ype Gas1p [Gas1p(HA)] o mu an Gas1*p [Gas1*p(HA)]
om hei endogenous p omo e s in wild- ype o in Pm 1–Pm 2
dele ion mu an (pm 1pm 2) cells. We gene ally obse ed a
lowe p o ein exp ession le el in pm 1pm 2 cells han in wild-
ype cells, which was also e lec ed in a educed g ow h a e (da a
no shown). As can be seen in Fig. 2A, Gas1*p showed an inc ease
in elec opho e ic mobili y in pm 1pm 2 cells as compa ed wi h
wild- ype cells, consis en wi h i being O-mannosyla ed (Fig. 2A,
compa e lanes 3 and 4, a ows). We occasionally obse ed a smea
abo e he majo p o ein bands, bu because he phenomenon was
a e we conside ed i unspeci ic. In con as o Gas1*p, bo h he
p ecu so and he ma u e o m o Gas1p mig a ed wi h simila
elec opho e ic mobili y in pm 1pm 2 and in wild- ype cells
(Fig. 2A, compa e lanes 1 and 2). To exclude he possibili y ha
he inc ease in elec opho e ic mobili y o Gas1*p in pm 1pm 2
cells esul s om de ec i e N-glycosyla ion a he han om lack
o O-mannosyla ion, we pe o med an addi ional se o expe imen s
using he de-N-glycanase PNGase F o emo e N-linked glycans
p io o SDS-PAGE (supplemen a y ma e ial Fig. S3) and co-
immunop ecipi a ion wi h he mannose-speci ic lec in
Concana aline A (supplemen a y ma e ial Fig. S4B). Finally, he
Table 1. Summa y o in e ac ing p o eins ha we e immunop ecipi a ed wi h CA- agged Pm 1p (Pm 1-CA) o CA- agged
Tagged p o ein (bai )
Pm 1-CA Pm 2-CA
In e ac ing
p o ein Func ional ca ego y Indi idual To al Indi idual To al
Pm 1p 33[1] 36/32 35[2] 39/34
Pm 2p O-mannosyla ion 28[2] 25/22 20[1] 22/21
Cdc48p ERAD – 1/2 – 18/3
E o1p P o ein olding – 2/2 – 3/2
Pdi1p P o ein olding – 2/1 – –/1
H d1p ERAD – 1/– – 2/–
Usa1p ERAD – 2/– – 2/2
Yos9p ERAD – –/– – 1/–
Ub 1p Ubiqui in ligase – –/5 – –/–
D m1p Cdc48p co ac o – –/– – 5/2
Cue4p Ubiqui in binding – –/2 – 1/3
Ted1p GPI emodeling – –/3 – –/2
IgG hc [3] [3]
IgG lc [4] [4]
Emp24p – 2/2 – 2/2
E 25p 1[5] 6/4 – 5/4
E p1p 1[5] 5/5 2[5] 4/3
E p2p
ER expo (p24 complex)
– 2/2 4[5] 1/1
Bound p o eins we e iden i ied using mass spec ome y. We analyzed ei he indi idual p o ein bands a e hei sepa a ion using SDS-PAGE and
s aining wi h Coomassie Blue (indi idual) o he o al p o ein elua e a e p ecipi a ion wi h ichlo oace ic acid ( o al). Numbe s indica e he numbe o
pep ides iden i ied by mass spec ome y. Fo mul iple expe imen s, numbe s a e sepa a ed by a solidus. The numbe s in squa e b acke s co espond o he
bands in Fig. 1A. See supplemen a y ma e ial Table S1 o he comple e se o mass spec ome y da a. Al hough Yos9p was only iden i ied wi h one
indi idual pep ide and would no pass ou c i e ia as a ue hi , we show i because i is a well-known H d1p complex componen . IgG hc, immunoglobulin
hea y chain; IgG lc, immunoglobulin ligh chain.
Pm 2p (Pm 2-CA)
Jou nal o Cell Science
a o emen ioned expe imen o es o he unc ionali y o agged
Pm 2p showed a Pm 2p-dependen change in elec opho e ic
mobili y o Gas1*p o e ime (supplemen a y ma e ial Fig. S1).
Toge he , hese da a con i m ha Gas1*p bu no Gas1p is modi ied
in a Pm 1p–Pm 2p complex-dependen manne , s ongly sugges ing
ha Gas1*p in e ac s di ec ly wi h he Pm 1p–Pm 2p complex and
is O-mannosyla ed.
Nex , we asked whe he Gas1*p is deg aded ia he H d1p
complex-dependen ERAD-L pa hway as p edic ed on he basis o
he ac ha he mis olded GPI-ancho ed p o ein is exposed en i ely
o he lumen o he ER. To his end, we exp essed Gas1*p in wild-
ype and dele ion mu an s o he H d1p complex and pe o med a
CHX-chase expe imen . As can be seen in Fig. 2B, lanes 1–4 and
g aph, when exp essed in wild- ype cells Gas1*p is deg aded wi h
a hal -li e o oughly 1.5 hou s, consis en wi h p e ious da a
(Fuji a e al., 2006). When we es ed dele ion mu an s o all
memb ane-bound H d1p complex componen s (H d1p, H d3p,
De 1p,and Usa1p), we ound ha hey s abilized Gas1*p whe eas
147
Pm 1p–Pm 2p complex in ER quali y con ol
dele ion o Doa10p ( he cen al componen o he ERAD-C
pa hway) did no (Fig. 2B,C). Dele ions o D m1p, Ub 1p o
Cue4p (o he binding pa ne s o he Pm 1p–Pm 2p complex; Table
1) did no in luence Gas1*p u no e (Fig. 2C and da a no shown).
