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Biosci.BiotechnoL Biochein.. 65 (10),
2226-2232, 2001

A Novel Membrane ProteinComplex the Reticulum

and Early Golgi Compartments in theon
YeastEndoplasmic
Saccharomyces cerevtsiaet"
Jung-Hwa CHo, Yoichi NoDA,Hiroyuki ADAcHI, and Koji YoDAT
Department ofBiotechnotogy. of7bkyo, Ydyoi,Bundyo-Ku,
the Uitiversity Tbtvo II3-865Z lapan

ReceivedApril3,2001;AcceptedJune 7, 2001

A novel protein,Ym]067c in the systemat-

membrane cles that have the early Golgi tSNARE, Sed5 protein,
ic ORF name, discovered as a component
was of im- on their surface.S) The essence of the method is to
munoisolated yesicles of the early Golgi compartment construct a strain that has a myc-tagged derivative of
of the yeast S2iccharomyces cerevisiae (Choet al. , FEBS the marker membrane protein and to pull down the
Lett. 469, 151-154 (2000)). Conseryed sequences haying tagged proteinfrom the cell lysateby an anti-myc
sequence similarity to Yml067c were widely distributed monoclonal antibody with a particularly high
in the eukaryotes and one of them, Yal042w, was found specificity. In the absence of detergents, the tagged
in the SZiccharomyces genome database. In the yeast proteinpullsdown the vesicles on the membrane of
cel], Yml067c and Yal042w were found to form a heter- which it is embedded, By detergentextraction, we
ooligomeric complex by imm"noprecipitation of their can obtain the component proteins of the vesicles.
tagged deriyativesfrom the detergent-solllbilized mem- The recovery of Sed5 protein was almost complete
brane.Cellfractionation and indirect immllnofluores- and the subunits of Golgi mannosyltransferases and
cent staining indicated that the majority of these pro- the p24-family proteins that shuttle between the ER
teins were localized on the ER membrane. Therefore, and the early Golgi compartment were also found in
the Yml067c-Yal042w complex sho"ld shuttle between the isolatedvesicles. As the ER membrane proteins
the ER and the early Golgi compartment as well as the and the Iate-Golgi Kex2 protease were excluded, the
p24-family proteins. isolated vesicles should mainly comprise the antero-
grade transport vesicles and the early Golgi compart-
Key words: yeast;endoplasmic reticulum (ER);Golgi ments, A previously uncharacterized protein,
compartment; Sed5 yesicle Yml067c in the systematic ORF naming, was identi-
fiedby the N-terminal sequence, XXLKTFDAF, of a
The vesicular transport is an essential system to 40-kDa protein,S) We focusedour study on this novel
deliverthe proteins and lipidsfrom the sites of their protein and found that Yml067c was mainly localized
synthesis to the proper places to reside and work in in the ER membrane as a detergent-soluble complex
the eukaryotic The Golgi apparatus
cell,"3) is at the with itscognate protein, Yal042w. The characteriza-
central position of this traMc between the endoplas- tion of these newly isolatedmembrane proteinsis
mic reticulum (ER)and the endosome, vacuole or describedin this paper.
plasma membrane.4) Although there are debatesbe-
tween the `vesicular transportlstatic cisternae' Materialsand Methods
the
`cisternal
models and progressionlmaturation'
models as for the intra-6olgi vesicle transport,2,i'7)it Strainsand media, S. cerevisiae KA3 1-1A (MA 7Zi,
is generally agreed that the COPII-coated vesicles Ahis3 Aleu2 Atrp1 ziur`L3), CJYI19 (asKA31-IA
and the COPI-coated vesictes are responsible for the also Asect5::l6nryc-SED5, tMA31)S), CJY146 (as
anterograde transport from the ER to the early Golgi KA31-IA also Ayml067t:;tYML067b-3HA, HISsy),
cornpartment and for the retrograde transport from CJY147 (as CJYI19 also Aymt067b::tYML067tr-
the 6olgi back to the ER, respectively,3) 3HA, HTSsp, CJY149 (as KA31-IA also
In our study to characterize the early Golgi com- Ayml067b::tYML067t-6hTyq IllS3D, CJY155 (as
partment of S. cerevisiae, we established a con- KA31-IA also Ayal042w:;t}LtlL042rv-6myc, HjrSij),
venient immunoisolation method to obtain the vesi- CJY153 (asKA31-IA also Ayal042w::l}C4L042w-

