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The Golgi-Localized Arabidopsis Endomembrane Protein12
Contains Both Endoplasmic Reticulum Export and Golgi
Retention Signals at Its C Terminus
C W
Caiji Gao, Christine K.Y. Yu, Song Qu, Melody Wan Yan San, Kwun Yee Li, Sze Wan Lo, and Liwen Jiang1
School of Life Sciences, Centre for Cell and Developmental Biology, The Chinese University of Hong Kong, Shatin, New Territories,
Hong Kong, China
Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and
mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short
cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their
protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12
in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent
protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus
of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in
Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif
and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of
EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting
signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in
plant cells.
INTRODUCTION
In the endomembrane system of eukaryotic cells, secretory
proteins start their journey from the endoplasmic reticulum (ER)
prior to reaching the Golgi apparatus for further sorting to postGolgi compartments, such as the trans-Golgi network (TGN) and
prevacuolar compartment (PVC) (Barlowe et al., 1994). Protein
traffic between the ER and Golgi apparatus is mediated by two
distinct coated vesicles (i.e., coat protein complex II (COPII) and
COPI), which mediate anterograde and retrograde transport,
respectively (Cosson and Letourneur, 1997; Kuehn et al., 1998;
Rabouille and Klumperman, 2005; Donohoe et al., 2007). Integral
membrane proteins that traffic between the ER and Golgi usually
contain specific sorting signals that are essential for selective
package into COPII vesicles for ER export or into COPI vesicles
for ER retrieval and Golgi retention (Barlowe, 2003; Beck et al.,
2009).
In mammals and yeast, various ER export signals have been
identified, including the diacidic motif (DXE) of vesicular stomatitis
virus glycoprotein, the dihydrophobic (LL) motif of ERGIC-53, and
the diaromatic motif (FF, YY) of 46-kD Endomembrane Protein
Precursor (Emp46p) (Nishimura and Balch, 1997; Nufer et al.,
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Liwen Jiang (ljiang@cuhk.
edu.hk).
C
Some figures in this article are displayed in color online but in black and
white in the print edition.
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.112.096057
2002; Sato and Nakano, 2002). All of these typical ER export
signals are found to reside on the cytosolic regions of transmembrane proteins that interact with COPII vesicles (Barlowe,
2003). Similarly, the dilysine motif, KKXX, is one of the best known
sorting signals required for retrograde Golgi-to-ER transport of
several type I membrane proteins that interact with COPI vesicles
(Cosson and Letourneur, 1994; Schröder et al., 1995; Harter and
Wieland, 1998; Gomez et al., 2000). Recently, the semiconserved
Phe-Leu-Ser-like motifs were identified as Golgi retention signals
in the cytoplasmic tails (CTs) of glycosyltransferases, a group of
Golgi-resident integral membrane proteins in yeast (Tu et al.,
2008). These motifs were shown to interact with COPI vesicles via
Vps74p to maintain the steady state Golgi localization of the
glycosyltransferases, thus providing the best known molecular
mechanism of Golgi retention of membrane proteins in eukaryotic
cells (Tu et al., 2008).
Several studies have been performed to analyze the targeting
mechanisms of integral membrane proteins in plant cells. The
transmembrane domain (TMD) and CT of binding protein 80 kD
(BP-80), a type I integral membrane protein belonging to the
vacuolar sorting receptor (VSR) family of proteins (Paris et al.,
1997; Li et al., 2002), were shown to be essential and sufficient
for its correct targeting to the PVC, which has been identified as
a multivesicular body (MVB) in plant cells (Jiang and Rogers,
1998; Tse et al., 2004, 2006; Robinson et al., 2008). In addition,
the length of the TMD may affect the subcellular localization of
an integral membrane protein, as green fluorescent protein
(GFP) fusions with different TMD sequences of 17, 20, or 23
amino acids in length showed distinct subcellular localization to
the ER, Golgi, and plasma membrane (PM), respectively
(Brandizzi et al., 2002). Similarly, the TMD length and cytosolic
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tail have been shown to be important for proper Golgi localization of glycosyltransferases, a group of type II integral membrane proteins in plant cells (Saint-Jore-Dupas et al., 2006).
