
The Rocke elle Uni e si y P ess, 0021-9525/2000/03/925/6 $5.00
The Jou nal o Cell Biology, Volume 148, Numbe 5, Ma ch 6, 2000 925–930
h p://www.jcb.o g 925
The Emp24 Complex Rec ui s a Speci ic Ca go Molecule in o Endoplasmic
Re iculum–de i ed Vesicles
Manuel Muñiz, Claude Nuo e , Hans-Pe e Hau i, and Howa d Riezman
Biozen um o he Uni e si y o Basel, CH-4056 Basel, Swi ze land
Abs ac .
Membe s o he yeas p24 amily, including
Emp24p and E 25p, o m a he e ome ic complex e-
qui ed o he e icien anspo o selec ed p o eins
om he endoplasmic e iculum (ER) o he Golgi ap-
pa a us. The speci ic unc ions and si es o ac ion o his
complex a e unknown. We show ha Emp24p is di-
ec ly equi ed o e icien packaging o a lumenal
ca go p o ein, Gas1p, in o ER-de i ed esicles.
Emp24p and E 25p can be di ec ly c oss-linked o
Gas1p in ER-de i ed esicles. Gap1p, which was no a -
ec ed by
emp24
mu a ion, was no c oss-linked. These
esul s sugges ha he Emp24 complex ac s as a ca go
ecep o in esicle biogenesis om he ER.
Key wo ds: COPII-coa ed esicles • ER • E 25p •
Saccha omyces ce e isiae •
p o ein so ing
In oduc ion
In euka yo ic cells, p o ein anspo be ween he o -
ganelles o he sec e o y pa hway is media ed by esicles
ha bud om a dono compa men and use wi h an ap-
p op ia e accep o compa men (Palade, 1975). The s a -
ing poin o he exocy ic ou e is he ER. The e, co ec ly
olded and assembled ca go molecules can en e COPII-
coa ed esicles o anspo o he
cis
-Golgi compa men
(Schekman and O ci, 1996). Many cy osolic and ans-
memb ane p o eins ha e been iden i ied ha media e his
esicula anspo s ep (Ro hman and Wieland, 1996;
Schekman and O ci, 1996).
p24 p o eins a e p esen in he e ome ic complexes ha
ha e been p oposed o cycle be ween ER and Golgi com-
pa men s because hey a e ound in bo h COPII and
COPI esicles in addi ion o ER and Golgi memb anes
(Schimmölle e al., 1995; S amnes e al., 1995; Belden
and Ba lowe, 1996; Sohn e al., 1996; Rojo e al., 1997;
Dominguez e al., 1998; Füllek ug e al., 1999; Ma zioch e
al., 1999). In mammals, unc ional da a sugges a ole o
hese p o eins in he o ma ion o ER exi si es (La oie e
al., 1999). In yeas , eigh genes encode p24 amily mem-
be s:
EMP24
,
ERV25
, and
ERP1
–
ERP6
(Ma zioch e al.,
1999). Mu an s in se e al o hese genes show selec i e
p o ein anspo de ec s. S ains wi h a single dele ion o
EMP24
,
ERV25
,
ERP1
, o
ERP2
show a delay in he ER
o Golgi anspo o Gas1p (Schimmölle e al., 1995;
Belden and Ba lowe, 1996; Ma zioch e al., 1999), a gly-
cosylphospha idylinosi ol (GPI)
1
-ancho ed p o ein. The
emp24
and
e 25
mu an s also show a anspo de ec o
in e ase, a soluble sec e ed p o ein. Se e al o he ca go
molecules, including
␣
ac o , a sec e ed phe omone, do
no equi e p24 p o eins o e icien anspo . Mu a ion
o se e al yeas p24 genes esul s in sec e ion o he ER lu-
menal p o ein, Ka 2p (El od-E ickson and Kaise , 1996;
Ma zioch e al., 1999).