These esul s show ha Gas1*p is indeed a ge ed o deg ada ion
by he ERAD-L pa hway. Ou esul s a e di e en om hose
published p e iously epo ing ha dele ing H d1p had no e ec
on Gas1*p u no e (Fuji a e al., 2006).
I has been epo ed ha he p24 p o ein complex has a ole in
deg ada ion o Gas1*p (Fuji a e al., 2006). Because he p24 p o ein
complex has an es ablished unc ion in ER expo o wild- ype
Gas1p, he simples scena io would be consis en wi h a p24 p o ein
complex-dependen ER exi o a ac ion o Gas1*p and i s a ge ing
o deg ada ion om a pos -ER compa men . Howe e , ou ea lie
esul s showed ha bo h he H d1p complex and he p24 p o ein
complex a e p esen as associa es o he Pm 1p–Pm 2p complex.
I could hus be ha he p24 p o ein complex in e ac s di ec ly wi h
he H d1p complex and has a ole in a ge ing o Gas1*p o he
Fig. 2. The mis olded model p o ein Gas1*p is a subs a e o he Pm 1p–Pm 2p complex and is in pa deg aded by he ERAD-L pa hway. (A)

Gas1p(HA)
o Gas1*p(HA) we e indi idually exp essed in wild- ype o in pm 1pm 2 cells. Equal amoun s o cells we e lysed and he p o eins analyzed by SDA-PAGE (5%
s anda d gel) ollowed by an i-HA immunoblo ing. A ows indica e he O-mannosyla ed ac ion o Gas1*p: p, p ecu so o m; m ma u e o m. (B,C)

The
deg ada ion o Gas1*p(HA) was measu ed in wild- ype cells o in cells ca ying he designa ed dele ions a e inhibi ion o p o ein syn hesis by addi ion o
200

g/ml cycloheximide. A he indica ed ime poin s, equal aliquo s o cells we e emo ed, lysed and he amoun o emaining Gas1*p(HA) was analyzed by
SDS-PAGE (4–20% g adien gel) ollowed by an i-HA immunoblo ing. The bands we e quan i ied by densi ome y and he esul s o a leas h ee independen
se s o expe imen s we e plo ed ( igh panels).
Jou nal o Cell Science
ERAD machine y. To check his idea, we es ed whe he we could
co-immunop ecipi a e membe s o he ERAD machine y di ec ly
wi h Emp24p. As can be seen in Fig. 3A, we could no co-
p ecipi a e Usa1p (a membe o he H d1p complex) wi h Emp24-
CA, whe eas he con ol eac ion using CA- agged De 1p
(De 1-CA), ano he membe o he H d1p complex, eadily b ough
down Usa1p (Fig. 3A, compa e lanes 4–6). Fu he mo e, we did
no ind H d1p complex componen s as binding pa ne s o Emp24-
CA when pe o ming a la ge-scale pull-down expe imen ollowed
by mass spec ome ic analysis, as desc ibed in Fig. 1 (V.G. and
A.M., unpublished da a). We hen di ec ly measu ed he deg ada ion
o Gas1*p in p24 p o ein complex dele ion mu an s. Dele ions o
ei he Emp24p o o E 25p led o s abiliza ion o Gas1*p (Fig.
3B, lanes 1–12), consis en wi h p e ious da a (Fuji a e al., 2006).
This ac ion was no sec e ed o a ge ed o he acuole (V.G. and
A.M., unpublished da a). When we addi ionally disabled he
ERAD-L pa hway using h d1emp24 dele ion mu an cells we
ound maximal s abiliza ion o Gas1*p (Fig. 3B, lanes 13–16 and
g aph). Toge he , hese da a sugges ha Gas1*p can be deg aded
by wo sepa a e pa hways: one is he ERAD-L pa hway, which
depends on he H d1p complex, and he o he depends on he p24
p o ein complex and is consis en wi h leading o p o ein
deg ada ion om a pos -ER compa men .
The Pm 1p–Pm 2p complex e ains Gas1*p and ul ima ely
a ge s i o he H d1p complex o deg ada ion
Nex , we di ec ly add essed he ole o he Pm 1p–Pm 2p complex
in deg ada ion o Gas1*p. The physical in e ac ion o he Pm 1p–
Pm 2p complex wi h he H d1p complex sugges s ha he Pm 1p–
148 Jou nal o Cell Science 124 (1)
Pm 2p complex migh a ge Gas1*p o he H d1p complex. In ha
case, absence o he Pm 1p–Pm 2p complex would lead o a
s abiliza ion o Gas1*p. I , al e na i ely, he Pm 1p–Pm 2p complex
e ains Gas1*p and p e en s i s e o ansloca ion by he H d1p
complex, i s absence should esul in as e deg ada ion o Gas1*p.
To dis inguish be ween hese possibili ies, we pe o med a CHX-
chase expe imen wi h dele ion mu an s o ei he Pm 1p o Pm 2p
o bo h. As can be seen in Fig. 4, dele ion o he Pm 1p–Pm 2p
complex d as ically inc eases he u no e a e o Gas1*p, bes
isible o he pm 1pm 2 cells, in which he hal li e was educed
o app oxima ely 45 minu es. This indica es ha he Pm 1p–Pm 2p
complex has an inhibi o y unc ion o he deg ada ion o Gas1*p,
consis en wi h an ER e en ion unc ion o he mis olded p o ein.
