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DOI 10.1002/pmic.200800803

Proteomics 2009, 9, 3036–3046

RESEARCH ARTICLE

A novel fractionation method of the rough ER integral
membrane proteins; Resident proteins versus exported
proteins?
Kumiko Sakai, Ryoji Hamanaka, Hideo Yuki and Makoto Watanabe
Department of Cell Biology, Faculty of Medicine, Oita University, Yufu-Shi, Oita, Japan

We treated the high salt-washed canine pancreatic rough ER (KRM) with 0.18% Triton X-100,
separated the extract from the residual membrane (0.18%Tx KRM), and processed the extract
with SM-2 beads to recover membrane proteins in proteoliposomes. To focus on integral
membrane proteins, KRM, 0.18%Tx KRM and proteoliposomes were subjected to sodium
carbonate treatment, and analyzed by 2-D gel electrophoresis. Consequently we found that a
distinct group of integral membrane protein of KRM preferentially extracted from themembrane and recovered in proteoliposomes did exist, while majority of KRM integral
membrane proteins were fractionated in 0.18%Tx KRM, which retained the basic structureand functions of KRM. Protein identification showed that the former group was enrichedwith proteins exported from the ER and the latter group comprised mostly of ER resident
proteins. This result will potentially affect the prevailing view of the ER membrane structure
as well as protein sorting from the ER.

Received: October 14, 2008
Revised: January 26, 2009
Accepted: February 18, 2009

Keywords:
2-D PAGE / ER / Membrane protein / Protein solubilization / Shotgun proteomics

1

Introduction

The ER is a site of lipid and protein biosynthesis. Its
membrane is the site of production of all the transmembrane proteins and lipids for most of the cell’s organelles.
The ER membrane synthesizes nearly all of the majorclasses of lipids, including both phospholipids and cholesterol. Newly synthesized proteins and lipids enter thesecretory pathway connected by vesicular transport. The
various organelles along the pathway display striking
differences in both their protein components and theCorrespondence: Dr. Makoto Watanabe, Department of Cell
Biology, Faculty of Medicine, Oita University, 1-1 Idaigaoka
Hasama-Machi, Yufu-Shi, Oita 879-5593, Japan
E-mail: makwatanabe@med.oita-u.ac.jp
Fax: 181-097-586-5665
Abbreviations: 0.18%Tx KRM, KRM extracted with 0.18% Tx-100
treatment; KRM, high salt-washed canine pancreatic roughendoplasmic reticulum; Sec61-a, Sec61-a subunit; TCR-a, T-cell
receptor a subunit; Tx-100, Triton X-100& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

composition of their membrane lipids. For example, theplasma membrane has a strongly asymmetric transbilayer
lipid distribution and contains high levels of sterols
and sphingolipids, in contrast to the situation of the ER,
which has symmetric one and contains low levels of
sphingolipids [1].
In addition to the heterogeneity of protein and lipid
contents from organelle to organelle, eukaryotic cell
membranes contain variety of phospholipids, sphingolipids
and sterols. This lipid heterogeneity leads to the possible
nonrandom mixing and microdomains. Studies in artificial
bilayers reveal that lipids have a strong self-organizing
capacity; lipid immiscibility can drive phase separation andgive rise to domains with unique lipid compositions andproperties. Current evidence supports the existence of suchphase-separated lipid domains in the cellular membranes[2]. Biophysical studies on model membranes firmly established that mixtures of sphingolipids, unsaturated glycolipids and cholesterol can segregate spontaneously into two
fluid phases, where the sphingolipids and part of thecholesterol coalesce into ‘‘liquid-ordered’’ domain and break
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Proteomics 2009, 9, 3036–3046

away from the unsaturated glycerolipids in a ‘‘liquid-disordered’’ phase [3]. In contrast to cytoplasmic membranes,
which are enriched in sphingolipids and sterols, the ER isvery low in cholesterol and sphingolipids, and is probably
present in liquid-disordered phase [3].
Protein translocation-integration into the ER membrane
has been also extensively studied, leading to the discovery oftranslocation machinery components such as signal-recognition particle receptor, translocon complex, signal peptidase complex and oligosaccharyl transferase complex.
Integral membrane proteins are immediately integrated into
the lipid bilayer as soon as their stop transfer signals reachtranslocons [4, 5]. Following the integration, protein transport from the ER along the secretory pathway begins withpackaging into COPII-coated transport vesicles, which canbe a selective process. The generation of COPII vesicles
requires a distinct set of coat proteins and secretory factors.
The sequential events of COPII complex assembly start with
Sar1p activation caused by membrane-bound Sec12p, whichcatalyzes GDP-GTP exchange on Sar1p [6]. This activation
leads to a functional prebudding formation by recruiting theSec23/24p complex. COPII-coated buds are formed at the
ER exit sites, microdomains continuous with the ER, but
ribosomeless, smooth patches of the rough ER. Although
Sec12p is presumably one of the most upstream components of the COPII budding process, some sort of a budding
domain scaffold is thought to exist from the disruption
experiment of the Sec12p function [7]. Little is known aboutsuch a scaffold [8].
The exit signals that direct proteins out of the ER fortransport to the Golgi and beyond are mostly not understood. Only a few membrane proteins’ signals working for
COPII recruitment have been known. One is a diacidic
motif, Asp-X-Glu (DXE, where X is any amino acid) [9–11]
and the other motif is the one binding on Sec23p/24p[12–15]. However, only a small number of integral
membrane proteins exported out of the ER have such exit
signals, but the rest great majority can get packaged in
vesicles without any known exit signals at a much faster rate
than ER resident proteins, which slowly leak out of the ER.
It is quite interesting to know whether and how these
proteins without known signals are recruited into the scaffold prior to or after Sec12p activation.
The process from protein integration at the translocon to
COPII formation started with Sec12p activation remains
unexploited at all.
We made a hypothesis that there are at least two distinct
microdomains in the ER, to which distinct subgroupsof ER integral membrane proteins with different fates areintegrated into respectively. This hypothesis could potentially provide a clue to elucidate the presence of the correlation between these two topics, ER membrane structure
and protein sorting prior to Sec12 activation. As a first step,
we treated ER membrane with various concentrations of
Triton X-100 (Tx-100), as treatment of cytoplasmic
membrane with 1% Tx-100 successfully showed the& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