To whorn should be addressed.

t
correspondence Tel: +81-3-5841-8138; Fax: +81-3-5g41-8008; E-rnail:asdig@mail.ecc.u-tokyo.ac.jp
tt The contents of this article was included in the Ph.D. thesis inJapanese of J.-H. Cho submitted to the University of Tokyo in July,
2000,
itlbbreviations: ER, endoplasmic reticulurll; HA, hemaggrutinin; ORF, open reading frame; P6K, phosphoglycerate kinase; SDS-PAGE,
sodium dodecylsulfate-polyacrylamide gel electrephoresjs

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A Novel Membrane Protein Complex on the Yeast ER and Golgi 2227

3HA, HIS3D, CJYIS7 (YML067tr-6hiyc }L4L042w- 200 mM Na2C03 (pH 11.5).After incubatingon ice
3HA), CJY163 (asKA31-iA also Ayml067b::H]rS3), for 30 min, the mixture were centrifuged at 100,OOO
CJYI64 (asKA31-IA also Ayai042w::LEU2), and ×
g for60 min at 40C to obtain the supernatant and
CJYI67 (as KA31-IA also Ayml067b::Hrs3 the pellet.Pelletswere suspended in B88 bufferin the
Ayal042w::LEtM) were used. The gene disruption same volume as the supernatant.
and epitope tagging were done by
homologous Proteins were fractionated by SDS-PAGE on 10%
recombination using appropriately constructed DNA polyacrylamide geli')and e]ectrically transferred
onto
fragments prepared by PCR. A]mt067b::HTS3 was PVDF membrane (Immobilontransfer membrane,
constructed with pRS303 (HTS3), the upstream frag- Millipore). After blocking with 5% skim milk in
ment amplified by using 5'-CGGGATCCAGAC- TTBS (25mM Tris-HCI, pH7.4, 150 mM NaCl, O.S%
TTGATGGAGAAGTAA and S'-CCTCTAGA- (wlv)Tween 20) for 1 h, the membrane was treated
CTCCTGAAGTCTCTGCCT and the downstream with an appropriate antibody (1/1000 dilutionwith
fragment amplified by using 5'-GCAGTATAC- TTBS) and then with the peroxidase-labe]led secon-
TCTAGATAACGTTTC and 5'-CTTGAAGTC- dary antibody. The rnonoclonal anti-myc antibody
AATGAATTCCTTGCTG. Ayal042w::LELL2 was 9EIO or anti-HA antibody 12CA5 (Berkeley Anti-
constructed with pRS305 (LEU2), the upstream body) was used to detectthe epitope tag. Anti-Sec221
BamHI-Miel O.3-kb fragment and the downstream Sly2 antibody was a kind giftof Dr, DieterGallwitz
Scal-JWiolO.3-kb fragmentprepared from a DNA (Max-PlanckInstitute, G6ttingen).Anti-PGK an-
amplified by using 5'-GTAACAATGGATCCAT- tibody was purchased from Molecular Probe, Inc.
CTCTGC and 5'-CCCTCGAGACTGGCTCTTC- (Oregon).
Anti-Sed5 and anti-Kex2 antisera were pre-
TTGCCCC. The chromosomal replacement was con- pared by immunizing rabbits with GST-fusion
firmed by PCR and Southern blotting. Escherichia recombinant proteins produced in E coli. Im-
coli DH5cr (F-,¢ 80laeZdMl5, supE44 AlacU169 munoreactive proteinbands were detectedby using
hsdRl7 rect4 l entL41 gynd96 thi-l retttl1) was used in SuperSignalWest Pico Chemiluminescent Substrate
plasmidpropagation.Yeast was grown in YEPD (1% (Pierce) and HyperfilmMP (Amersham Pharmacia).
Bacto yeast extract (Difco), 2% Bacto peptone
(Difco),and 2% glucose) medium or in SD (O.67% Ihimunoprecipitation.
The cleared yeast lysatewas
Bacto yeast nitrogen base without amino acids prepared as describedabove and mixed with the same
(Difco)),2% glucose, and appropriate supplements) yolume of B88 bufiercontaining 2% Triton X-100.
medium, Solidmedia were made with 2% agar. After 20 min on ice,the mixture were centrifuged at
100,OOO g for 60 min at 40C to obtain the super-
×