The Emp24 complex in yeas comp ises Emp24p, E 25p,
and mos likely E p1p and E p2p. Dele ion o
EMP24
causes a s ong educ ion in he le els o he o he h ee
p o eins o he complex (Belden and Ba lowe, 1996; Ma -
zioch e al., 1999). In his s udy we p o ide e idence ha
he Emp24 complex is di ec ly equi ed o e icien pack-
aging o Gas1p in o ER-de i ed esicles. Two subuni s o
his complex can be di ec ly c oss-linked o he ca go p o-
ein in pu i ied ER-de i ed esicles, consis en wi h he
hypo hesis ha he Emp24 complex plays a ole as a ca go
ecep o in ER o Golgi anspo .
Ma e ials and Me hods
S ains
An myc epi ope was in oduced a he NH
2
e minus o ma u e Emp24p.
The myc- agged
emp24-E178A
mu an was cons uc ed by subs i u ing an
app op ia e agmen o
EMP24
wi h he sequenced mu an e sion ob-
ained by PCR echniques. RH4443 (
MAT
␣
, u a3, leu2, his4, ba 1, emp24::
KanMx
) was ob ained by eplacing he en i e
EMP24
coding sequence o
RH1959 (
MAT
␣
,
u a3, leu2, his4, ba 1
) wi h a
KanMx
casse e.
EMP24
al-
leles we e cloned in o a YCplac111 (CEN/ARS) plasmid. The
S. ce e isiae
s ain RH696-2B (
MAT
␣
, sec18-20 gap1
⌬
::LEU2 u a3 ade2 leu2 lys2
⌬
201
pPL269) was ob ained by c ossing PLY129 (pPL269) (
MAT
␣
gap1
⌬
::
LEU2 u a3 ade2 leu2 lys2
⌬
201
pPL269) (Kuehn e al., 1996), wi h RH478
(
MAT
␣
sec18-20 leu2 his4
). Cy osols we e p epa ed om RH732 (
MAT
␣
his4 leu2 u a3 lys2 pep4::URA3 ba 1
) and RH2043 (
MAT
␣
sec18-20 his4
leu2 u a3 pep4::URA3 ba 1
).
Add ess co espondence o Howa d Riezman, Biozen um o he Uni e -
si y o Basel, Klingelbe gs asse 70, CH-4056 Basel, Swi ze land. Tel.: 41-
61-267-2160. Fax: 41-61-267-2149. E-mail: [email p o ec ed]
1
Abb e ia ions used in his pape :
DSG, disuccinimidyl glu a a e; DSP,
di hiobis(succinimidylp opiona e); gp
␣
F, glycosyla ed p o
␣
ac o ; GPI,
glycosylphospha idylinosi ol.
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Published Ma ch 6, 2000
The Jou nal o Cell Biology, Volume 148, 2000 926
P o ein Techniques
P o ein le els we e de e mined by ex ac ion o log phase cul u es and
Wes e n blo ing (Sü e lin e al., 1997) using an ibodies aised agains he
Emp24p cy osolic ail o he myc epi ope. Analysis o Gas1p anspo in
i o was by a pulse-chase p o ocol (Sü e lin e al., 1997). Gas1p ma u a-
ion was analyzed using a 4-min pulse and subsequen chase a 30
⬚
C, ol-
lowed by immunop ecipi a ion, SDS-PAGE, and luo og aphy. Imma u e
(105 kD) and ma u e (125 kD) o ms o Gas1p we e quan i ied using a
densi ome e . The pe cen age o ma u e Gas1p was used as an indica ion
o anspo o he Golgi appa a us. De e mina ion o pa i ioning o
Gas1p be ween aqueous and de e gen phases o T i on X-114 was pe -
o med as desc ibed (Nuo e e al., 1993), excep ha he media ac ions
we e no analyzed. A e sepa a ion in o de e gen and aqueous phases,
Gas1p was dena u ed, immunop ecipi a ed, esol ed by SDS-PAGE, i-
sualized, and quan i ied using a Phospho Image .