presence of ‘‘raft’’ in the cytoplasmic membrane [3]. We
found that by the treatment of high salt-washed canine
pancreatic rough ER (KRM) vesicles with Tx-100 of up to
0.24% concentration more than half of KRM vesicles
retained their structure and their functions such as proteintranslocation and core-glycosylation [16]. In this study wetreated KRM vesicles with 0.18% Tx-100, recovered
membrane components, if any, in the extract to proteoliposomes by SM-2 beads treatment, and analyzed integral
membrane protein fractions obtained by sodium carbonate
treatment together with the analysis by 2-D gel electrophoresis. This novel combination followed by protein identification by LC-MS/MS led to the first demonstration that a
distinct group of ER integral membrane proteins is extracted
by 0.18% Tx-100 treatment without solubilizing the KRM
membrane.

2

Materials and methods

2.1 Tx-100 extraction of KRM
Rough microsomes were prepared from the canine
pancreas, as described by Walter and Blobel [17]. The Oita
University animal experiment committee approved theexperiment, which was carried out according to the
University and national guidelines and regulations. Microsomes were nuclease-treated after EDTA washing andsubsequent high salt washing by the procedure of Walterand Blobel [17], and are referred to as KRM. The extraction
mixture contained 3–5 mg/mL KRM protein, 100 mM KCl,and 0.18% of Tx-100. The mixture was incubated on ice for
20 min, applied on a cushion (0.4 M sucrose, 500 mM KOAc,
and 5 mM Mg(OAc)2) and centrifuged for 20 min at
424 000gav in the Beckman TLA100.3 rotor. After centrifugation, the supernatant and the pellet were separated. The
membrane vesicles pelleted were resuspended in themembrane buffer (0.25 M sucrose, 50 mM triethanolamine
acetate pH 7.5, and 1 mM DTT) and are referred to as
0.18%Tx KRM (KRM extracted with 0.18% Tx-100 treatment).

2.2 Proteoliposome reconstitution
The supernatant prepared as described above was incubatedon ice with SM-2 beads (Bio-Rad) by batch method for
30 min. The amount of beads used depended on the amountof Tx-100 and was determined according to the manufacturer’s instruction. After incubation, the flow-throughwas diluted with an equal volume of ice-cold water andcentrifuged for 10 min at 424 000  gav in the Beckman
TLA100.3 to collect proteoliposomes. The supernatantcontaining unreconstituted materials was incubated withtrichloroacetic acid with a final concentration of 10% to yield
the unreconstituted materials.
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K. Sakai et al.

2.3 Analysis of 2-D Electrophoresis
Membrane vesicles were resuspended in the 2-D sample
buffer (8 M urea, 2.5 M thiourea, 4% CHAPS, 100 mM DTT,
0.2% w/v Bio-Lyte 3/10, and 0.001% bromophenol blue).
The resuspended sample was applied on an IPG ready strip
pH 3–10 (Bio-Rad) and processed using Bio-Rad ProteanIEF Cell (Bio-Rad) according to the manufacturer’s
instruction. After IEF, the IPG strip was subjected onto
10–20% SDS-PAGE (Bio-Rad). The gel was stained with
CBB R-250. Three independent experiments were carriedout, and each spot of the proteoliposome fraction wasquantitatively compared with the corresponding spotof 0.18% Tx KRM by PDQuest version 8.0 (Bio-Rad). Spotsthat were two or more times larger than the corresponding
spots from 0.18% Tx KRM were selected and were considered to be proteins specifically fractionated in the proteoliposome fraction. All such spots were excised from the gel,pooled, and sent to Hitachi Science Systems (Hitachinaka
Ibaraki, Japan) for LC-MS/MS analysis. After sodium
carbonate treatment 0.18% Tx KRM was also sent to Hitachi
Science Systems. The subsequent processing of samples
was provided by them as described below.