As men ioned ea lie , some classical ER chape ones such as
BiP and calnexin a e known o ha e mul iple unc ions and play
a ole in p o ein olding, p o ein ER e en ion and in a ge ing o
p o ein deg ada ion. A simila unc ion o he Pm 1p–Pm 2p
complex o a ge subs a es o he ERAD machine y o o he p24
p o ein complex could be masked by he e en ion e ec ha we
obse ed. We hus cons uc ed iple mu an s in which we dele ed
H d1p o Emp24p in a pm 1pm 2 backg ound and measu ed
he deg ee o s abiliza ion o Gas1*p. This allowed us o compa e
he amoun o Gas1*p deg aded by ei he pa hway in he p esence
and absence o he Pm 1p–Pm 2p complex. As can be seen in Fig.
4B, bo h iple mu an s s abilized Gas1*p as compa ed wi h
pm 1pm 2 cells. Howe e , he ela i e amoun o Gas1*p
deg aded ia he ERAD-L pa hway was ma kedly educed in he
absence o he Pm 1p–Pm 2p complex, whe eas mos o he
Gas1*p was deg aded ia a p24 complex-dependen pa hway (Fig.
Fig. 3. The p24 p o ein complex does no in e ac wi h he H d1p complex and is pa o an ERAD-L independen deg ada ion pa hway o a ac ion o
Gas1*p. (A)

Wild- ype cells (lanes 1 and 4) o cells exp essing a agged e sion o De 1p (De 1-CA; lanes 2 and 5) o Emp24-CA (lanes 3 and 6) we e lysed and
samples we e analyzed as o Fig. 1B excep ha memb anes we e immunos ained wi h an i-Usa1p an ibodies. The SDS-PAGE was pe o med using a 7% s anda d
gel. The a owhead indica es co-immunop ecipi a ed ac ion (no e ha he signal o Usa1p in he inpu ac ions is low; lanes 1–3). S a s indica e Emp24-CA and
De 1-CA ha we e also ecognized by he seconda y an ibody. The b acke indica es IgG hea y chains ha we e ecognized by he seconda y an ibody. (B)

The
deg ada ion o Gas1*p(HA) was measu ed, quan i ied and plo ed in wild- ype cells o in cells ca ying he designa ed dele ions as desc ibed in Fig. 2. The SDS-
PAGE was pe o med using a 7% s anda d gel.
Jou nal o Cell Science

4C). Toge he wi h ou da a o physical in e ac ions (Fig. 1C),
hese esul s sugges ha he Pm 1p–Pm 2p complex has a dual
ole in he a e o Gas1*p: i s , i e ains he p o ein and p e en s
i s o he wise apid deg ada ion; and second, i ul ima ely deli e s
he p o ein o he H d1p complex-dependen ERAD-L pa hway
o deg ada ion.
The Pm 1p–Pm 2p complex is equi ed o he as ER exi
o wild- ype Gas1p
Because o he associa ion o he Pm 1p–Pm 2p complex wi h he
p24 p o ein complex, we p edic ed ha he Pm 1p–Pm 2p complex
plays a ole in ER exi o e en wild- ype Gas1p. I he Pm 1p–
Pm 2p complex possesses a chape one unc ion, i should p omo e
ER exi o Gas1p. We i s es ed whe he Gas1p would be s able
in he absence o he Pm 1p–Pm 2p complex, which would be an
indica ion o i s p ope olding. To his end, we exp essed
ch omosomally HA- agged Gas1p in wild- ype cells and in
pm 1pm 2 cells and pe o med a CHX-chase expe imen (Fig.
5A, lanes 5–12). As can be seen, Gas1p ma u ed and was s able
o e a pe iod o se e al hou s in wild- ype and in pm 1pm 2
cells, which was consis en wi h he wild- ype p o ein being olded
p ope ly e en in he absence o he Pm 1p–Pm 2p complex. As a
con ol, we show he ypical pa e n o Gas1*p ha is being O-
mannosyla ed and deg aded in wild- ype cells (Fig. 5A, lanes 1–
4). We we e awa e o a minu e dec ease in elec opho e ic mobili y
o he ma u e o m o Gas1p in pm 1pm 2 cells compa ed wi h
wild- ype cells, he eason o which is cu en ly unknown (Fig.
5A, compa e lanes 5–8 wi h 9–12). Nex , we di ec ly add essed he
ER exi kine ics o Gas1p in wild- ype cells and in pm 1pm 2
cells. We pe o med a adioac i e pulse-labeling and chase
149
Pm 1p–Pm 2p complex in ER quali y con ol
expe imen ollowed by immunop ecipi a ion o Gas1p (Fig. 5B).
As can be seen, he ER exi o Gas1p was ma kedly dec eased in
pm 1pm 2 cells compa ed wi h wild- ype cells, as judged by he
slowe con e sion o he p ecu so o m in o he ma u e o m.
Whe eas in wild- ype cells abou hal o he Gas1p was con e ed
in o he ma u e o m wi hin 10 minu es, he same p ocess ook
abou 22 minu es in pm 1pm 2 cells (Fig. 5B). By con as , he
ER exi o endogenous ca boxypep idase Y (CPY) was undis u bed
in pm 1pm 2 cells compa ed wi h wild- ype cells, showing ha
he o e all kine ics o ER exi we e no gene ally a ec ed (Fig.
5C). These esul s demons a e ha he Pm 1p–Pm 2p complex
p omo es ER exi o wild- ype Gas1p.