Generaimethods. Standardgenetic and biochemi- natant. The detergent-solubiLized lysatewas mixed

cal techniques were used.9,iO) with Protein A-Sepharose beads and Totated for 1 h
at 4eC to remove nonspecifically binding materials.
SubceUular.fractionation and PVesternblotting. A The precleared lysatewas mixed with anti-myc 9EIO
50-ml culture of the yeast strain was grown in SD and incubatedfor 2h at 40C. Protein A-Sepharose
medium to an OD6oonm of 1.0.The cells were collected beads (10ul bead volumeltube) was then added and
by centrifugation, washed in distilled water, and rotated overnight at 40C. The beads were washed 6
resuspended in B88 bufier(10mM HEPES, pH 6.8, times in the B88 buffercontaining 1% Triton X-100.
150 mM potassiumacetate, 5 mM magnesium acetate, Bound materiais were eluted by boilingin the Laem-
200mM sorbitol) with a protease inhibitorcocktail mli sample buiTerand fractionatedby SDS-PAGE
(PIC;1 mM phenylmethylsulfonyl fluoride(PMSF), under non-reducing conditions. The gelswere im-
5mM 1,10-phenanthroline,2ptM pepstatin A, munoblotted or stained with silver or Coomassie bril-
2 ptg!ml aprotinin, O.5 ptglml leupeptin). Glassbeads liantblue.
were added to the mixture, which were vortexed four
times for 1 min with 1-min incubation on ice between immunoj7uotwscencemicroscopy. Tenmloflog
each burst.The crude lysatewas immediately cen- phase cells at an OD6oonm of 1.0 were fixed by adding
trifuged at 500 × g for5 minutes at 4eC to remove un- 1 ml of 37% forrnaldehydeand 330 pt1of 2% NaN3
broken cells. The cleared lysate (lysate) was cen- directlyto the media. The cells were centrifuged and
trifuged at 10,OOOxg for1O min at 4eC and the pellet fixedwith 3.7% formaldehyde in bufferA (100 mM

was collected (PIO). The supernatant was then cen- potassium phosphate buffer,pH 7.3, 10mM NaN3)
trifuged at 100,OOO × g for 60 min at 40C to generate for90 min. Cellswere washed two times with buffer
high-speedsupernatant (SIOO) and pellet(P1oo)frac- A and one time with bufferB (1.2 M sorbitol, 1OO mM
tions. Pelletswere suspended in B88 bufferin the potassium phosphate buffer,pH 7.3, 10mM NaN3)
same volume as the supernatant. before being resuspended in 5 volumes of buffbrB
For characterization of proteins,the
membrane containing 25 mM 2-mercaptoethanoland O.1mglml
cleared lysatewas mixed with a same of B88 volume Zymolyase 100-T (Seikagaku Kogyo, Tokyo). Then
bufferwith none, 2% Triton X-100, 2M NaCl, or they were incubatedfor 30 min at 37eC to digestthe

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2228 J.-H.CHo et al.

cell wall. Cells were washed with buffer B and localization,

teristics, and functional
role in compari-
resuspended in bufierB with O,1% TritonX-10e and son with the cognate proteinYal042w.
then were incubatedfor 10min on ice.After being
washed with buffer B, pipetted onto poly-
cells were Membrane of Yml067b
localization and Ylxl042w
lysine-coated microscope incubated with
slides and We constructed yeast strains that could produce
the anti-myc monoclonal antibody 9EIO (Berkeley the epitope-tagged derivativesof Yml067c or
Antibody) or rabbit polyclonal anti-Kar2 (yeast BiP) Yal042w by chromosomal replacement as described

antibody (a kind gift of Dr. Masao Tokunaga, in Materials and Methods. In order to find whether
Kagoshima University)that was dilutedto 1110 in these proteins are membrane proteins as predicted by
buffer C (1mgfml skim milk, O.1% Tween 20 in their hydropathy profilesor not, cell lysateswere pre-
PBS) for 60min at room ternperature, Slides were pared using glassbeads and treated with agents to
washed and incubatedin the appropriate secondary characterize their solubility. Both Yml067c-6myc and
antibody(FITC-or Texas Red-conjugatedgoat IgG, Yal042w-3HA were completely recovered in the
dilutedto 1!400, Cappel) for 30 min, washed with precipitate even after a high salt or alkaline treat-
bufferC, and mounted, Images were obtained using ment, whereas a considerable portionwas solubilized
an AX-80 microscope (Olympus). in 1% Triton X-100 (Fig, 4). This result indicates
these proteins are the integralmembrane proteins
Resultsand Discussion which should have membrane-spanning regions.