2.4 Methods of sample preparation for LC-MS/MS
Isolated protein spots from proteoliposomes stained with
CBB were excised from the gel, pooled, washed with 50 mM
NH4HCO3 in 50% v/v ACN twice, and dehydrated in 100%
ACN. Reduction of protein sample was achieved with
10 mM DTT in 100 mM NH4HCO3 for 1 h at 561C andalkylation was performed with 55 mM iodoacetamide in
100 mM NH4HCO3 for 45 min at room temperature in thedark. Subsequently, the gel pieces were washed twice for5 min alternatively with 100 mM ammonium carbonate and
ACN, and then completely dried up under reduced pressure.
Appropriate volumes of trypsin solution (25 m/ml in 50 mM
NH4HCO3) were added to the dried gel pieces. After incubation overnight at 371C, the supernatant containing
digested peptides was transferred to a new tube. The gelpieces were washed with 20 mM NH4HCO3 and 50% v/v
ACN in 5% v/v formic acid. The supernatants were collectedto the new tube and dried up under reduced-pressure
up to 10 mL.
One molar of DTT was added to 0.18% Tx KRM to a final
concentration of 10 mM, incubated at 371C for 1 h, andalkylation was performed by adding 0.5 M iodoacetamide toa final concentration of 25 mM and incubated for 30 min atroom temperature followed by the incubation at 1001C for
10 min. After the sample was cooled, appropriate volumes oftrypsin solution (25 mg/ml in 50 mM NH4HCO3) were added
and incubated at 371C for 2 h. The reaction was stopped byadding equal volume of 0.1% TFA.
The solution contained peptides mix was analyzed withan LC-ESI-MS/MS LCQdecaXP (Thermo Fisher Scientific& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2009, 9, 3036–3046

K.K., Yokohama, Japan) in positive ion mode. The spectrum
data were submitted for protein identification and databasesearching was performed with MASCOT ver1.9 (Matrix
Science, London, UK) in NCBInr. The searching parameters
were as indicated below, mammals as taxonomy, enzyme oftrypsin, one miss cleavage, fixed modification of carbamidomethyl (C), variable modification of oxidation (M), peptide
tolerance of 2.0 Da, MS/MS tolerance of 0.8 Da, peptide
charge of 11, 21and 31, monoisotopic. Only significant
hits, as defined by the MASCOT probability analysis(po0.05), were accepted.

2.5 Integration of T-cell receptora subunit (TCR-a)synthesized in vitro into KRM membrane andfractionation in proteoliposomes
TCR-a or Hsp47 were synthesized in Rabbit Reticulocyte
Lysate System (Promega) and subsequently subjected to an
integration assay, as described [17]. KRM vesicles harboring
TCR-a or Hsp47 were collected and resuspended into themembrane buffer (0.25 M sucrose, 50 mM triethanolamine
acetate pH 7.5, and 1 mM DTT). Carrier KRM vesicles wereadded to the suspension to adjust the protein concentrationof the extraction mixture to 3–5 mg/mL. The extraction andthe reconstitution experiments were performed as describedabove.

2.6 Materials
Chemicals and solvents were of analytic grade and obtained
from Wako Pure Chemical Industries, (Tokyo, Japan). Antidog C-terminal calnexin rabbit polyclonal antiserum was
obtained from Stressgen Bioreagents; anti-mouse ERp72
rabbit polyclonal antiserum, from GENETEX; anti-human
Sec61 a subunit (Sec61-a) rabbit polyclonal antiserum, from
Upstate USA; and ECL Western Blotting Detection System,from GE Healthcare.

3

Results

3.1 A novel fractionation of microsomal proteins
We treated KRM with 0.18% Tx-100 and separated theextracted materials (extract in Fig. 1A) from the residual
membranes (extracted membranes; 0.18% Tx KRM) byultracentrifugation through a high-salt sucrose cushion. In
order to examine which cell components were extracted by
0.18% Tx-100 treatment, we further fractionated the extracted materials by SM-2 bead treatment into two fractions: aproteoliposome fraction to which extracted membraneproteins, if any, were expected to be incorporated and afraction of proteins not recovered in proteoliposomes whichwere expected to be ER luminal proteins. A membrane
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3039

Figure 1. Fractionation of KRM
vesicular protein. (A) Schemeof KRM vesicular protein fractionation. After KRM wasincubated with 0.18% Tx-100,
the extract and residual
membrane (0.18% Tx KRM)
were separated by ultracentrifugation. The extract
obtained was processed using
SM-2 beads to reconstituteproteoliposome,andtheproteins not recovered in
the proteoliposome (luminal
proteins) were separated byultracentrifugation. (B) Electron Microscopic picture ofKRM (left panel), 0.18% Tx
KRM (middle panel), andproteoliposomes (right panel)(C) Protein profile of eachfraction. KRM (lane 1) was
fractionated into 0.18% Tx
KRM (lane 2), proteoliposome(lane 3) and luminal proteins(lane 4) as shown in panel A.
(D) Immunoblotting of KRM
fractions shown in panel B byanti-calnexin,
anti-Sec61-a,
and anti-ERp72 antisera. (E)Quantitation of the results of
the immunoblotting results
shown in panel D.

floatation experiment confirmed that the proteoliposome
fraction was composed of membrane vesicles (unpublisheddata). This extraction–proteoliposome reconstitution treatment resulted in the fractionation of KRM proteins into
three fractions: 0.18% Tx KRM, proteoliposomes, and aluminal protein fraction (Fig. 1A).
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electron microscopic examination showed that theproteoliposomes were indeed formed and remarkably
different in appearance either from the KRM or from 0.18%
Tx KRM (Fig. 1B, compare right panel with left and middle
panels), indicating that the solubilization–reconstitution
treatment yielded vesicles which varied considerably in size.
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K. Sakai et al.