Similar to yeast and mammalian membrane proteins, various ER
export signals, such as the diacidic DXE motif and dibasic motif,
have been identified in different classes of transmembrane
proteins in plant cells (Hanton et al., 2005; Yuasa et al., 2005;
Mikosch et al., 2006; Schoberer et al., 2009; Zelazny et al.,
2009). In addition, the dilysine motif present in the Arabidopsis
thaliana p24 (At p24) homolog has also been shown to bind
with the COPI subunit to maintain the ER localization of p24
(Contreras et al., 2004b; Langhans et al., 2008). More recently,
the rice (Oryza sativa) SECRETORY CARRIER MEMBRANE PROTEIN1 (SCAMP1), a polytopic PM protein with four TMDs (Lam
et al., 2007a, 2007b, 2008; Law et al., 2012), was shown to reach
the PM via an ER-Golgi-TGN-PM pathway in tobacco (Nicotiana
tabacum) Bright Yellow-2 cells (Cai et al., 2011). Interestingly, both
its cytosolic sequences and TMDs regulated the proper trafficking
steps from ER to PM owing to the presence of various signals
within the SCAMP1, including the ER export signal in the cytosolic
N terminus, the Golgi export signal within the TMD2-TMD3, and the
TGN-PM targeting signal in TMD1 (Cai et al., 2011). However, the
underlying mechanisms remain elusive.
Endomembrane proteins (EMPs) belong to a family of integral
membrane proteins with nine TMDs, a large lumenal N terminus,
and a short (10 to 17 amino acids) C terminus located in the
cytosol. Three EMP members are found in Dictyostelium discoideum (Phg1A to Phg1C) (Cornillon et al., 2000) and Saccharomyces cerevisiae (Tmn1 to Tmn3) (Froquet et al., 2008) and
four members in human (TM9SF1 to TM9SF4) (Lozupone et al.,
2009). Loss of Phg1A function in slime mold D. discoideum led
to a defect in cellular adhesion and inefficient phagocytosis
(Cornillon et al., 2000); similarly, Phg1b played a synergistic but
not redundant role in controlling cellular adhesion and phagocytosis (Benghezal et al., 2003). EMPs in yeast were also found
to be required for cell adhesion and filamentous growth under
nitrogen starvation (Froquet et al., 2008). Interestingly, the human EMP protein, TM9SF4, was highly expressed in human
malignant melanoma cells from metastatic lesions but was undetectable in healthy human tissues and cells, thus serving as
a marker for malignancy (Lozupone et al., 2009). In addition,
studies on the subcellular localization of EMPs in yeast and
human cells yielded different conclusions. For example, myctagged human EMP p76 was found predominantly in the
endosomes (Schimmöller et al., 1998), and an N-terminal GFPtagged human EMP (GFP-TM9SF4) was also shown to colocalize with the Rab5 protein in the early endosome (Lozupone
et al., 2009). By contrast, yeast EMP Yer113c was shown to
localize to the Golgi apparatus (Huh et al., 2003), whereas
a C-terminal GFP-tagged yeast EMP (TMN2-GFP) was recently
shown to localize to the endosome and vacuole (Aguilar et al.,
2010).
The Arabidopsis genome contains 12 EMP isoforms (termed
EMP1 to EMP12 in this study), but little is known about their
protein subcellular localization and function in plants. As a first
step to study the biology of EMPs in plants, we performed
studies on the subcellular localization and targeting mechanisms of EMP12 in Arabidopsis. We demonstrated that both
endogenous EMP12 and GFP-EMP12 fusion (as detected by
EMP12 and GFP antibodies, respectively) were localized to the
Golgi apparatus in wild-type and transgenic Arabidopsis plants
and in transiently expressed Arabidopsis protoplasts. We further
showed that the cytosolic C terminus of EMP12 contained both
ER export and Golgi retention signals that interact with COPII
and COPI subunits, respectively. In addition, this Golgi retention
signal of EMP12 trapped several post-Golgi membrane proteins
within the Golgi apparatus in a gain-of-function analysis using
transiently expressed Arabidopsis protoplasts. It seems that
these targeting features of EMP12 are conserved among the
plant EMPs for their correct targeting to the Golgi apparatus in
plant cells.
RESULTS
EMP12 Is a Golgi-Localized Protein with Multiple TMDs
All 12 isoforms of Arabidopsis EMP proteins (termed EMP1 to
EMP12 in this study; see Supplemental Figure 1 online) share
high similarity at the amino acid level and in predicted topology.
All At EMPs are predicted to have a large lumenal N-terminal
domain, followed by nine TMDs and a short CT (Figure 1A).
Eleven At EMP members (excluding EMP1) are predicted to
have a signal peptide (SP) in their N terminus. As a first step to
understanding the biology of EMPs in plants, we analyzed the
subcellular localization and trafficking route of EMP12 in Arabidopsis.