In Vi o ER-budding Assay
The pe meabilized, cell-based, ER ca go packaging assay was pe o med
as desc ibed (Kuehn e al., 1996) wi h a ew modi ica ions. S ains
RH1959, RH4438, and RH696-2B we e ans o med wi h pCNYG1 o
o e exp ess Gas1p (app oxima ely i e old) (Nuo e e al., 1991). O e -
exp ession did no a ec Gas1p anspo kine ics o dependence on
EMP24
. 10 mM 2-me cap oe hanesul onic acid eplaced 10 mM di hio-
h ei ol. Budding eac ions con ained 30
␮
l o memb anes ( om 12
⫻
10
7
cells), 300
␮
g c ude cy osol, 3
␮
g Sa 1p, 1
⫻
ATP mix, and 0.2 mM GTP in
a 150-
␮
l olume. A e 2 h a 25
⬚
C, a po ion o he sample was emo ed
o analysis ( o al), he emaining aliquo was sedimen ed (14,000 pm, 2
min, 4
⬚
C), and 125
␮
l o he supe na an collec ed and subjec ed o lo a-
ion on a Nycodenz
®
s ep g adien (Ba lowe e al., 1994). 100
␮
l om he
op was disca ded and he nex 800
␮
l ans e ed o a new ube. 400
␮
l
was dilu ed h ee old wi h B88 and cen i uged (100,000
g
, 1 h, 4
⬚
C). The
memb ane pelle was dissol ed in 1% SDS in TEPI bu e (Sü e lin e al.,
1997) o 10 min a 55
⬚
C, subjec ed o immunop ecipi a ion, and analyzed
by SDS-PAGE wi h subsequen exposu e and quan i a ion using a Phos-
pho Image .
Vesicle Immunoisola ion
Vesicles we e p oduced in a budding eac ion using
sec18
memb anes and
cy osol. The
sec18
memb anes we e p epa ed as abo e, excep ha he
las 5 min o deple ion and he pulse-labeling we e a 32
⬚
C. The
sec18
cy-
osol was p eincuba ed (32
⬚
C, 10 min) be o e use. Vesicles we e immu-
noisola ed wi h o wi hou Emp24p an i- ail an ibody and p ocessed ac-
co ding o Kuehn e al. (1996).
C oss-Linking
Nycodenz-pu i ied esicles p oduced in a budding eac ion we e adjus ed
o 2.5 M u ea in B88 and incuba ed wi h 1 mM di hiobis(succinimidylp o-
piona e) (DSP) o a ious amoun s o disuccinimidyl glu a a e (DSG;
Pie ce) (20
⬚
C, 20 min). The c oss-linking eac ion was quenched by addi-
ion o glycine (50 mM inal, 5 min, 20
⬚
C). Vesicles we e sedimen ed a
100,000
g
(1 h, 4
⬚
C), dissol ed wi h 1% SDS in TEPI (5 min, 95
⬚
C o
Gas1p and glycosyla ed p o
␣
ac o [gp
␣
F], o 55
⬚
C o Gap1p), and im-
munop ecipi a ed wi h Emp24p an i- ail an ibody o E 25 an ibody
(Belden and Ba lowe, 1996) and p o ein A–Sepha ose. P ecipi a ed ma e-
ial was elu ed om he Sepha ose beads by incuba ion wi h 1% SDS in
TEPI (5 min, 95 o 55
⬚
C) and eimmunop ecipi a ed wi h an i-Gas1p o
an i-Gap1p an ibody.
Resul s
Emp24p Is Requi ed o E icien Rec ui men o Gas1p
in o ER-de i ed Vesicles
To in es iga e whe he Emp24p plays a ole in ca go exi
om he ER we quan i ied he packaging o di e en
sec e o y p o eins in o esicles ha we e gene a ed om
wild- ype and
emp24
mu an ER memb anes in i o. Bud-
ding o Gas1p was much less e icien (
⬎
70% less) om
emp24
⌬
memb anes han om wild- ype memb anes,
whe eas he budding e iciencies o gp
␣
F and an amino
acid pe mease (Gap1p) we e simila om he wo mem-
b ane sou ces (Fig. 1). gp
␣
F is an es ablished con ol o
selec i i y, because i s anspo om he ER o he Golgi
is no a ec ed in
emp24
⌬
cells (Schimmölle e al., 1995).
Gap1p was also in es iga ed because i has been shown o
be e icien ly inco po a ed in o COPII-coa ed esicles in
i o (Kuehn e al., 1996). The e o e, Emp24p is equi ed
o e icien packaging o a leas one sec e o y p o ein
in o COPII-coa ed esicles. The magni ude o he de ec
in Gas1p budding om he ER is consis en wi h he ER
o Golgi anspo de ec seen in i o (Schimmölle e al.,
1995) and could, he e o e, be he en i e explana ion o
he anspo delay.