On the other hand, there was no significant difference
between KRM and 0.18% Tx KRM (Fig. 1B, compare middle
panel with left panel), while no membrane structure wasobserved when treated by 1% Tx-100 to solubilize the KRM
membrane completely [16]. This suggested that KRM vesicles maintained the basic membrane structure in spite of
0.18% Tx-100 treatment and were sedimented in a pellet as
0.18% Tx KRM by ultracentrifugation. Protein profiles ofthese fractions showed that the majority of KRM proteins
were recovered in the 0.18% Tx KRM (Fig. 1C, compare lane
2 with lane 1) and a great part of the extracted proteins from
KRM, especially those of higher molecular range, wererecovered in the luminal proteins fraction (Fig. 1C, compare
lane 4 with lane 1). The results of these electron microscopy
and protein profiles suggested that by 0.18% Tx-100 treatment, the majority of KRM vesicles retained their basic
membrane structure and membrane proteins, but were leftin a leaky state, releasing luminal proteins. Under theconditions of our experiment, the effect of the 0.18% Tx-100
treatment, which is higher than the CMC, probably occurredbecause the ratio of detergent to membrane lipids was toolow. This was consistent with the result of our previous
functional study, in which we showed that 0.18% Tx KRM
still retained protein translocation and core-glycosylation
activities when included in an in vitro protein synthesis
system [16]. The functional study suggested that the
majority of integral membrane proteins involved in those
activities were retained in the 0.18% Tx KRM membrane,
but also kept their structures to retain their functions inspite of the treatment.
In contrast with 0.18% Tx KRM, only a small amount(less than 15%) of KRM proteins was recovered in theproteoliposome fraction (Fig. 1C, lane 3). When each band
was compared between KRM or 0.18% Tx KRM, andproteoliposomes (Fig. 1C, compare lane 3 with lane 1 orlane 2), the amounts of almost all bands of the proteoliposome fraction were considerably decreased from those ofcorresponding bands of KRM or of 0.18% Tx KRM. From
this small amount of recovery of each protein to proteoliposomes and the electron microscopic picture, we considered that a small population of KRM vesicles had beennonspecifically solubilized, and membrane proteins together with membrane lipids of solubilized vesicles formed
proteoliposomes by SM-2 beads treatment. Nonspecificalsolubilization of KRM vesicles inevitably occurred when
0.5% Tx-100 was added to the extraction mixture to obtain a
final concentration of 0.18%.
Immunoblotting analysis showed that Sec61-a, a
component of translocation machinery, and calnexin, bothof which were representatives of KRM membrane proteins,mainly were not extracted, then were recovered in the 0.18%
Tx KRM fraction (Fig. 1D, compare lane 1 with lane 2). The
relatively small amounts (one-third at most) of both of theproteins that were extracted were mostly recovered byincorporating into the proteoliposome fraction (Fig. 1D,
lane 3), and trace amounts (less than 5%) of the proteins& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2009, 9, 3036–3046

were detected in the luminal protein fraction (Fig. 1D,
lane 4), probably because of incomplete reconstitution
efficiency. Quantitation of the band in each fraction
showed that practically neither of the proteins was lost in
this reconstitution process by binding to SM beads(unpublished data). In contrast to these integral membraneproteins, ERp72 – an ER luminal protein – was almost
entirely extracted and recovered in the luminal protein
fraction (Fig. 1D, compare lane 1 with lane 2 and lane 4).
These results supported that most of the membraneproteins of KRM were fractionated in 0.18% Tx KRM.
Their small portions contained in the extract were recoveredin the proteoliposomes, and most of the luminal proteins
were extracted from the KRM and fractionated in theluminal protein fraction. The quantitation data are shown in
Fig. 1E.
Close inspection of the protein profile, however, suggested that some proteins appeared to be specifically extracted
from KRM membrane and recovered in the proteoliposome
fraction (Fig. 1C, compare lane 3 with lane 2 and lane 4), in
contrast to those that were expected to be released from thesmall, nonspecifically solubilized population of KRM vesicles, and recovered in proteoliposomes, as observed in
Sec61-a and calnexin fractionation (Fig. 1D and E).