Characterization of the amino acid sequence of Subcetlular.fractionation of Ymt067lr and Ydl042w

Yml067c The localizations of these proteins were examined
As describedpreviously, we have isolated a defined by subcellular fractionationand Western blotting
fractionof membrane vesicles that had the early using appropriate antibodies (Fig. 5).As the control
Golgi tSNARE, Sed5 protein,on their surface.8) A markers, phosphoglyceratekinase(PGK)was taken
previously uncharacterized protein, Yml067c, was for the cytosolic protein, Sed5 protein for the early
found in the Triton X-100 extract of the vesicles. Golgi compartrnent, Sec22 proteinforthe transport
Yml067c is composed of 352 amino acids and has a vesicles, 3HA-Sec71 protein for the ER membrane,
calculated molecular mass of 40.7 kDa. In search for and Kex2 endopeptidase forthe lateGolgi compart-
similar sequences in thedatabaseby the FASTA pro- ment. Both Yml067c-6myc and Yal042w-3HA were
gram,i2)we found that ORFs with a sequence identity recovered in the P10
PIOO fractions.Apparently
and

of about 25-30% to Yml067c and size of around 400 these proteins abundant
were in PIO than in
rnore

amino acids were widely distributed in the eukaryotes PIOO, as in the case of the ER-marker 3HA-Sec71
(Fig. 1):Homo sapiens, LOC51290 and LOC51614, protein.This result suggested that a majority of

the insectDrosophila melanogaster EG:65Fl.l and Yml067c and Yal042w might be in the ER membrane
CG7011, the plant Arabidopsis thatiana, F5J5.4, although a portion of them are in the compartment
the nematode Caenorhabditis elegans, K09E9.2, having Sed5 proteinon the surface,
the fission yeast Schizosaccharomyces pombe
SPAC24Bll,08C and SPBC2G5,04C. In S. indirect immunoj7uorescent staining pattern of
cerevisiae itself,Yal042w (415amino acids) had 23% Yml0671r
identityin 366 amino acids. When a phylogenic tree To make the compartment visible where the
was constructed using CLUSTAL Wi3) and Tree- majority of Yml067c are loca]ized, the cells of
View,i4} four proteins including Yml067c were CJY149 (YML067b-6myc)were marked by indirect
grouped in a subpopulation of a common ancestor immunofluorescent staining with rnouse anti-myc
(Fig. 2). monoclonal antibody and rabbit anti-Kar2 antibody.
Figure3 shows the hydropathy profilesof Yml067c As shown in Fig. 6, Yml067c-6myc was Iocalized in a
and Yal042w according to the method of Kyte and perinuclear ring and threads along the contour of the
Doolittle.iS)The homologous sequences described cell. This staining patternof Yml067c-6myc coincid-
above gave similar hydropathy profiles.Two highly ed with that of Kar2p (yeast BiP) that localizes in the
hydrophobic regions, whichrepresent the
should lumen of the ER. Therefore, the majority of
transmembrane domains,located near the N-
were Yml067c are in the ER membrane as suggested by the
and C-terminals of the polypeptides. These polypep- subcellular fractionation experiment. The p24-family
tides would be embedded in the membrane vesicles at proteins, encoded by EMM4, ERV25, and ERPI-
these two terminal regions and the central large ERP6 in S. eerevisiae, are similarly distributed be-
region should be in the lumenal space. tween the ER and Golgi compartment. They form a
AIthough Yml067c proteinwas recovered in the heteromericcomplex and are suggested to functionas
Sed5 vesicles in a largeenough amount to be detected a partof the cargo selection machinery in the COPII
by Coomassie brilliant blue staining, ithad not been vesicles and shuttle between the ER and early Golgi
studied in detail. We planned to examine itscharac- compartment.`,i6'iS} Complete lossof the eight mem-

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A Noyel Membrane Protein Complex on the Yeast ER and Golgi

te ze ]e 4e se 6e 7e se ee lee2229
Ymle6T[SPBC2G5.enC --ANRK El88sgBSS6B4ss899fiS5
P
H,s.Lecsnge N
H.S.LeCSIE14
C,E.Ke9E9.Z
Tatas4ZltD.M.CG7e!1A.T.FSJS,4D.M.EGT55Fl
--KS--