I has been p oposed ha Emp24p may be a nega i e
egula o o esicle o ma ion (El od-E ickson and Kaise ,
1996) and ha i s absence would esul in inc eased Ka 2p
exi om he ER. To add ess his poin , we compa ed he
inco po a ion o Ka 2p in o esicles using wild- ype and
emp24
⌬
memb anes. Ka 2p was poo ly packaged in o ER-
de i ed esicles using ei he memb ane sou ce (Fig. 1).
The e o e, i does no seem ha Emp24p is equi ed o
excluding Ka 2p om ER-de i ed esicles. This sugges s,
bu does no p o e, ha he eason o Ka 2p sec e ion in
he
emp24
mu an is a de ec in e og ade anspo .
Figu e 1. In i o budding o ER-de i ed esicles om wild- ype
and emp24⌬ memb anes. Sphe oplas s om wild- ype (EMP24)
and mu an (emp24⌬) cells we e pulse-labeled (3 min), pe me-
abilized, and incuba ed wi h an ene gy sou ce wi h o wi hou cy-
osol (2 h, 25⬚C). Vesicles we e pu i ied by di e en ial cen i-
uga ion and lo a ion on Nycodenz® g adien s. Budding o
indica ed p o eins was assayed by immunop ecipi a ion, SDS-
PAGE, isualiza ion, and quan i ica ion using a Phospho Image .
The budding e iciency o a gi en p o ein e e s o he pe cen -
age o he o al adiolabeled p o ein ha was eco e ed in he
pu i ied esicula ac ion.
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Published Ma ch 6, 2000
Muñiz e al.
Ca go Rec ui men in o ER-de i ed Vesicles
927
Emp24p Is Di ec ly In ol ed in he Selec i e Packaging
o Ca go Molecules
Since Emp24p could play a ole in e og ade anspo i
is possible ha he ine icien budding o Gas1p om
emp24
⌬
memb anes is an indi ec consequence o a e o-
g ade anspo de ec . To add ess his poin , we used an i-
bodies agains he cy osolic ail o Emp24p o inhibi he
unc ion o Emp24p in wild- ype memb anes. P eincuba-
ion o memb anes wi h an ibodies on ice educed he
budding e iciency o Gas1p g ea e han h ee old (Fig. 2
A). This inhibi ion was speci ic o Gas1p, because he
packaging o Gap1p and gp
␣
F was no a ec ed. As a con-
ol o he speci ici y o ou an i- ail an ibodies, we used
an
emp24
mu an wi h an E178A subs i u ion in he cy o-
solic ail. This poin mu a ion does no a ec he unc ion
o Emp24p (Fig. 2 B), bu he an i- ail an ibodies no
longe ecognize he mu an p o ein (Fig. 2 B). P eincuba-
ion o memb anes de i ed om
emp24
-
E178A
cells wi h
an i- ail an ibodies had no e ec on budding e iciencies o
Gas1p, gp
␣
F, o Gap1p (Fig. 2 A). The e o e, we conclude
ha Emp24p is equi ed di ec ly o he e icien inco po-
a ion o Gas1p in o ER-de i ed esicles.
One equi emen o Gas1p exi om he ER is he a -
achmen o he GPI ancho (Doe ing and Schekman,
1996). To es whe he he GPI ancho is e icien ly a -
ached o Gas1p in he
emp24
⌬
mu an , we pe o med a
pulse-chase analysis ollowed by sepa a ion o he p o ein
ex ac s in o de e gen and aqueous phases using T i on
X-114. GPI-ancho ed Gas1p pa i ions e icien ly in o he
de e gen phase, whe eas he unancho ed p o ein pa i-
ions app oxima ely equally be ween de e gen and aque-
ous phases (Nuo e e al., 1993). A e he 4-min pulse-
labeling,
ⵑ
90% o he Gas1p was al eady ound in he
de e gen phase in bo h wild- ype and
emp24
⌬
mu an
s ains (Fig. 2 C), demons a ing ha he GPI ancho is e -
icien ly a ached o Gas1p in he
emp24
⌬
mu an . The e-
o e, a de ec in GPI ancho ing canno explain he ans-
po de ec o Gas1p in he
emp24
⌬
mu an .