Discussion
We ha e shown ha he Pm 1p–Pm 2p complex possesses
p e iously unknown cellula unc ions ha a e eminiscen o hose
collec i ely e med ‘ER p o ein quali y con ol’. The iden i ica ion
o i s binding pa ne s and subsequen unc ional da a p o ide a
ela i ely simple pic u e and sugges a model o how he Pm 1p–
Pm 2p complex pe o ms quali y con ol o ER p o eins.
Ou da a also cla i y some puzzling esul s wi h ega d o he
deg ada ion o mis olded GPI-ancho ed p o eins. Fo ins ance,
al hough i was epo ed ha Gas1*p is la gely deg aded by he
p o easome, a pa hway o he p o easome could no be iden i ied
(Fuji a e al., 2006). Fu he mo e, al hough i was shown ha he
Pm 1p–Pm 2p complex O-mannosyla es Gas1*p, i has emained
unclea why and whe e O-mannosyla ed Gas1*p is a ge ed o
deg ada ion (Hi ayama e al., 2008).
In he model depic ed in Fig. 6, he Pm 1p–Pm 2p complex
plays he cen al and mo e uni e sal ole, whe eas he unc ion o
Fig. 4. The Pm 1p–Pm 2p complex has a
dual ole in he a e o Gas1*p: ini ial
e en ion o he p o ein and i s ul ima e
a ge ing o he H d1p complex.
(A,B)

Deg ada ion o Gas1*p(HA) in cells
ca ying he indica ed dele ions was
measu ed, quan i ied and plo ed as
desc ibed in Fig. 2. The SDS-PAGE was
pe o med using a 4–20% g adien gel (A)
o 5% s anda d gel (B). (C)

The deg ee o
Gas1*p(HA) s abiliza ion when deple ing
he H d1p complex was compa ed wi h
ha when deple ing he p24 p o ein
complex, bo h in he absence and p esence
o he Pm 1p–Pm 2p complex (see
Ma e ials and Me hods o calcula ion).
Jou nal o Cell Science
he p24 p o ein complex is es ic ed o GPI-ancho ed p o eins,
like ou model p o eins Gas1p and Gas1*p. We base he la e
assump ion on he ac ha he p24 p o ein complex is associa ed
wi h Gas1p h oughou i s ER esidence ime and also du ing i s
ER exi (Manuel Muñiz, pe sonal communica ion). Acco dingly,
we p opose ha he Pm 1p–Pm 2p complex binds o Gas1p ha is
al eady associa ed wi h he p24 p o ein complex. Because he
p esence o he Pm 1p–Pm 2p complex p omo es as ER exi o
Gas1p (Fig. 5B), he simples scena io would be ha he complex
di ec ly o indi ec ly aids in p o ein olding (Fig. 6A). This is
suppo ed by wo obse a ions: i s , E o1p and Pdi1p, which
media e oxida i e p o ein olding, a e amongs he binding pa ne s
o he Pm 1p–Pm 2p complex (Table 1); and second, mammalian
cells exp ess a highly UPR- egula ed soluble p o ein in he ER
wi h homology o he la ges luminal loop, loop 5, o he amily o
PMTs (Fukuda e al., 2001; Hamada e al., 1996). This p o ein,
SDF2L1 in mouse, is also ound in complex wi h o he ER esiden
chape ones (Meunie e al., 2002). Ve y in e es ingly, on he basis
o he egion o homology be ween SDF2L1, Pm 1p and Pm 2p
and on unc ional s udies wi h Pm 1p (Gi bach e al., 2000),
SDF2L1 should be incapable o media ing O-mannosyla ion. This
s ongly sugges s ha he p oposed chape one-like unc ion o he
Pm 1p–Pm 2p complex is dis inc om i s O-mannosyla ion
ac i i y. Finally, p24 p o ein complex-dependen ER expo o
co ec ly olded Gas1p occu s a e dissocia ion om he Pm 1p–
150 Jou nal o Cell Science 124 (1)
Pm 2p complex, which, in con as o he p24 p o ein complex,
emains in he ER (Haselbeck and Tanne , 1983; Huh e al., 2003).
Like Gas1p, Gas1*p binds o he p24 p o ein complex and
subsequen ly o he Pm 1p–Pm 2p complex (Fig. 6B). Howe e ,
since Gas1*p canno be olded p ope ly i con inuously ebinds o
he Pm 1p–Pm 2p complex and is hus e ained by i in he ER
(Fig. 4 and Fig. 6B). This e en ion has a leas wo consequences:
i s , Gas1*p is inc easingly O-mannosyla ed wi h ime in a Pm 1p–
Pm 2p complex-dependen manne (Fig. 2, Fig. 5A, Fig. 6B;
supplemen a y ma e ial Fig. S1 and Fig. S3); and second, he
Pm 1p–Pm 2p complex e en ually a ge s Gas1*p o he H d1p
complex o ERAD (Fig. 4 and Fig. 6B). The p ecise molecula
ole o O-mannans in Pm 1p–Pm 2p complex-media ed p o ein
e en ion and H d1p complex-media ed p o ein deg ada ion is an
exci ing subjec o be add essed nex . Because he Pm 1p–Pm 2p
complex can associa e wi h he H d1p complex p o ein, O-
mannosyla ion should ha e a unc ion di e en om a ge ing
p o eins o he ERAD machine y. Consis en ly, O-mannosyla ion
is no s ic ly equi ed o ERAD-dependen deg ada ion o Gas1*p
because deg ada ion ia he H d1p complex s ill occu s in he
absence o he Pm 1p–Pm 2p complex (Fig. 4B). We es ed whe he
he Pm 1p–Pm 2p complex migh egula e subs a e deg ada ion
by O-mannosyla ing ERAD machine y componen s bu ound ha
his is no he case (supplemen a y ma e ial Fig. S4). In ano he
model, inc easing O-mannosyla ion o he subs a e migh help o
Fig. 5. The Pm 1p–Pm 2p complex p omo es
ER exi o Gas1p. (A)

Gas1*p(HA) o
Gas1p(HA) we e indi idually exp essed in
wild- ype o in pm 1pm 2 cells and analyzed
as desc ibed in Fig. 2. p, p ecu so o m; m,
ma u e o m. The SDS-PAGE was pe o med
using a 5% s anda d gel. (B)

Yeas cells
exp essing Gas1p(HA) in wild- ype o in
pm 1pm 2 cells we e labeled wi h
[35S]me hionine o 10 minu es and chased o
he indica ed imes. The cells we e lysed and
Gas1p(HA) was immunop ecipi a ed using an i-
HA an ibodies and analyzed by SDS-PAGE
(5% s anda d gel) and au o adiog aphy. P o ein
ma u a ion was plo ed o e ime on he basis o
he ob ained ac ion o ma u e p o ein o m (m)
om o al p o ein a indica ed imes ( igh
panel). Values we e ob ained using a
Phospo Image . The appa en a e o
ma u a ion o Gas1p was calcula ed and shown
wi h linea eg ession a e inclusion o he
alue ze o a ime –10 (s a o pulse). (C)

The
same expe imen wi h iden ical cells was
pe o med bu he acuola pep idase CPY was
immunop ecipi a ed using an i-CPY an ibodies.