We first used a GFP fusion approach by making a GFPEMP12 construct in which a SP-GFP was used to replace the
SP of EMP12 (Figure 1B). To test if this new fusion maintains
the identical topology as EMP12, we performed a protease
protection assay using microsomes isolated from Arabidopsis
cells expressing the GFP-EMP12 fusion, followed by immunoblot analysis using GFP antibody (Figure 1C). The N terminus of
GFP-EMP12 was found to be resistant to protease digestion
because the 47-kD GFP-TMD1 fragment remained intact in the
presence of trypsin, whereas the 75-kD full-length GFP-EMP12
fusion did not (Figure 1C, lanes 1 and 2), indicating the lumenal
location of the GFP tag as predicted (Figure 1A). The reliability of
such a protease protection assay was further verified by an
identical experiment using a GFP fusion of the type I integral
membrane protein GFP-VSR2 as a control, in which the lumenal
GFP remained intact upon protease digestion (Figure 1C, lanes
4 and 5; Cai et al., 2011).
To study the subcellular localization of EMP12 and GFPEMP12, we next generated EMP12 antibodies and transgenic
Arabidopsis plants expressing GFP-EMP12 for immunoblot
analysis. As shown in Figure 2A, GFP antibody detected a single
protein band around 75 kD, likely representing GFP-EMP12 in
the cell membrane (CM) fraction but not the cell-soluble (CS)
fraction of the transgenic Arabidopsis seedlings expressing
GFP-EMP12, whereas no such protein band was detected in
wild-type plants. By contrast, anti-EMP12 antibodies detected
the putative 50-kD endogenous EMP protein band in the CM
fraction of both wild-type and GFP-EMP12 plants (Figure 2A,
lanes 6 and 8, asterisk) as well as the GFP-EMP12 fusion (lane 8,
Targeting and Subcellular Localization of EMP12
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Figure 1. Topology Analysis of EMP12.
(A) Predicated topology of EMP12 using TMHMM Server 2.0.
(B) Construction of GFP-EMP12 and GFP-VSR2 fusions.
(C) Protease protection assay. Microsomes were isolated from the Arabidopsis (At) protoplasts expressing GFP-EMP12 or GFP-VSR2 for trypsin
digestion, followed by protein extraction and separation via SDS-PAGE for immunoblot analysis using GFP antibody. M, molecular weight marker in
kilodaltons.
[See online article for color version of this figure.]
arrowhead). A subcellular fractionation study using a continuous
sucrose gradient and wild-type Arabidopsis cells showed that
anti-EMP12 had an identical distribution pattern as the cis-Golgi
marker anti-Man1 (Tse et al., 2004; Lam et al., 2007b), thus indicating a possible Golgi localization of EMP12 (Figure 2B). To
further prove this, confocal immunofluorescence labeling was
next performed in root cells of transgenic Arabidopsis plants
expressing GFP-EMP12. As shown in Figure 2C, the punctate
GFP signals of GFP-EMP12 were fully colocalized with antiEMP12. In addition, anti-EMP12 was found to fully colocalize
with the cis-Golgi marker yellow fluorescent protein (YFP)SYP32 but was largely separated from the PVC marker YFPARA7 (Geldner et al., 2009) in these transgenic Arabidopsis root
cells (Figure 2C), indicating that both endogenous EMP12 and
GFP-EMP12 fusion are localized to the Golgi apparatus in
Arabidopsis cells. Thus, GFP-EMP12 can be used to study the
subcellular localization and trafficking of EMP12 in plant cells.
To further confirm the Golgi localization of EMP12 and GFPEMP12 proteins from the above confocal immunofluorescence
study, we next performed immunogold electron microscopy
(EM) using either EMP12 or GFP antibodies on ultrathin sections
prepared from high-pressure frozen/freeze-substituted root tips
of wild-type or transgenic GFP-EMP12 Arabidopsis plants. As
shown in Figure 3, in wild-type Arabidopsis root cells, antiEMP12 labeled the Golgi apparatus in which the majority of the
gold particles (>80%) were found in the cis- and medial-Golgi
(Figure 3A). Similar patterns of Golgi localization were also obtained in GFP-EMP12–transformed Arabidopsis root cells using
anti-GFP antibodies (Figure 3B). In addition, Golgi labeling with
EMP12 or GFP antibodies was specific, as little labeling was
seen in other organelles, including TGN, MVB, and mitochondria
in these experiments (Figure 3, Table 1). Taken together, these
confocal immunofluorescence and immunogold EM studies
demonstrated that EMP12 and GFP-EMP12 localized to the
Golgi apparatus with preferential localization to the cis- and
medial-Golgi in Arabidopsis cells.