3.2 Demonstration of integral membrane proteinsspecifically fractionated in proteoliposomes by
2-D gel analysis
To focus on the integral membrane proteins of each fraction, we treated each fraction, except the luminal protein
fraction, with sodium carbonate. This treatment is a wellestablished method for eliminating soluble and peripheral
membrane proteins from ER membrane preparations [18].
The extraction–reconstitution followed by sodium carbonate
treatment enabled the fractionation of KRM integral
membrane proteins into two fractions: 0.18% Tx KRM andproteoliposomes. The fractions obtained were subjected to
2-D gel electrophoresis. As expected from the protein profile
and immunoblotting results shown in Fig. 1, most of the
spots obtained from the KRM fraction were also detected in
the 0.18% Tx KRM fraction (Fig. 2, compare panel A withpanel B), although the spots appeared to be decreased in size
to some extent. Many of the spots obtained from both the
KRM and 0.18% Tx KRM did not have sharp, well-defined
shapes but were streaky or fuzzy. This was considered to bea result of their hydrophobic membrane-spanning domains;the more hydrophobic membrane-spanning domains they
have, the streakier are their shapes. In our experiment, thepresence of numerous spots such as these suggested that
proteins with several membrane-spanning domains weresolubilized well and visualized in the 2-D gel. This
ill-defined spot characteristic resulted in the overlap ofthese spots, as well as overlap with the well-defined spots(see Fig. 2A and B), making spot by spot quantitative
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3041

Figure 2. Analysis of KRM integral membrane protein fractionation by 2-D gel electrophoresis. KRM (A) and its fractions 0.18% Tx KRM (B) andproteoliposome (C) were treated with alkaline solution, andanalyzed by 2-D gel electrophoresis. Dashed rectangles inpanels B and C are enlarged
in panels D and E, respectively.
In panels D and E, the spots
from proteoliposome (E), thatwere two or more times larger
than the corresponding spots(dashed circle) from 0.18% Tx
KRM (D), are circled.

assessment very difficult. Consequently, after sodium
carbonate treatment, the 0.18% Tx KRM fraction was
directly subjected to the shotgun analysis by LC-MS/MS
without 2-D gel analysis. This led to the overestimation of
0.18% Tx KRM proteins, since even if the larger portion is
extracted and recovered in the proteoliposomes, the small
amount of a protein left in the 0.18% Tx KRM membrane,
might be sufficient for its identification.
In contrast with those proteins that were retained mainlyin the 0.18% Tx KRM fraction, 2-D gel analysis demonstrated that the other types of proteoliposome spots larger
than those corresponding to the spots in 0.18% Tx KRM
existed (Fig. 2, compare panel D with panel E). In otherwords, they were specifically fractionated in the proteoliposome fraction. The presence of such proteins indicated thatthe proteins recovered in the proteoliposomes werecomposed of two distinct groups; one derived from thenonspecifically solubilized minor population of KRM vesicles; the other specifically extracted from the majority of& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

unsolubilized KRM vesicles. In order to select proteins thatwere primarily recovered in the proteoliposome fraction, we
had to quantitatively assess each spot that was obtained
from proteoliposomes in 2-D gel, comparing them with thecorresponding spot from the 0.18% Tx KRM. However, 2-D
gel electrophoresis of the proteoliposome fraction revealed
that many protein spots were streaky and did not show
well-defined patterns like those obtained from KRM
or from 0.18% Tx KRM. In this study, we focused on
quantitative assessment of the well-defined spots, inspite of the risk of contamination by neighboring streaky
spots. We selected well-defined protein spots obtained fromproteoliposomes, especially those that were at least two or
more times larger than the corresponding spots from 0.18%
Tx KRM (semiquantitative information by PDQuest version
8.0 (Bio-Rad) in Fig. S1 in the Supporting Information),excised them from the gel, pooled them, and subjected themto the shotgun analysis by LC-MS/MS, as described in
Section 2.
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K. Sakai et al.

3.3 Proteins identified
The proteins identified after subtracting contaminantsoluble proteins and mitochondrial proteins are summarized in Table 1. A total of 32 integral membrane proteinsProteomics 2009, 9, 3036–3046

were identified in the 0.18% Tx KRM fraction. Twenty-fourof 32 proteins that were identified as resident integral
membrane proteins of the ER, and more than half ofthem are involved in the protein translocation process,such as signal-recognition particle receptor, translocon

Table 1. Integral membrane proteins identified in 0.18%Tx KRM and in proteoliposomesgiProtein

0.18%Tx KRM
50978924
73991908
73984484
50979164
57112873
73963665
50979076
73969959
73945996
47523726
73957446
11345462
553184950979098
73954525
50979162
57101164
73990468
73962567
57112339
73996823
57087817
76639219
50979014
50979146
73945930
57098751
73990585
73980965
21759777
57112869
50979070

Ribosome-binding protein 1
224
Ribophorin II precursor isoform 3
200
Dolichyl-diphosphooligosaccharide—protein glycosyltransferase 67 kDa subunit 186
Dolichyl-diphosphooligosaccharide-protein glycosyltransferase
169
Translocon-associated protein, delta subunit precursor (TRAP-delta)
131
Transmembrane trafficking protein isoform 1
91
Signal sequence receptor, alpha88
Cytoskeleton-associated protein 4375
DC2 protein
54
Ribophorin I
261
Transmembrane emp24 protein transport domain containing 6
158
Signal peptidase complex subunit 3
156
SURF-4 isoform
116
Glycoprotein 25L
116
Oligosaccharyl transferase STT3 subunit homolog (B5)138
Signal peptidase complex subunit 2 homolog
72
Microsomal signal peptidase 12 kDa subunit
118
Signal sequence receptor, gamma isoform 2
70
Defender against cell death 1
67
Progesterone receptor membrane component
62
FK506-binding protein 11 precursor
76
Vitamin K epoxide reductase complex, subunit 1 isoform 1
56
Protein KIAA0152
77
Calnexin
240
Sec61 alpha 1 subunit
77
ERGIC-53 protein precursor
60
SEC22 vesicle trafficking protein-like 1
104
Signal recognition particle receptor beta subunit
77
Mannosyl-oligosaccharide glucosidase
47
ENTPD4 protein
28
B-cell receptor-associated protein 31
39
Translocation associated membrane protein 1
35