SPAC24Bll.e8C
geD

agt
ue 13e !6e 17e 18e 1ee zez
-.-PNKAma
.....t----157143151!SlISI1581418
ymte67cSPBCZGS,exH.S.Loc5129e
Iine pt--iei?
I･Iww.,iue
'"' EATPA

ecS
EK LD------TR SFFDESD--N
-t.---.---
---.FRKKNN
..--.-----
-e

g,,iiilll,i
,ppa.m-5VtSIpt.=I:

as-lpT,ee,I
H.S.LOC51614 DTKYN-DItwEl

me.,, eeiftsilli.
C.E.KooE9.2
--------de
.--abNEpt4
yoletLzwD,M,CG7ellA.T.FS]S.4D,M.EG:6SFI
-------HN

SPAC24Bll.eSC
Teeta.,,ISi,igim,
ye,ew't
ge-mai ,,ww.ARES Ii:'III:eiagie
21e 22e z3e z4e z6e ne 2se 2ge 3eezel19e21S2442432S2232z7g24S2

gX
""-----'
iii[:iii:!
Ymle67cSPB[2G5.e4[ EL ITAKS-
--.T ----T-----Nvv
II[-ige"'25e L TAP--

zz
H.S.Loc5129e T
H.S.LO[516Z4 --- V
P
[.E.KooE9.2
YA "J
YaZe42wD.M,CGTellA,T,FS)S.4O.M.EG:6SFt
IRQF ---
v ---
SPACZ4Bll.osC maN RIGDV"Fdiwa･GKNee L ptgww 4.f

x]QS"SKLLGN
Ble 32e ]]e se 3Ge 37e 38e 3ge 4ee
----KLN
Ymle67cspBc2Gs.e4c --------n
.--.---t-L ec ---asFAEv
asFE
NDIgetttw
G
D::I
-----
27426128711611334g3a63

l,!iv･.I!-
L"-----TYK IS INH
H.S.LO[S129M
-i?itw AN-eL ---
H.S,LOCS1614
---FRQS-----
C.E,Ke9ES.2
Yate42wD.M.[GlellA.T,F5)S,4D,M,EG:65Fl
IE"----DKRHGGIsell ,tw
;::kgG-I SH
R RDKDHpmTLH
--RER-----
-vEwr
SPAC24Bli.esC
SGR=---.--
Las----
S-----seHy
FN.-.fi-'--------FAK

s
V IHEE
KVS
N-
-T
YR-Y
V IH-----QTF
MFdeLSKST
L
T AF
Y
, E st,ct=I
REEKVpmHVN

41eDL 4zeFIV 43e 44e 4se 4Se 47e 48e 49e

---K
[･A,Y,illl!!$,-[-[-:--k
Ym!e6T[SPBC2G5.e4C DVR-L SFLVY:tw 3]7BZZ3"3S9Ss6]9534941
PN ST
-----vptP ptT---Tt-
FEsee
H.S.Le[S129e
H.S.LOCS1614 LYRNWS
MFV
-EIIEYt
C.E.KeeE9.Z
YaLe4ZwD.M.[G7elln.T.FSJ5.4D.M.EG:S5Fl
v
ysFI c

spAcz4Bn.esc F QF
NDR-DHLV
ERQVRMdTR]
T I
NVL L
ISGagN,!e,,,,,, E"iRS::PN4PSLstLS
[eelie(;V:i AK

sle s2e s3e

YmTe6]cSPBC2G5,e4C --"-----fi'Nr'--MLG
------YQPD ptrLDR 3S233S3TS3SS38e41S37S"e441sge
LLGajE'--' rrT'r"'TE C31jStdentity in ]2e)351)3fi8)sgs)SG6)3fi8)tS9)3")37])
H.S.LOC5129e '-- NHLP LLENNTHJ-- -"-"fi'r C27NCzesCZ6NCZ3N(23%(31XCZ6SGC24%
inin:･:l･::.:
H.S.LOCS1614 DL ----
C.E.KeeE9.Z T HKSRIAG ---..
Yale4Z-D,".CG7ellA.LFS]5AD.M.Eg/fiSFI
TV A G". ----
VNNW ----
IF ---
NLMA SV TT LQAGL
SPltCZ4Bll.e8C sF GyEFv dyL----- - ---
Fig. 1. The Amino Acid Sequence Alignment of the Proteins Related to S. cerevisiae Yrn1067c Protein.
The analyzed proteins were, frorn the top, S. cerevisiae Ymle67c, Strhtzosaccharonryces pombe SPBC2G5.e4C, Hbmo sapiens
H.S.LOC51290, H, sapiens H.S.LOC51614, Cbenorhabditis elegans C.E.K09E9.2,S. cerevisiae Yal042w, Drosophiia melanogaster
D.M.CG7011, Arabidopsis thatiana A,T.F5J5.4, D, melanogaster D.M.EG:65Fl,1,ancl S,potnbe SPAC24B11.08C, The highly con-
served amino acids arnong these 1O sequences are indicated by the reverse characters. The total numbers of their arnino acids and the %
identityto the sequence of Yrn1067c are alsoindicated.