p1, p ecu so o m 1, p2, p ecu so o m 2.
Plo ing and calcula ions we e done as o B.
Jou nal o Cell Science
dissocia e i om he Pm 1p–Pm 2p complex and allow ans e o
he associa ed ERAD machine y. This model can be es ed in he
u u e h ough he gene a ion and u iliza ion o sui able O-
mannosyla ion mu an s o he Pm 1p–Pm 2p complex.
Deple ion o he Pm 1p–Pm 2p complex leads o an ‘escape’ o
mos o he Gas1*p om H d1p complex-media ed ERAD o a
p24-dependen , p obably pos -ER, deg ada ion (Figs 4 and 6).
Redundan quali y con ol in he ER, al hough abundan , is p obably
no oo s ingen o a oid cos ly des uc ion o olding in e media es.
Howe e , i becomes clea ha he cell possesses addi ional and
less well cha ac e ized quali y con ol sys ems in he sec e o y
pa hway downs eam o he ER ha can a ge p o eins o
deg ada ion (Caldwell e al., 2001; Haynes e al., 2002; Vashis e
al., 2001; Wang and Ng, 2010). Al hough we canno ule ou wi h
ce ain y ha he p24 complex is a membe o such a pos -ER
151
Pm 1p–Pm 2p complex in ER quali y con ol
quali y con ol sys em, we did no ind componen s wi h known
unc ional links o he p o easome o o he acuole when we
analyzed he binding pa ne s o Emp24p (V.G. and A.M.,
unpublished da a). Thus, he pa hway o p24 p o ein-dependen
Gas1*p deg ada ion emains o be iden i ied.
Apa om Gas1*p, he Pm 1p–Pm 2p complex has been shown
o O-mannosyla e se e al o he mis olded soluble o memb ane-
bound p o eins. Examples include KHN, KWW, mu an aspa ic
p o ease I and mu an - ac o , none o which a e GPI-ancho ed
bu all o which a e subjec ed o H d1p complex-dependen ERAD
(Ha y e al., 2001; Naka sukasa e al., 2004; Vashis e al., 2001;
Wahlman e al., 2007). An o e all inc ease in p o ein O-
mannosyla ion has also been obse ed upon inhibi ion o N-
glycosyla ion, which induces p o ein mis olding (Ha y e al.,
2001).
We he e o e sugges ha he Pm 1p–Pm 2p complex can a ge
a wide a ie y o mis olded p o eins o he H d1p complex. One
explana ion why his conclusion has been missed so a and why
he e is con o e sy abou he a e o O-mannosyla ed mis olded
p o eins is ha classical es s o in ol emen o cellula
componen s in p o ein deg ada ion measu e he deg ee o inhibi ion
o subs a e deg ada ion in dele ion mu an s. Howe e , ou da a
clea ly show ha he Pm 1p–Pm 2p complex has a e en ion
unc ion on op o i s a ge ing unc ion o he ERAD machine y,
which complica es ma e s. In addi ion, he cell possesses mul iple
and appa en ly e y dynamic pa hways o p o ein deg ada ion,
which can lead o he a ge ing o mis olded p o eins o di e en
ou es upon deple ion o one pa hway. Conside ing his, a mo e
combina o ial app oach using mu an s wi h dis up ions in mul iple
pa hways was needed o ob ain be e insigh in o he se e al
unc ions o he Pm 1p–Pm 2p complex (Fig. 4). The same
a gumen s eadily explain he kine ic al e a ions obse ed o ER
expo o Gas1p and o he deg ada ion o Gas1*p in he absence
o he Pm 1p–Pm 2p complex. Whe eas he expo a e o Gas1p
was dec eased (Fig. 5), he appa en deg ada ion a e o Gas1*p
was inc eased (Fig. 4). Wi h espec o Gas1p, he delay o ER
expo in he absence o he Pm 1p–Pm 2p complex is consis en
wi h loss o a chape one ha helps apid olding o Gas1p (Fig.