Emp24p and Gas1p Can be Found in he Same P o ein
Complex in ER-de i ed Vesicles
Since Emp24p is e icien ly inco po a ed in o ER-de i ed
esicles (Schimmölle e al., 1995) and is di ec ly equi ed
o Gas1p ec ui men , we in es iga ed i Gas1p could be
ound in a p o ein complex wi h Emp24p in ER-de i ed
esicles. ER-de i ed esicles we e gene a ed in i o and
uncoa ed esicles we e exposed o he clea able mem-
b ane-pe meable c oss-linke DSP. A e lysis and dena-
u a ion, he samples we e immunop ecipi a ed using an i-
Emp24p ail an ibodies. The p ecipi a es we e dena u ed
and subjec ed o a second immunop ecipi a ion using an i-
bodies agains Gas1p. The c oss-linke was clea ed and
he samples we e analyzed by SDS-PAGE
.
Gas1p was
c oss-linked o Emp24p wi h good e iciency (Fig. 3). No
Gas1p was eco e ed i c oss-linke was omi ed. Mo e-
o e , Gas1p could no be de ec ed when he c oss-linking
was pe o med on esicles gene a ed om
emp24
-
E178A
memb anes (Fig. 3), p o ing ha Gas1p was eco e ed as
pa o a complex con aining Emp24p. This complex is
speci ic because ano he ca go molecule, Gap1p, whose
budding was no educed om
emp24
mu an memb anes,
Figu e 2. Inhibi ion o Gas1p budding by an i-Emp24p ail an i-
bodies. (A) Pe meabilized sphe oplas s we e p epa ed om
wild- ype (EMP24) and emp24-E178A (mE178A) cells as in Fig.
1. They we e p eincuba ed in he p esence o absence o an ibod-
ies agains he cy osolic domain o Emp24p (1 h, 4⬚C). Budding
eac ions we e pe o med and analyzed as in Fig. 1. (B) The a e
o Gas1p ma u a ion in wild- ype and emp24 mu an s was ana-
lyzed a e a pulse-chase immunop ecipi a ion p o ocol. Emp24p
was isualized by Wes e n blo ing using an ibodies agains he
c-myc epi ope o agains he cy osolic ail o Emp24p. Mu an
(emp24⌬) cells we e ans o med wi h emp y ec o (emp24⌬),
he wild- ype allele (EMP24), myc- agged Emp24p (mEMP24),
o he myc- agged emp24-E178A (mE178A) allele. (C) A e
pulse-chase labeling o wild- ype (EMP24) and mu an (emp24⌬)
s ains, cells we e lysed and ex ac ed wi h T i on X-114 ollowed
by de e gen phase sepa a ion. Aqueous (A) and de e gen (D)
phases we e subjec ed o immunop ecipi a ion and SDS-PAGE.
Gas1p (bo h o ms combined) was quan i ied using a Phospho -
Image . Imma u e Gas1p (105) was con e ed o he ma u e
o m (125) wi h delayed kine ics in he emp24⌬ mu an , bu was
e icien ly ex ac ed in o he de e gen phase.
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Published Ma ch 6, 2000
The Jou nal o Cell Biology, Volume 148, 2000 928
was no c oss-linked o Emp24p. These da a clea ly dem-
ons a e ha Emp24p and Gas1p can be ound in he same
p o ein complex in ER-de i ed esicles and sugges ha
his complex is speci ic.
An al e na i e explana ion o he absences o a ole o
Emp24p in Gap1p budding and o Gap1p om he c oss-
linked Emp24 complex would be i Emp24p and Gap1p
we e ound in di e en ER-de i ed esicles. To es his
we p oduced esicles using memb anes and cy osol om a
sec18 mu an . SEC18 encodes yeas N-e hylmaleimide–
sensi i e usion p o ein, and i s inac i a ion unde he con-
di ions o his expe imen blocks usion o ER-de i ed
esicles wi h he Golgi appa a us (Muñiz, M., and H. Riez-
man, manusc ip in p epa a ion). These condi ions ensu e
ha we a e analyzing p ima y ER-de i ed esicles. The
esicles we e immunoisola ed using an ibody agains he
cy osolic ail o Emp24p. 83% o he Gas1p and 96% o
he Gap1p copu i ied wi h he immunoisola ed esicles
(Fig. 4). Vi ually no ca go p o eins we e isola ed when
Emp24p ail an ibody was omi ed. The e o e, Emp24p is
ound in he same esicles as Gap1p and Gas1p. The lack
o e ec o he emp24 mu a ion on Gap1p budding and
Gap1p absence om he c oss-linked Emp24 complex
canno be due hei p esence in dis inc esicles.