We next wanted to test if the established transient expression
system of Arabidopsis protoplasts (Miao and Jiang, 2007) could
be used to study the trafficking of GFP-EMP12. Consistent with
the above immunofluorescence and immunogold EM studies in
transgenic Arabidopsis root cells (Figures 2 and 3), GFP-EMP12
was found to be fully colocalized with the cis-Golgi marker
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Figure 2. Golgi Localization of Endogenous EMP12 and GFP-EMP12 Fusion in Arabidopsis.
(A) Characterization of EMP12 antibody. Proteins were isolated from wild-type (WT) or transgenic Arabidopsis plants expressing GFP-EMP12, followed
by SDS-PAGE and immunoblot analysis using GFP or EMP12 antibodies. Both anti-GFP and anti-EMP12 recognized the full-length GFP-EMP12 fusion
(lanes 4 and 8), whereas anti-EMP12 also detected the endogenous EMP12 protein in both wild-type and transgenic Arabidopsis plants (lanes 6 and 8).
Arrowhead and asterisk indicate GFP-EMP12 fusion and endogenous EMP12, respectively.
(B) Subcellular fractionation analysis of endogenous EMP12. The microsomes prepared from wild-type Arabidopsis (At) cells were fractionated by
ultracentrifugation on a continuous (25 to 50%) Suc gradient. Proteins extracted from individual fractions were probed with anti-EMP12, anti-Man1
(Golgi marker), and anti-VSR (PVC marker) as indicated.
(C) Golgi localization of EMP12 and GFP-EMP12 fusion in transgenic Arabidopsis plants. Confocal immunofluorescence labeling showing full colocalization of anti-EMP12 with GFP-EMP12 (panel 1) or Golgi marker YFP-SYP32 (panel 2) in root cells of transgenic Arabidopsis plants expressing these
two XFP fusions but distinct localization from the PVC marker YFP-ARA7 in root cells of a transgenic Arabidopsis plant (panel 3). The insets represent
a three times enlargement of a randomly selected area. DIC, differential interference contrast. Bars = 10 mm.
Man1-mRFP (for monomeric red fluorescent protein; Tse et al.,
2004; Lam et al., 2007b) when they were transiently coexpressed together in Arabidopsis protoplasts (Figure 4A). By
contrast, GFP-EMP12 was largely separated from the TGN
marker mRFP-SYP61 and the PVC marker mRFP-VSR2 in
Arabidopsis protoplasts (Figures 4B and 4C). In addition, when
the ER-to-Golgi traffic was blocked by overexpression of
Sec12p, a Sar1p-specific guanosine nucleotide exchange factor
for COPII vesicle formation (Pimpl et al., 2003; Cai et al., 2011),
both GFP-EMP12 and Man1-mRFP were trapped and colocalized within the ER (Figure 4D), indicating that GFP-EMP12
reached the Golgi via the COPII-dependent ER-to-Golgi sorting
route, a result consistent with the Golgi localization of Man1mRFP (Langhans et al., 2008).
The Cytosolically Exposed C-Terminal Region Contains
Essential ER Export Signals for the Trafficking of EMP12
from the ER to the Golgi
Since GFP-EMP12 is fully colocalized with the endogenous
EMP12 (as detected by anti-EMP12) to the Golgi apparatus in
Arabidopsis cells (Figure 2) or with the Golgi marker in Arabidopsis protoplasts (Figure 4), we thus used Arabidopsis protoplasts to study the targeting of GFP-EMP12. Since little is
known about the Golgi targeting of EMPs with nine TMDs, we
first tested whether the large lumenal N terminus or the short
cytosolic C terminus are essential for Golgi localization of GFPEMP12 by generating two truncated constructs, GFP-EMP12
(DN) and GFP-EMP12(DC), in which either the lumenal N terminus
Targeting and Subcellular Localization of EMP12
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Figure 3. Immunogold EM Localization of EMP12 and GFP-EMP12.