5B). Wi h espec o Gas1*p, as e deg ada ion by a pa hway
o he han ERAD in he absence o he Pm 1p–Pm 2p complex is
consis en wi h loss o bo h ER e en ion and a ge ing o ERAD
(Fig. 4).
Besides i s gene al ole in ER p o ein e en ion, ERAD and ER
expo , he Pm 1p–Pm 2p complex migh possess an addi ional
and mo e speci ic unc ion o he olding and ER expo o GPI-
ancho ed p o eins. This is sugges ed by he physical link o he p24
p o ein complex, as only one o many known ER expo ac o s.
In e es ingly, we also iden i ied Ted1p as a binding pa ne o
he Pm 1p–Pm 2p complex bu no o he p24 p o ein complex
(Table 1; V.G. and A.M., unpublished da a). Ted1p was i s
iden i ied in a sc een o p o eins impai ing he su ace exp ession
o mammalian G-p o ein-ac i a ed Ki channel GIRK2 in yeas
(Haass e al., 2007). Likewise, he p24 p o ein complex componen s
Emp24p and E 25p we e among he only six o he hi s om he
376 es ed dele ions. I was p e iously shown ha Ted1p and he
p24 complex p o eins clus e in an epis asis mini-a ay (E-MAP),
which sugges ed a common biological unc ion (Schuldine e al.,
2005). Indeed, he ma u a ion o Gas1p was delayed in a Ted1p
dele ion s ain simila o an Emp24p dele ion s ain (Haass e al.,
2007). The mammalian o holog o Ted1p, PAGP5, is a GPI-
ancho emodeling enzyme and was ecen ly shown o emo e he
Fig. 6. A model o he ole o he Pm 1p–Pm 2p complex in ER p o ein
quali y con ol. The scheme illus a es he p oposed ole o he Pm 1p–Pm 2p
complex in ER p o ein quali y con ol o he es ed model p o eins Gas1p and
Gas1*p. The basic p inciple should also be applicable o o he subs a es (see
Discussion). (A)

ER ma u a ion o Gas1p (solid black a ows). A e ER
ansloca ion and GPI-ancho ans e and p io o ER expo , Gas1p olds and
has i s GPI ancho emodeled. Gas1p is p obably associa ed o he p24 p o ein
complex h oughou i s ER esidence ime. Because he Pm 1p–Pm 2p
complex possesses some cha ac e is ics o classical chape ones and aids in
apid ER expo o Gas1p, i could be in ol ed in p o ein olding and/o GPI-
ancho emodeling ( hick g ay a ows). (B)

ER e en ion and deg ada ion o
Gas1*p (dashed black a ows). Like Gas1p, Gas1*p associa es wi h he p24
p o ein complex. Howe e , an ER expo compe en old canno be achie ed,
which esul s in ex ended associa ion wi h he Pm 1p–Pm 2p complex
(“Re en ion”), p o ein O-mannosyla ion (“O-mannosyla ion”) and subsequen
ans e o he H d1p complex (“Ta ge ing o ERAD”) o e o ansloca ion
and p o easomal deg ada ion (see Discussion o de ails). Those Gas1*p
a ian s ha can escape his p ima y ER quali y con ol ( he amoun will
d as ically inc ease when he Pm 1p–Pm 2p complex is absen ) will be
subjec ed o a second, as ye uncha ac e ized, con ol sys em, p obably in a
pos -ER compa men (ques ion ma k).
Jou nal o Cell Science
152 Jou nal o Cell Science 124 (1)
side-chain e hanolamine phospha e o he second mannose a ached
o he GPI-ancho backbone. This ac i i y is p e equisi e o
e icien ER exi o GPI-ancho ed p o eins (Fuji a e al., 2009). In
a specula i e model, he Pm 1p–Pm 2p complex migh egula e
access o he Gas1p–p24 p o ein complex o GPI-ancho
emodeling enzymes and hus couple p o ein olding wi h GPI-
ancho emodeling. This scena io could be pa o Pm 1p–Pm 2p
complex-media ed Gas1p ‘ olding’, as shown in Fig. 6A.
Ma e ials and Me hods
Yeas s ains and plasmids
The s ains used we e isogenic o W303 (MATa leu2-3,112 p1-1 can1-100 u a3-1
ade2-1 his3-11,15) excep hose used in expe imen s shown in Fig. 1, Fig. 3A and
supplemen a y ma e ial Fig. S2, which we e isogenic o BY4741 (MATa his3leu2
u a3). Tagging o p o eins o genomic gene dele ions we e pe o med using
s anda d PCR-based homologous ecombina ion echniques. Gas1*p(HA) was
in eg a ed in o he URA3 locus o yeas cells using he in eg a i e plasmid pMF616,
which was a gi om Mo ihisa Fuji a (Fuji a e al., 2006). Gas1p(HA) was de i ed
om pMF616 by back mu a ion o he single base exchange using s anda d PCR-
based mu agenesis and in eg a ed in o he URA3 locus o yeas cells. All cons uc s
we e sequenced.
P o ein complex pu i ica ion and co-immunop ecipi a ion
Fo mass spec ome y analysis, app oxima ely 15 g o cells we e lysed by g inding
in liquid ni ogen and he memb anes sedimen ed. Memb anes we e solubilized in
bu e con aining 1% digi onin (Calbiochem). The ex ac was incuba ed o se e al
hou s wi h IgG-coupled magne ic beads (Dynal). A e washing and elu ion, he
elua e was ei he p ecipi a ed wi h ichlo oace ic acid (TCA) o sepa a ed by SDS-
PAGE, ollowed by Coomassie Blue s aining and excision o indi idual bands. In
bo h cases, he p o eins we e iden i ied by mass spec ome y. Fo co-
immunop ecipi a ion, essen ially he same p o ocol was u ilized, wi h he excep ion
ha ma e ial om 200 ml cul u es we e used and cells we e lysed using bead
bea ing. A e SDS-PAGE, p o eins we e analyzed by immunblo ing wi h an ibodies
o HA (Roche) o Usa1p (Ca alho e al., 2006), o wi h abbi IgG (Sigma).