bersof yeast p24-family proteins was not lethaland a ldentptcation ofProteinsin the Hbteromeric Com-
transport delay as in the single disruptantwas ob- plex with YinI067b
served.i") As Yml067c and Yal042w were detectedin In order to findany proteinsthat physically inter-
the ER and early Golgi compartment, they may be act with Yml067c, we did immunoprecipitationfrom
also shuttle in the early part of vesicular traMcking as a detergent-solubilizedlysateof CJY149 (YML067ic-
p24-familyproteins. 6hiyc)containing 1% Triten X-1OO with anti-myc an-
tibody and analyzed the precipitate by SDS-PAGE.

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223e J.-H. CHo et aJ.

.08C bufferTX-IOO Na2C03 NeCl

psps psps
21
Yml067c6myc

LOC5ri290
Yal042w-3HA
ee}pmeiesops$
el wa va .
de}pme}ee-ee.
'
ge'ilgggi.as
Fig. 4. SolubilizationTest of Yml067c and Yal042w Proteins.
D.M. EG165Fl The cleared lysates ef CJY149 (}QS4L067b-6nryc) and CJY153
(}14L042w-3rm) were mixed with a same volume of B88 buffer

with none (buffer), 2% Triton X-1OO (TX-100), 200 mM Na2C03

BC2G5.e4C
(Na2COi}, or 2 M NaC] (NaCl)and ineubated on ice for 30 min.

Yml067c The precipitate(P) and selubre fraction (S)were separatecl by

centrifugation at 1OO,OOO x g for 60 min and analyzed by SDS-
PAGE and Western blotting.
1O%sequencedivergence

Fig. 2. A Phylogenic Tree ofthe Proteins Related to S, cerevisiae lysatePtO PIOO SIOO
Yrn1067c Protein.
Using the defaultsetting of CLUSTAL W 1.7,i])thc se-
vapt /ewwog
' fu.nt''ttv.
Yml067c-6mycw fiaj1teeetw
quences were aligned, and a bootstrap
treefile was created. The eft
phylogram tree was drawn with TreeVeiw.i4}Bar represents 1O%
gmpe
S･etesSiSl,
sequence diyergence. Yal042w;3HA-i-,ii,ksc
- ns #h ee .'
I if
fitsside/i ;ec
ee
.. ms mde

Yml067c PGK･" .maii'i#:eV"Siglnv, ,.g

･eqseegeeidi-vaethlmpe.e'$l,el:
3
Sed5ttttttt･- {
tsv.!.9 Sec22 ･ew;" tmpriSdeleci･$g;g
ttcaaee.,,vatt.eeitf:,g,si
a,
£ o
3HA-Sec71
a9Vh=
tw , eetpmgasteg'ggeeg
Kex2wu'-g'isi
kt
-31 Fig.5. Subcellular Fractionatjon of Ymt067c Protein.
The cleared lysates(lysate) of CJYI49 (wwL06k-6nu,c) and
1oo 200-]3eo
CJYI53 (-4L042w-31Ltl)were centrifuged at 10,OOOXg for
10min at 40C and the pellet was collected (PIO). The super-
natant was then centrifuged at leO,OOOxg for60 min at 4OC to
generate high-speed supernatant (SIOO)and pellet(P100)frac-
Yal042w tions.Fractions were analyzed by SDS-PAGE and Western blot-
ting using appropriate The control marker proteins
antibodies.
were, phosphoglycerate kinase CPGK)fer the cytosolic protein,
3
Sed5 protein for the
early Golgi compartment, Sec22protein for
the transport vesicles, 3HA-Sec71 protein for the ER rnem-
xv,g.y=tta9g= brane, and Kex2 endopeptidase fer the late Goigicompartrnent.