Proteoliposomes
73959043
Nodal modulator 2 isoform 227696122
DOK7 protein
50979076
Signal sequence receptor, a73994752
Protein KIAA0152 precursor isoform 1
73970263
Prenylcysteine oxidase 150979234
Zymogen granule membrane glycoprotein 273945930
ERGIC-53 protein precursor
73989836
Adipocyte plasma membrane-associated protein
73963665
Transmembrane trafficking protein isoform 1
57112873
Translocon-associated protein, delta subunit precursor
73957263
ATPase, H1transporting, lysosomal, V0 subunit D isoform 1
73994418
Coated vesicle membrane protein isoform 3

Score Subcellular location

75
22
63
29
521
293
234
437
209
229
119
116

ER
ER
ER
ER
ER
Transport vesicle
ER
Transport vesicle
ER
ER
Transport vesicle
ER
ER
Transport vesicle
ER
ER
ER
ER
ER
ER
ER
ER
Transport vesicle
ER
ER
ER-golgi intermediate
Transport vesicle
ER
ER
ER
ER
ER
ER
Plasma membrane
ER
Transport vesicle
Lysosome
Zymogen granule
ER-golgi intermediate
Secretary vesicle
Transport vesicle
ER
Lysosome
Transport vesicle

KRM was fractionated into proteoliposomes and 0.18%Tx KRM. After sodium carbonate treatment, 0.18% Tx KRM was directly subjected
to shot gun analysis by LC-MS/MS. Also after sodium carbonate treatment, proteoliposomes and 0.18% Tx KRM were analyzed by 2-D gel,and proteoliposome spots the amounts of which were twice or more than corresponding those of 0.18% Tx KRM were selected, excised,pooled, and subjected to shot gun analysis by LC-MS/MS. The results are summarized in table after soluble proteins and mitochondorial
proteins were eliminated as contaminations.

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Proteomics 2009, 9, 3036–3046

complex, signal peptidase complex, and oligosaccharyltransferase complex and its related proteins. So that ourexperiment successfully identified main members ofrough ER integral membrane proteins in the 0.18% Tx KRM
was also consistent with our previous functional assay [16].
Only eight proteins in this fraction were identified asproteins exported out of the ER. No glycosylphosphatidylinositol-anchored protein was identified. In
contrast, 9 of the 12 proteins that were identified in
the proteoliposome fraction were exported out of the ER.
Only three ER resident proteins were identified in thisfraction. The ratio of proteins exported out of the ER
increased significantly from 25% for 0.18% Tx KRM toapproximately 75%, for the proteoliposome fraction, indicating that our extraction–reconstitution method successfully enriched the proteoliposome fraction with integral
membrane proteins exported out of the ER. Conversely, theratio of ER resident proteins decreased remarkably from75% for 0.18% Tx KRM to approximately 25% for theproteoliposome fraction. These results indicated that two
distinct groups of KRM integral membrane proteins exist
according to varying sensitivity to 0.18% Tx-100 treatment.
Other information such as number of peptides used to
identify a protein, the sequence and charge state of each
peptide, and MS/MS spectra of some low score proteins arein the Supporting Information Table S1 and the Supporting
Information data.

3.4 Confirmation by an in vitro system
To confirm integral membrane protein extraction from the
KRM membrane by 0.18% Tx-100 treatment we used an
in vitro system, wherein synthesized integral membraneproteins were indeed integrated into KRM vesicularmembranes included in the system. It is well known that
only interaction between the membrane-spanning domain
and membrane lipids determines whether the protein is
integrated into the membrane. TCR-a is an integral
membrane protein that has one membrane-spanningdomain. We used TCR-a as a representative of the integral
membrane proteins exported out of the ER. We previously
showed that TCR-a synthesized in the in vitro system wastranslocated into KRM membrane and core-glycosylated[16]. We performed a sodium carbonate treatment to test
whether the TCR-a synthesized in the system, in thepresence of KRM, was integrated into the vesicularmembranes. As a control, Hsp47, a luminal protein, wassynthesized. Hsp47 synthesized in the presence of KRM wascore-glycosylated at translocation through the KRM
membrane. In fact, two products moving slower than the
precursor in SDS-PAGE (Fig. 3A, compare lane 2 with lane
1 in the upper panel) were confirmed to be the core-glycosylation products by an EndoH digestion experiment(unpublished data). After the synthesis, vesicles harboringtranslocated Hsp47 were sedimented in neutral pH condi-