Gas1*p(HA) and Gas1p(HA) deg ada ion expe imen s
Cycloheximide shu -o expe imen s we e pe o med in exponen ially g owing cells.
The shu -o was s a ed by addi ion o CHX o a inal concen a ion o 200 g/ml.
Equal olume aliquo s o cell cul u e we e emo ed a indica ed ime poin s and
mo ed o ice. Cells we e lysed using 150 mM NaOH ollowed by addi ion o sample
bu e con aining 1% SDS and hea ing. Cellula Gas1*p(HA) o Gas1p(HA) con en
was p obed by SDS-PAGE ollowed by wes e n blo ing using an i-HA an ibodies.
To quan i y bands wi h he Odyssey In a ed Imaging Sys em (LI-COR Biosciences)
a dye-coupled seconda y an ibody was used (Fig. 2B,C, Fig. 4A; supplemen a y
ma e ial Fig. S2). To quan i y bands wi h a LAS-3000 Imaging Sys em (Fuji ilm
Li escience) and Mul i-Gauge So wa e, a pe oxidase-coupled seconda y an ibody
was used (Fig. 3B, Fig. 4B; supplemen a y ma e ial Fig. S1).
Radioac i e pulse-chase and immunop ecipi a ion
Fo in i o pulse labeling, an o e nigh yeas cul u e was dilu ed and g own o an
op ical densi y (OD) o ~0.8 a 600 nm. Cells equi alen o 5 OD we e esuspended
in 1 ml medium lacking me hionine, incuba ed o 15 minu es a 30°C, and labeled
o 10 minu es wi h 100 Ci/ml [35S]me hionine (Pe kin Elme s). Cells we e dilu ed
o OD 0.8, supplemen ed wi h me hionine and 200 g/ml CHX. Aliquo s we e aken
a indica ed imes, cells we e mo ed o ice and supplemen ed wi h 10 mM azide,
pelle ed, esuspended in 50 mM T is pH 7.5, 5 mM EDTA, 1 mM PMSF, and lysed
wi h glass beads o 7 minu es in a bead-bea e , supplemen ed wi h 1% SDS, and
hea ed a 65°C o 10 minu es. Cell emnan s we e emo ed by cen i uga ion o 10
minu es in a mic o uge, and he supe na an used o immunop ecipi a ion using
an i-HA an ibodies. Immune complexes we e isola ed wi h P o ein G-Sepha ose (GE
Heal hca e) and analyzed by SDS-gel elec opho esis and au o adiog aphy using a
Phospho Image (Fuji).
Calcula ing Gas1*p s abiliza ion in h d1 and emp24 cells in p esence o
absence o he Pm 1/2p complex as shown in Fig. 4C
Gas1*p u no e in wild- ype cells was de e mined om a leas h ee indi idual
expe imen s by measu ing emaining Gas1*p by wes e n blo ing as shown in Fig.
2B. The ob ained alues o indi idual imepoin s we e used o linea eg ession
using he leas squa e me hod (LINEST unc ion in EXCEL). We ob ained R2 alues
o 0.9344 o highe o each da a se . The m alues (ymx+b) o each indi idual
eg ession se we e calcula ed, as well as he mean alue and i s s anda d de ia ion.
We hen compa ed he m alue ob ained wi h wild- ype cells wi h hose ob ained
wi h single dele ion mu an s (emp24 and h d1). The de ia ion in m alues o
mu an s om hose ob ained wi h wild- ype cells was aken as deg ee o s abiliza ion
(s abmmu /mw ). Because we ound Gas1*p is maximally s able in emp24h d1
cells compa ed o he indi idual mu an s (Fig. 3B), we assumed sepa a e deg ada ion
pa hways and combined he indi idual de e mined deg ees o s abiliza ion o 100%
(s abemp24+s abh d1100). Las ly, we plo ed he deg ee o Gas1*p s abiliza ion o
each mu an as a pe cen age o o al, ei he in he p esence o he Pm 1p–Pm 2p
complex (h d1 s emp24) o in i s absence (h d1pm 1pm 2 s
emp24pm 1pm 2).
We would like o hank Ped o Ca alho (CRG, Ba celona, Spain)
and Manuel Muñiz (Uni e si y o Se ille, Se ille, Spain) o an ibodies,
Mo ihisa Fuji a (Osaka Uni e si y, Osaka, Japan) o plasmids and
Ross Tomaino (Ha a d Medical School, Bos on, MA) o excellen
mass spec ome ic analysis, Alex Palazzo, Tom Rapopo , Ma in
Spiess and Ka l E landson o c i ical eading o an ea lie e sion o
he manusc ip ; and Manuel Muñiz and Le icia Lemus o e y ui ul
discussions h oughou he wo k. V.G. is suppo ed by a G an o he
Spanish Minis y o Science, BFU2009-07290. V.G. is a Ramon y
Cajal ellow.
Supplemen a y ma e ial a ailable online a
h p://jcs.biologis s.o g/cgi/con en / ull/124/1/144/DC1
Re e ences
Balli , B. A., Roux, P. P., Ge be , S. A., MacKeigan, J. P., Blenis, J. and Gygi, S. P.