o
As shown in Fig.7, lane 3, at leastfour protein
bands with apparent molecular mass of 42 kDa (band

a), 47 kDa (bandb), 55 kDa (bandc) and 88 kDa

"1 (band d) were detccted reproducibly by Coomassie
brilliantblue staining, The 55-kDa band reacted with
1OO 200 300 400
anti-myc antibody and should represent Yml067c-
Fig. 3. The Hydropathy Profitesof Yml067c and Yal042w Pro- (Fig.7, bandeof
6myc itself lane 5).We analyzed
teins. the N-terminal amino acid sequences of the other
The hydropathywas calculated according to Kyte and Doolit- bands. The 47-kDa protein(band b) had the N-termi-
tle'S)using the scan wjndow of 20.
nal amino acid sequence of MKRSTLLSLD and thus

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Membrane Pretein Complex on the YeastER and Golgi 2231

Yml067c-6mycANovel controi These results that Yml067c and Yal042w
suggested

were components heteromeric membrane

of a protein
complex. To confirm that thetwo membrane proteins
have a physical interaction,we next did im-
anti-mvc munoprecipitation of Yml067c-6myc from a deter-
gent-solubilized lysateof CJY157 (YML067b-6hiyc
}14L042w-3HA), Western blotting of the im-
munoprecipitate indicatedthe presenceof Yal042w-
3HA (Fig 7, band f of lane7).Immunoprecipitation
of Yal042-6myc gave similar resuits that confirmed

entl-KaMp
physical interactionbetween these membrane pro-
teins(datanot shown). There are several examples of
heteromeric proteins the subunits of which have a se-
quence similarity inthe ER-Golgi membrane proteins
of S. cerevisiae.The sequences of eight rnembers of
Fig. 6. Indirect Double Irnmunofluorescent Staining Pattern of the p24 familyproteins are conserved.4) The subunits
Yml067c-6myc Protein and Kar2 <yeastBiP) in the Yeast Cells, of the Golgi mannosyltrans ferases, Mnn9, Van1, and
The cells of CJYI49 (}Z4L06k-6myc) or KA31-IA (control) Anpl, have a conserved sequence, and while Mnn9
were fixed and processed for indirect immunofluorescent stain-

ing with anti-myc and anti-Kar2 antibodies. Top panels (a,b) and Vanl form a dimeric enzyme complex, Mnn9
show staining patterns with mouse anti-myc rnonoelonal an- and Anpl are components of another pentameric
tibody and FITC-labeledgoat secondary antibody. Bottom complex.4･oo･2i) Two-hybrid and substitution experi-
panels (c, d) show those with rabbit anti-Kar2 antibody and Tex-
ments of the transmembrane anchor region among
as Red-labeled goat secondary antibody. Panela and c; CJY149
the subunits suggested that the lumenal domains are
(nvL067b-6inyc), b and d; KA31-IA (control).
important to the assembly of these functionalcom-
plexes.22)Molecular dissection experiments are neces-
1 2 3 45 67 sary to evaluate the role of the hydrophilicconserved
kDa175.S3 kDeIT5-
regions in the function and structure of these pro-
teins.