Figure 3. Integration of TCR-a synthesized in an in vitro system into KRM and its fractionation into proteoliposome. (A) Integration of TCRa synthesized in an in vitro system into KRM membrane. Hsp47 mRNA (upper panel of A) and TCR-a mRNA (lower panel of B) wastranslated in an in vitro system in the absence (lane 1) or presence of KRM (lanes 2–6). After the translation, membrane vesicles weresedimented at pH 7.5 to separate the supernatant (lane 3) from the pellet (lane 4). The pellet was resuspended, and treated with alkaline
solution (pH 12.5), and then sedimented to separate the supernatant (lane 5) from the pellet (lane 6). m; mature form, p; precursor.
(B) Fractionation of TCR-a into proteoliposomes KRM vesicles harboring TCR-a or Hsp47 (lane 1 of B) were fractionated into 0.18% Tx KRM(lane 2), proteoliposome (lane 3), and luminal fractions (lane 4). (C) Quantitation of the fractionation results is shown in panel B.

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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K. Sakai et al.

tions and sodium carbonate treatment was subsequently
performed. Mature forms of products were sedimented
with KRM vesicles at a neutral pH (Fig. 3A, lane 4 inupper panel), but extracted from the vesicles at alkaline pH(Fig. 3A, lane 5 in upper panel), showing that the sodium
carbonate treatment acted as expected. On the other
hand, TCR-a synthesized in the presence of KRM wasglycosylated to a single mature product that was probably
fully glycosylated at its putative glycosylation sites (Fig. 3A,
compare lane 2 with lane 1 in the lower panel). Further, theresistance of this product to sodium carbonate treatment(Fig. 3A, lane 6 in the lower panel) indicated that thisproduct was indeed integrated into the KRM vesicularmembranes.
KRM vesicles harboring either TCR-a or Hsp47 synthesized using the in vitro system were collected and subjectedto the extraction–reconstitution experiment carried out inthe manner illustrated in Fig. 1. Approximately 80% of
TCR-a was extracted from the KRM vesicular membrane(Fig. 3B, compare lane 2 with lane 1 in the upper panel)and recovered in the proteoliposome fraction (Fig. 3Blane 3), while approximately 70% of Hsp47 was recovered in
the luminal fraction (Fig. 3B lane 4). This showed that alarge portion of TCR-a, once integrated into the KRM
membrane, was extracted by 0.18% Tx-100 treatment,strongly supporting the possibility that a group of integral
membrane proteins was preferentially extracted from the
KRM membrane itself. In addition, no obvious loss of TCRa by binding to SM-2 beads was observed. The quantitation
data are shown in Fig. 3C.

4

Discussion

To our knowledge, this is the first 2-D gel analysis of roughER integral membrane proteins. This enabled us to evaluate
our novel fractionation method of rough ER integral
membrane proteins, leading to the successful demonstration of preferential extraction of a distinct group of integral
membrane proteins from KRM vesicular membrane by
0.18% Tx-100 treatment. In addition, the group of extracted
proteins was interestingly enriched with proteins exported
from the ER.
Electron microscopic examination, protein profiles, andimmunoblotting results illustrated in Fig. 1, suggested thatthe majority of KRM vesicles retained their basic membrane
structure and membrane proteins in spite of 0.18% Tx-100
treatment, and were recovered in the pellet as 0.18% Tx
KRM by ultracentrifugation. This was consistent with ourprevious functional assay of 0.18% Tx KRM [16]. On theother hand, KRM membrane appeared to be in a leaky state,
as a result of 0.18% Tx-100 treatment, releasing theirluminal proteins recovered as the extract in the supernatant
after ultracentrifugation. To examine whether any
membrane components besides soluble components were
contained in the extract, we treated the extract with SM-2
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2009, 9, 3036–3046