(2005). Quan i a i e phospho yla ion p o iling o he ERK/p90 ibosomal S6 kinase-
signaling casse e and i s a ge s, he ube ous scle osis umo supp esso s. P oc. Na l.
Acad. Sci. USA 102, 667-672.
Ba lowe, C. (2003). Signals o COPII-dependen expo om he ER: wha ’s he icke
ou ? T ends Cell. Biol. 13, 295-300.
B achmann, C. B., Da ies, A., Cos , G. J., Capu o, E., Li, J., Hie e , P. and Boeke, J.
D. (1998). Designe dele ion s ains de i ed om Saccha omyces ce e isiae S288C: a
use ul se o s ains and plasmids o PCR-media ed gene dis up ion and o he
applica ions. Yeas 14, 115-132.
B odsky, J. L., We ne , E. D., Dubas, M. E., Goeckele , J. L., K use, K. B. and
McC acken, A. A. (1999). The equi emen o molecula chape ones du ing
endoplasmic e iculum-associa ed p o ein deg ada ion demons a es ha p o ein expo
and impo a e mechanis ically dis inc . J. Biol. Chem. 274, 3453-3460.
Caldwell, S. R., Hill, K. J. and Coope , A. A. (2001). Deg ada ion o endoplasmic
e iculum (ER) quali y con ol subs a es equi es anspo be ween he ER and Golgi.
J. Biol. Chem. 276, 23296-23303.
Ca alho, P., Gode , V. and Rapopo , T. A. (2006). Dis inc ubiqui in-ligase complexes
de ine con e gen pa hways o he deg ada ion o ER p o eins. Cell 126, 361-373.
Denic, V., Quan, E. M. and Weissman, J. S. (2006). A luminal su eillance complex ha
selec s mis olded glycop o eins o ER-associa ed deg ada ion. Cell 126, 349-359.
Dieguez-Acuna, F. J., Ge be , S. A., Kodama, S., Elias, J. E., Beausoleil, S. A.,
Faus man, D. and Gygi, S. P. (2005). Cha ac e iza ion o mouse spleen cells by
sub ac i e p o eomics. Mol. Cell P o eomics 4, 1459-1470.
Ellgaa d, L. and Helenius, A. (2003). Quali y con ol in he endoplasmic e iculum. Na .
Re . Mol. Cell Biol. 4, 181-191.
F a, A. M., Fagioli, C., Finazzi, D., Si ia, R. and Albe ini, C. M. (1993). Quali y con ol
o ER syn hesized p o eins: an exposed hiol g oup as a h ee-way swi ch media ing
assembly, e en ion and deg ada ion. EMBO J. 12, 4755-4761.
Fuji a, M., Yoko, O. T. and Jigami, Y. (2006). Inosi ol deacyla ion by Bs 1p is equi ed
o he quali y con ol o glycosylphospha idylinosi ol-ancho ed p o eins. Mol. Biol.
Cell 17, 834-850.
Fuji a, M., Maeda, Y., Ra, M., Yamaguchi, Y., Taguchi, R. and Kinoshi a, T. (2009).
GPI glycan emodeling by PGAP5 egula es anspo o GPI-ancho ed p o eins om
he ER o he Golgi. Cell 139, 352-365.
Fukuda, S., Sumii, M., Masuda, Y., Takahashi, M., Koike, N., Teishima, J., Yasumo o,
H., I amo o, T., Asaha a, T., Dohi, K. e al. (2001). Mu ine and human SDF2L1 is an
endoplasmic e iculum s ess-inducible gene and encodes a new membe o he Pm /
p o ein amily. Biochem. Biophys. Res. Commun. 280, 407-414.
Gi bach, V. and S ahl, S. (2003). Membe s o he e olu iona ily conse ed PMT amily
o p o ein O-mannosyl ans e ases o m dis inc p o ein complexes among hemsel es.
J. Biol. Chem. 278, 12554-12562.
Gi bach, V., Zelle , T., P iesmeie , M. and S ahl-Bolsinge , S. (2000). S uc u e-
unc ion analysis o he dolichyl phospha e-mannose: p o ein O-mannosyl ans e ase
ScPm 1p. J. Biol. Chem. 275, 19288-19296.
Gode , V., Ca alho, P. and Rapopo , T. A. (2008). The ER-associa ed deg ada ion
componen De 1p and i s homolog D m1p a e con ained in complexes wi h dis inc
co ac o s o he ATPase Cdc48p. FEBS Le 582, 1575-1580.
Haass, F. A., Jonikas, M., Wal e , P., Weissman, J. S., Jan, Y. N., Jan, L. Y. and
Schuldine , M. (2007). Iden i ica ion o yeas p o eins necessa y o cell-su ace
unc ion o a po assium channel. P oc. Na l. Acad. Sci. USA 104, 18079-18084.
Hamada, T., Tashi o, K., Tada, H., Inazawa, J., Shi ozu, M., Shibaha a, K., Nakamu a,
T., Ma ina, N., Nakano, T. and Honjo, T. (1996). Isola ion and cha ac e iza ion o a
no el sec e o y p o ein, s omal cell-de i ed ac o -2 (SDF-2) using he signal sequence
ap me hod. Gene 176, 211-214.
Hammond, C., B aakman, I. and Helenius, A. (1994). Role o N-linked oligosaccha ide
ecogni ion, glucose imming, and calnexin in glycop o ein olding and quali y con ol.
P oc. Na l. Acad. Sci. USA 91, 913-917.
Jou nal o Cell Science