Emp24p and E 25p Can be Di ec ly C oss-Linked o
Gas1p in ER-de i ed Vesicles
The c oss-linking esul s p esen ed abo e sugges ha
Emp24p may be pa o a ca go ecep o ha imp o es he
e iciency o ca go en y in o ER-de i ed esicles. How-
e e , hey do no show ha Emp24p binds he ca go mole-
cule di ec ly. To add ess his issue we used a nonclea able
c oss-linke , DSG, and pe o med simila c oss-linking
s udies on ER-de i ed esicles. Emp24p was c oss-linked
o Gas1p as seen by a smea s a ing a a ound 25,000
highe appa en molecula mass han he ER o m o
Gas1p (Fig. 5 A). Addi ional bands we e seen a highe
molecula weigh s. The band a 130 kD mus ep esen a
di ec ly c oss-linked p oduc be ween Emp24p (24 kD)
and Gas1p (105 kD). All o he p ecipi a ed ma e ial was
speci ic because i was no seen when he esicles we e
gene a ed om emp24-E178A mu an memb anes.
Nex , we immunop ecipi a ed he c oss-linked ma e ial
wi h an ibodies agains E 25p, ollowed by an ibodies
agains Gas1p. A s ong band appea ed a he posi ion ex-
pec ed o a di ec ly c oss-linked p oduc be ween he wo
molecules. The e o e, we conclude ha E 25p can also
be di ec ly and e icien ly c oss-linked o Gas1p in isola ed
ER-de i ed esicles. We could also de ec his c oss-linked
p oduc in esicles de i ed om emp24-E178A mu an
(da a no shown), con i ming ha esicle budding oc-
cu ed using hese memb anes.
Finally, we pe o med a i a ion o DSG on he isola ed
esicles. When c oss-linke was omi ed, no E 25p-Gas1p
p oduc could be de ec ed (Fig. 5 B). A low concen a-
ions o c oss-linke he di ec ly c oss-linked p oduc was
by a he main p oduc seen. A highe concen a ions o
c oss-linke mo e c oss-linked p oduc s we e seen and he
bands a highe molecula weigh became mo e appa en .
The c oss-linking s udies shown abo e demons a e ha
Gas1p can be speci ically and di ec ly c oss-linked o he
Figu e 3. C oss-linking o Emp24p and Gas1p. (A) ER-de i ed
esicles gene a ed in i o om wild- ype (EMP24) and emp24-
E178A mu an (mE178A) memb anes we e incuba ed wi h DSP.
The samples we e dena u ed and immunop ecipi a ed wi h an i-
bodies agains he ail o Emp24p. The p ecipi a es we e dena-
u ed and ep ecipi a ed wi h an ibody agains Gas1p o Gap1p
(C). 10 o 20% o he esicle p epa a ion was immunop ecipi-
a ed di ec ly wi h an ibodies agains Gas1p o Gap1p, wi hou
addi ion o DSP, o use as a s anda d (S) o eco e y. In con ol
expe imen s, c oss-linking wi h DSP did no a ec eco e y o
Gap1p by immunop ecipi a ion. Samples we e incuba ed wi h
5% 2-me cap oe hanol, analyzed by SDS-PAGE, and isualized
using a Phospho Image . (B) Vesicles gene a ed om wild- ype
memb anes we e incuba ed wi h o wi hou DSP and p ocessed
as abo e.
Figu e 4. Immunoisola ion o Emp24p con aining esicles. ER-
de i ed esicles gene a ed in i o om sec18 memb anes and cy-
osol we e immunoisola ed wi h o wi hou Emp24p an i- ail an-
ibody. The supe na an s (S) and pelle s (P) we e p ocessed o
immunop ecipi a ion.