Ultrathin sections were prepared from high-pressure frozen/freeze-substituted samples of wild-type (WT) or transgenic GFP-EMP12 Arabidopsis root
tips, followed by immunogold labeling using either EMP12 antibodies (A) or GFP antibodies (B), respectively. The relative percentage distribution of
gold particles among cis-, medial-, and trans-Golgi was also quantified. Arrows indicate examples of gold particles present in the Golgi. CW, cell wall; G,
Golgi; M, MVB; V, vacuole. Bars = 100 nm.
or the cytosolic CT was deleted, respectively. As shown in
Supplemental Figure 2 online, upon transient expression in
Arabidopsis protoplasts, GFP-EMP12(DN) remained in the Golgi
as it colocalized with the Golgi marker, indicating that the N
terminus of EMP12 was not essential for its Golgi localization.
By contrast, CT deletion resulted in ER localization of EMP12
because GFP-EMP12(DC) fully colocalized with the ER marker
Calnexin-mRFP upon their coexpression, indicating the possible
existence of an ER export signal at the CT of EMP12.
So far, various ER export signals have been identified at the
cytoplasmic part of integral membrane proteins. Best known
examples are the diacidic motif DXE within the vesicular stomatitis virus glycoprotein cytosolic tail, the dihydrophobic motifs
(FF, YY, LL, or FY) found in the p24 family proteins, and the
Tyro-based signals identified in the tail sequence of ERGIC53/
Emp47p family proteins (Nishimura and Balch, 1997; Nufer et al.,
2002; Sato and Nakano, 2002; Langhans et al., 2008). Alignment
analysis of CTs of Arabidopsis EMPs indicates the existence of
a putative dihydrophobic motif and a Tyr residue in all At EMPs
(see Supplemental Figure 1 online). To find out if the putative
dihydrophobic motif and Tyr residue in the EMP12 CT
(SNLFVRRIYRNIKCD) are important for the ER export of EMP12,
Table 1. Distribution of Immunogold Particles with EMP12 and GFP Antibodies in Wild-Type and Transgenic GFP-EMP12 Arabidopsis Root Cells
Organelle
Wild type (Anti-EMP12)
Golgi
TGN
MVB
Mitochondrion
GFP-EMP12 (anti-GFP)
Golgi
TGN
MVB
Mitochondrion
No. of Organelles
No. of GPs
GPs per Organelle
30
30
30
30
94
7
5
6
3.03**
0.23**
0.17
0.20
30
30
30
30
128
8
4
5
4.27**
0.27**
0.13
0.17
Significant difference between the distribution in the Golgi and TGN was analyzed using the two-tailed paired t test (**P < 0.001). Data were collected
and analyzed from three independent labeling experiments. GP, immunogold particles.
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Figure 4. Golgi Localization of GFP-EMP12 in Arabidopsis Protoplasts.
GFP-EMP12 was coexpressed with various known organelle markers as indicated ([A] to [C]) in Arabidopsis protoplasts, followed by confocal imaging
at 12 to 14 h after transfection, whereas overexpression of Sec12p was used to inhibit the ER export of GFP-EMP12 and Man1-mRFP (D). Bars = 10 mm.
we performed mutagenesis analysis on the dihydrophobic or Tyr
motifs of GFP-EMP12 CT (Figure 5A). Mutations of dihydrophobic combined with Tyr motifs totally block the ER export
of GFP-EMP12, which was fully colocalized with the ER marker
Calnexin-mRFP (Figure 5B). Interestingly, the solo Tyr mutation
only partially inhibited the ER export of GFP-EMP12 (Figures 5C
and 5D). However, the combined mutations of Tyr with one
hydrophobic amino acid, Phe or Val, effectively blocked the ER
export of GFP-EMP12 (Figures 6A and 6B), indicating that the
dihydrophobic residue synergistically combined with Tyr to drive
the ER export of EMP12. This conclusion was further confirmed
by double mutation of the dihydrophobic motif, Phe and Val, that
only partially blocked the ER export of EMP12 as they colocalized with both Golgi and ER markers (Figures 6C and 6D);
however, single mutation on one hydrophobic amino acid, Phe
or Val, had no effect on the ER export of EMP12 (Figures 5E and
6E). Mutations of other amino acids besides the dihydrophobic
or Tyr residues did not affect the ER export of GFP-EMP12 (see
Supplemental Figure 3 online). Taken together, these results
support the conclusion that both the hydrophobic motif and Tyr
residue in the cytosolic tail play essential roles in the ER export
of EMP12.