beads to recover the former ones in proteoliposomes. As
shown in Fig. 1, proteoliposomes were formed from theextract, indicating that the extract obviously contained
enough membrane lipids and proteins to form proteoliposomes without affecting the structure and functionsof the residual membrane, 0.18% Tx KRM, so much. Thus,
our concern lay in determining what type of lipids andproteins were extracted. Some portion of the extract
came from the nonspecifical solubilization of the minor
portion of KRM vesicles as already described. But in thiscase, each spot obtained from proteoliposomes in
the 2-D gel should be smaller than the corresponding
one from the 0.18% Tx KRM. However, we showedthat the other kind of spots, each of which was larger thanthe corresponding one from 0.18% Tx KRM, did exist in the
2-D gel obtained from the proteoliposomes. Based on
numerous experiments under various conditions to confirm
the reproducibility of the occurrence of these spots, we
picked up and subjected them to the protein identification
by LC-MS/MS as described in Section 2. These spots
never come from the minor subpopulation of KRM vesicles
that became low density by 0.18% Tx-100 treatment andwere recovered in the supernatant, and thereby in proteoliposomes.
Another problem was whether the proteins recovered in
the proteoliposome fraction originated from the KRM
or from contaminant intracellular membrane systems
other than KRM. The main contaminants were expected
to be mitochondria, because KRM was prepared frompost-mitochondrial supernatants by sedimentation througha 1.35 M sucrose cushion [17] that could almost completely
eliminate low-density intracellular membrane systems
especially of secretory pathway. We estimated that
contaminants other than mitochondria, if any, wereconfined to a small percentage of the KRM. Proteins identified in the proteoliposome fraction were detected as
Coomassie-stained spots, and they were unexpectedly
large to have originated from contaminants. In fact,
no peroxisomal, proteins not part of the secretory
pathway, were identified in 0.18% Tx KRM or in theproteoliposome fraction. At the very last moment of thispaper preparation we succeeded in showing that the
majority of glycoproteins recovered in proteoliposomes wereof high mannose type, sensitive to EndoH digestion(unpublished data). This indicated that membraneproteins recovered as proteoliposomes were derived from
KRM itself rather than the contaminated intracellular
membrane system. In addition, the in vitro system provided
strong supporting evidence that a protein, once integrated
into the KRM membrane, could be extracted from themembrane by 0.18% Tx-100 treatment, and recovered inproteoliposomes. Thus, we concluded that we did establish a
novel fractionation method to divide KRM integral
membrane proteins into two distinct groups, based onwhether or not they can be extracted by low-concentration
Tx-100 attack.
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In addition, we found that the subgroup of integral
membrane proteins specifically fractionated in the proteoliposomes was enriched with integral membrane proteins
exported from the ER. The amount of total proteins in 0.18%
Tx KRM was considerably greater than that in the proteolipososmes; hence, the former was a rate-limiting amountapplied on the 2-D gel, and it limited the latter to a small
quantity. The amount of each protein exported from the ER is
generally expected to be very small because it stays in the ER
only transiently. In spite of these unfavorable aspects, thesuccessful identification of ten proteins exported from the ER
in the proteoliposome fraction was estimated to be unexpectedly significant. Furthermore, because of the aforementioned
difficulties in the 2-D gel analysis of integral membraneproteins and recovery of the vast majority of KRM proteins in
the 0.18% Tx KRM, we subjected 0.18% Tx KRM directly to
LC-MS/MS. Thus, it may be possible to interpret as follows:
the eight proteins exported out of the ER in 0.18% Tx KRM
might have been identified due to their small portions retainedin this fraction, although their major portions were recoveredin the proteoliposomes. Note the presence of many streaky
spots in the proteoliposome fraction which were larger thanthe corresponding ones in 0.18% Tx KRM (Fig. 2C and E).
This suggested that proteins with several membrane-spanningdomains were also recovered in the proteoliposome fraction.
However, proteins identified in the proteoliposome fraction
have only one or two membrane-spanning domains, probably
because we selected clear spots for the purpose of comparative
quantitation reason. Although 2-D gel analysis was indispensable to show that a group of integral membrane proteins
was specifically fractionated in the proteoliposomes, asystematic immunoblotting analysis of numerous candidate
proteins is required. This procedure must serve to show much
more proteins specifically recovered in the proteoliposomes
and allow detection of proteins showing streaky or poorly
circumscribed spots. It will also prove that our method fractionates the rough ER integral membrane proteins into clearcut fractions (such as resident protein fraction versus a fractionof proteins exported from the ER).
It remains to be elucidated how the group of integral
membrane proteins is extracted from KRM membrane by
0.18% Tx-100 treatment without affecting its basic structureand functions. Some may argue that proteins with a few
membrane-spanning domains are easily extracted from
KRM membrane even by 0.18% Tx-100 treatment, andrecovered in proteoliposomes. Nevertheless, this is unlikely,
because 24 out of 32 identified proteins in the 0.18% Tx
KRM fraction have one or two membrane-spanningdomains. The preferential extraction would be attributed tothe presence of at least two types of microdomains into
which integral membrane proteins were selectively integrated as soon as they were released from the translocons in
the rough ER. By 0.18% Tx-100 treatment one microdomain
was extracted from the ER membrane as a Triton-extractablesoluble structure, and the other was inextractable andremained in the ER membrane. Thus, our findings will
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

potentially raise a serious doubt regarding the prevailingview of the ER membrane structure, which has been
considered to be in a homogeneous ‘‘liquid-disordered’’state because of its low sphingolipid and cholesterol
concentrations [3]. Another significant result of this study
was the successful enrichment of the proteoliposome fraction with integral membrane proteins exported out of the
ER. The protein segregation into the Triton-extractablemicrodomain might correspond to the segregation into thescaffold prior to Sec12p activation [8]. Both protein integration into the ER membrane at the translocon and protein
exit from the ER by COPII vesicle formation (that has been
considered to be the first selection step of proteins exportedout of the ER) have been extensively studied. The area
between those steps remains unexplored.
That a distinct group of integral membrane proteins was
extracted from the rough ER membrane without affecting
the basic structure and the function of the membrane, andthat the group was enriched with proteins exported out of
the ER are totally unexpected and very interesting results per
se. Furthermore, they will potentially have a huge impact on
ER membrane biogenesis and protein trafficking.
We thank Takashi Saito (Chiba University) for the gift ofTCR-a cDNA and Kazuhiro Nagata (Kyoto University) for thegift of Hsp47 cDNA.
The authors have declared no conflict of interest.

5

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