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Muñiz e al. Ca go Rec ui men in o ER-de i ed Vesicles 929
Emp24 complex. These esul s a e consis en wi h he hy-
po hesis ha he Emp24 complex is a ca go ecep o e-
qui ed o he e icien inco po a ion o Gas1p in o ER-
de i ed esicles.
Discussion
A majo inding o his s udy is ha Emp24p is di ec ly
equi ed o e icien inco po a ion o Gas1p in o ER-
de i ed esicles in i o. Two independen assays we e
used o show his. One used mu an memb anes de-
oid o Emp24p. The o he used an ibodies o inhibi
Emp24p unc ion di ec ly in wild- ype memb anes. Bo h
assays showed ha Gas1p budding depended s ongly on
Emp24p, whe eas budding o o he p o eins did no . This
s iking speci ici y sugges s ha his complex has a highly
speci ic ole in packaging o Gas1p in o ER-de i ed esi-
cles.
This di ec ole o Emp24p in selec i e budding om he
ER can be explained in h ee ways. Fi s , Emp24p could
be equi ed o e icien ancho a achmen o Gas1p. We
ha e shown ha his is no he case. Second, Emp24p
could be equi ed o he o ma ion o ER-de i ed esi-
cles. I has been sugges ed ecen ly ha ␣2p24, a mamma-
lian membe o he p24 amily, plays a ole in he o ma-
ion o ER ca go exi si es (La oie e al., 1999). Howe e ,
in yeas , he absence o Emp24p o he inhibi ion o i s
unc ion by he ail an ibody does no lead o a gene al de-
ec in esicle o ma ion because budding o o he ca go
p o eins was no a ec ed. Also, mu an memb anes e i-
cien ly package he SNARE Sec22p (Belden and Ba -
lowe, 1996). Mo eo e , since Emp24p is ound in esicles
wi h Gas1p as well as wi h Gap1p, i is highly unlikely ha
Emp24p is equi ed only o o ma ion o esicles con ain-
ing Gas1p.
The hi d explana ion o he di ec ole o Emp24p in
selec i e budding om he ER is o conside Emp24p as
pa o a ca go ecep o ha is equi ed o he packaging
o a e y small subse o ca go p o eins. This ca go ecep-
o would ec ui speci ic p o eins in o he ER-de i ed
esicles, inc easing he e iciency o packaging. A second
majo inding o ou s udy, ha Emp24p and E 25p can
be di ec ly c oss-linked o Gas1p, suppo s his hypo hesis.
This model aises wo issues. Why does mu a ion o
EMP24 o o he genes encoding membe s o he p24 am-
ily a ec he anspo o so ew p o eins? And why is
anspo o hese p o eins no comple ely de ec i e in
emp24 mu an cells?
One possibili y o explain he small numbe o p o eins
a ec ed is ha he e could be a la ge and he e ogeneous
amily o ca go ecep o s wi h o e lapping unc ion. E i-
dence exis s o ano he ype o ca go ecep o ac ing in
ER o Golgi anspo , namely ERGIC 53, a lec in ha
also appa en ly acili a es anspo o some glycop o eins
(Appenzelle e al., 1999). I is in e es ing o no e ha mu-
a ion o ei he ecep o only a ec s a small numbe o
ca go molecules in a highly speci ic manne . The e is a
la ge numbe o p o eins ha cycle be ween ER and Golgi
compa men s. Many o hese could ha e ca go ecep o
unc ions in di ec o indi ec ways.
Ano he way o explain he small numbe o p o eins a -
ec ed in a ca go ecep o mu an would be ha only a
small subse o p o eins equi es a ecep o a his s ep. I
seems clea ha some e y abundan p o eins in special-
ized sec e o y cells a e no concen a ed in COPII-coa ed
esicles (Ma ínez-Mená guez e al., 1999). The e o e, no
all sec e o y p o eins equi e a ecep o o e icien ER
exi . So why would some p o eins equi e ecep o s o
his anspo s ep? Some p o eins may bind wi h low a -
ini y o o he p o eins ha a e e ained in he ER lumen.