-fi2 d
e3- Gene disruptionof YML067tr and X4L042w
.4T.5- 62-47,5- Null mutant alleles of YML067b and X4L042w
'cbe e
were constructed in the diploid KA31 by chro-
mosornal replacement using the DNA fragments of
S2,5- Ayml067b::lflrS3 and Ayal042w::LECM as described
in Materials and Methods, respectively. Spores were
dissectedat 25eC on YEPD and the tetrad progenies
were examined for the presenceof the null allele by
their auxotrophy. A double null mutant strain was
Fig. 7. Detectionof Proteinsin the HeterooligomeTic Complex
with Yrn1067c, constructed by crossing the appropriate yeast
Adetergent-solubilizedlysateofKA31-1A(lane2,control)or pairs. The chromosomal struetures of CJY163
CJY149 (lane 3. }Cit4L067b-6inyc)containing 1% TritonX-100 (tlymi067t::H17S3),
CJY164 (Ayal042w::LEU2),
and
was processed to immunoprecipitation with anti-rnyc antibody. CJY167 (z]yml067b::HirS3
Ayat042w::LEU2) were
The precipitatewas analyzed by SDS-PAGE without adding a
reducing agent to prevent the appearance of the dissociatedim-
confirmed by bothPCR amplification and Seuthern
munoglobulin subunit polypeptides, Protein bands weTe stained
blottinganalysis. These null mutants showed no dis-
by Ceomassiebrilliantblue. The reproducibly ebserved protein tinct phenotype in comparison with KA31-IA, in-
bands are indicated by arrowheads a-d. A similar gel was im- cluding growth at 15OC, 25eC, 35eC on YEPD or SD
munoblotted using anti-myc antibedy (lanes 4, KA31-IA; lane
plate.The effects of adding 1 M sorbitol, 1 M NaCl,
5, CJY149 (}wrL06k-6inyc)) or anti-HA antibocly (lane 6,
KA31-IA; lane7, CJY157 (wwL067b-6no,c}14L042w-3IL4)),
O.5M KCI, O.oo3% SDS, 50ptg!ml Congo red, or
Molecular mass inkDa isindicated at the leftef the panels(lane 50 uglml Calcofluorwhite didnot show any distinct
1, molecular mass standard proteins). difference between the disruptantsand the parent.
The kinetics of carboxypeptidase Y at 25 OC was exa-
mined by pulse-chase experiments, but no significant
was found to be Yal042cprotein.The 42-kDa protein difference was detected(data not shown).
(banda) had the same arnino acid sequence and Recently, Otte et al.23)reported the identification
of
therefore should degradationpToduct of
represent a proteinsin the purifiedCOPII vesicles. There were
Yal042c that losta C-terminalportion. The identifi- Yml067c and Yal042w among the identifiedproteins
cation of the 88-kDa protein (bandd of lane 3) was and the names Erv41 and Erv46 were proposed for
unsuccessful: them, respectively, in theirpaper. Their biochemical

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2232 J.-H. CHo et at.

characterization of these proteins was basically coin- Harbor, New York. (1989).
cident with our results. A main inconsistency is the 11) Laemmli, U, K,, Cleavage of structural proteins
result of the phenotype of the null mutants. during the assembly of the head of bacteriophageT4.

Although we could not findany distinct IVbture,227, 680-685 (1970).

phenotype,
12) Pearson, W. R., and Lipman, D. J,,Improved tools
their disruptantsshowed reduced growth rates at
for biologicalsequence comparison. Proc. Ndtl.
160C, especially in the double knockout strain. The Acad. Sci,U.S.A., 85, 2444-2448 (1988),
reason of this inconsistency is not clear at present, 13) Thompson, J.D., Higgins,D. G., and Gibson, T, J,,
however, a difference of the genetic background of CLUSTAL W: improving the sensitivity of progres-
the strains might be concerned, In combination with sive multiple sequenee alignment through sequence
certain genetic alleles, the loss of these proteins that weighting, position-specific
gap penaltiesand weight
are localized in the ER and early Golgi might be una- matrix choice. Nucleic Acids Res,, 22, 4673-4680
ble to be compensated for and this might reduce the (1994).Page,
growth rate. 14) R. D.. TreeView: an application to display
phylogenetictrees on personal computers. Comput.
Alrpl.Biosci.,12, 357-358 (1996).
Acknowledgments 15) Kyte, J., and Doolittle,R. F., A simple method for
displayingthe hydropathic character of a protein.J.
We thank Dieter Gallwitzand Masao Tokunaga Mbl, Biol,,268, 10558-10563 (1993).
forantisera, Ken Sato and Akihiko Nakano forplas- 16) Schimm611er, F., Singer-KrUger,B,, SchrOder, S.,
mids, and ShinjiNagata for valuable advice and dis- KrUger, U,, Barlowe, C., and Riezman, H., The
cussion, This work was supported by a grant-in-aid absence of Emp24p, a component of ER-derived
for ScientificResearch from the Ministry of Educa- COPII-coat vesicles, causes a defectin transport of
tion, Science,Sports,and Culture of Japan, grants selected proteins to the Golgi. EMBO J,, 14,
for ``Biodesign
Research" and
``Bioarchitect
1329-1339 (1995),
Research''from the Instituteof Physicaland Chemi- 17) Belden,W. J.,and Barlowe, C,, Erv25p, a eornpo-

cal Research (RIKEN),and a grant from the Kato nent of COPII-coated vesicles, forms a complex with

Memorial Bioscience Foundation (to Y, N.) Emp24p that is required for erncient endoplasmic
reticulum to Golgi transport. J. Biol, Chem., 271,
26939-26946 (1996).
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