These ca go molecules would equi e a highe a ini y in-
e ac ion o libe a e hem om he ER. Al e na i ely,
some p o eins may equi e high a es o sec e ion, o ex-
ample, a p o ein wi h an ac i i y ha would be de imen al
in he ER. Gas1p p obably has a unc ion in c oss-linking
␤ 1,3 glucans wi h ␤ 1,6 glucans and chi in in he cell wall
(Popolo and Vai, 1999). Such an ac i i y may ha e nega-
i e consequences in he ER.
P e iously, a bulk low mechanism (Wieland e al.,
1987) whe e p o eins lea e he ER simply by being a ail-
able o en y in o he o ming esicle, simila o he p o-
cess o luid-phase pinocy osis, has been p oposed. This
could explain why mu a ion o genes encoding membe s o
he p24 amily a ec s anspo o some ca go p o eins
s ongly, bu no comple ely. Wi hou a ecep o , he ca go
p o ein would s ill be able o en e ER-de i ed esicles
by bulk low. When he unc ion o o he ca go ecep-
o s, such as he mannose-6-phospha e ecep o (Ko n-
eld, 1992), o yeas Vps10p (Ma cusson e al., 1994) is
blocked, he lysosomal o acuola p o eins ha a e a -
ec ed s ill exi he Golgi. Howe e , hey a e sec e ed
a he han a ge ed o he lysosome o acuole. Since, as
a as we know, he e is only one des ina ion o ER-
de i ed esicles, namely he Golgi appa a us, p o ein mis-
so ing would no occu in he absence o ecep o s in he
ER. This may also help o explain why only a small subse
o p o eins ac ually needs a ecep o . As long as hey can
exi he ER hey will go o he Golgi compa men .
A ole in speci ic ca go ec ui men is no likely o be
he only ole o he Emp24 complex. Sec e ion o Ka 2p
caused by mu a ions in subuni s o his complex implies a
ole in he e og ade anspo om he Golgi compa -
men o he ER (El od-E ickson and Kaise , 1996; Ma zi-
och e al., 1999). Consis en wi h his, p24 ails om animal
Figu e 5. Di ec c oss-linking o Emp24p and E 25p wi h
Gas1p. (A) Vesicles gene a ed in i o om wild- ype (EMP24)
and emp24-E178A mu an (mE178A) memb anes we e incu-
ba ed wi h 0.25 mM o DSG and p ocessed as in Fig. 3. (B) Vesi-
cles gene a ed om wild- ype memb anes we e incuba ed wi h
he indica ed amoun s o DSG, dena u ed, immunop ecipi a ed
wi h an ibody agains E 25p, hen ep ecipi a ed wi h an ibody
agains Gas1p and p ocessed as in Fig. 3.
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Published Ma ch 6, 2000

The Jou nal o Cell Biology, Volume 148, 2000 930
cells can bind COPI p o eins (Fiedle e al., 1996; Sohn e
al., 1996; Dominguez e al., 1998) and when displayed on
liposomes hey can s imula e he o ma ion o COPI-
coa ed esicles (B emse e al., 1999). Quali y con ol in
he ER in ol es ecycling om he Golgi compa men o
he ER o mis olded p o eins (Hammond and Helenius,
1994). In e es ingly, a mu a ion in a p24 p o ein om C. el-
egans, SEL-9, allows some mis olded p o eins o escape
he quali y con ol mechanisms in he ea ly sec e o y pa h-
way (Wen and G eenwald, 1999) which could pe haps be
explained by a e og ade de ec . S udies ha can di ec ly
add ess he e og ade o o he pu a i e unc ions o p24
p o eins in esicula a ic would be desi able.
We hank C. Ba lowe, M.J. Kuehn, and R. Schekman o ma e ials and
ad ice; B. And é o an ibody; Riezman labo a o y membe s and M.I.
Geli o c i ical eading o he manusc ip ; and T. Aus and T. Ebe le o
echnical assis ance.
This wo k was suppo ed by Swiss Na ional Founda ion g an s ( o H.
Riezman and H.-P. Hau i), Fede a ion o Eu opean Biochemical Socie ies
and Human F on ie s Science P og am O ganiza ion ellowships ( o M.
Muñiz).
Submi ed: 29 No embe 1999
Re ised: 14 Janua y 2000
Accep ed: 19 Janua y 2000
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