Transmembrane cargos usually possess a binding affinity with
Sar1–Sec23–Sec24 prebudding complexes through their ER
export signals in order to be included in COPII vesicles for ER
export (Bi et al., 2002; Barlowe, 2003). We next wanted to find
out if COPII subunits interact with the ER export motifs in the
EMP12 CT using in vitro peptide binding experiments (Contreras
et al., 2004b). The soluble fractions isolated from Arabidopsis
protoplasts expressing YFP-Sec24 were used as a source of
COPII subunits (Hanton et al., 2009) for binding with columns
conjugated with synthetic peptides of EMP12 CT or its mutation
on the dihydrophobic/Tyr residues. As shown in Figure 7, the
expressed YFP-Sec24 was retained in the column with the
EMP12 CT peptides (lane 3), but such binding was dramatically
reduced in the mutated column (lane 4), indicating that the dihydrophobic /Tyr residues of EMP12 CT preferably bound to the
COPII Sec24 subunit.
The C-Terminal-Fused GFP Tag Causes Mislocalization of
the EMP12-GFP Fusion to the TGN, PVC, and Vacuole
Interestingly, when GFP was fused at the C terminus of EMP12
(Figure 8A), the resulting EMP12-GFP was found to localize to
Targeting and Subcellular Localization of EMP12
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Figure 5. The EMP12 C Terminus Contained Multiple ER Export Signals.
(A) The GFP-EMP12 C terminus mutation constructs and their subcellular localization in Arabidopsis protoplasts. CT, C terminus; NT, N terminus; T,
TMD; WT, the wild type.
(B) to (E) Typical subcellular localization patterns of four selective GFP-EMP12 C terminus mutation fusions upon their coexpression with known
markers (as indicated) in Arabidopsis protoplasts. Confocal images were collected from cells at 12 to 14 h after transfection. Bars = 10 mm.
both the vacuole and punctate structures distinct from the Golgi
marker Man1-mRFP (Figure 8B), but partially colocalized with
the TGN marker mRFP-SYP61 and the PVC marker mRFP-VSR2
(Figures 8C and 8D) in Arabidopsis protoplasts, indicating that
GFP fusion at the C terminus of EMP12 caused it to move from
the Golgi to the vacuole via TGN/PVC. In addition, EMP12-GFP
also passed through the ER-Golgi sorting route via the COPII
vesicle en route to the vacuole because overexpression of
Sec12p trapped it within the ER (Figure 8E), indicating that the
C-terminal GFP fusion did not affect the ER-to-Golgi transport
but caused mislocalization of EMP12-GFP to post-Golgi compartments, including TGN, PVC, and the vacuole.
The EMP12 CT Contains a Novel KXD/E Motif for
Golgi Retention
To further find out if the EMP12 CT is essential for its Golgi
retention, we used a gain-of-function approach and added an
additional EMP12 CT sequence to the C terminus of EMP12GFP (Figure 9A). Indeed, the resulting EMP12-GFP-CT was
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Figure 6. Subcellular Localization of GFP-EMP12 Harboring Mutations on the ER Export Signals.
These various GFP fusions were coexpressed with known markers (as indicated) in Arabidopsis protoplasts. Confocal images were collected from cells
at 12 to 14 h after transfection. Bars = 10 mm.
found to remain in the Golgi as it colocalized with the Golgi
marker Man1-mRFP but largely separated from the TGN and
PVC markers upon their transient coexpression in Arabidopsis
protoplasts (see Supplemental Figure 4 online). Further mutagenesis (deletion) analysis of the EMP12 CT demonstrated that
the last six amino acids RNIKCD (EMP12-GFP-RNIKCD; Figure
9B) or even the last three residues KCD (EMP12-GFP-KCD; see
Supplemental Figure 4 online) were sufficient to retain the
EMP12-GFP within the Golgi or efficiently retrieve EMP12-GFP
back to the Golgi apparatus.
Interestingly, sequence alignment analysis of the CTs of EMP
homologs from Arabidopsis, rice, and maize (Zea mays) showed
the presence of a highly conserved Lys in the –3 position and an
acidic residue (Asp or Glu) in the –1 position (see Supplemental
Figure 1D online). To test if this conserved Lys-based motif KXD/
E was essential for Golgi retention, we performed point mutation
analysis on this motif via substitution with Ala residues. Double
point mutations on Lys and Asp (EMP12-GFP-RNIACA; see
Supplemental Figure 5 online) or single mutation on Lys
(EMP12-GFP-RNIACD; Figure 9C) or Asp (EMP12-GFP-RNIKCA; Figure 9D) all resulted in mislocalization of these GFP fusions to vacuole and post-Golgi PVC organelles that were
largely distinct from the Golgi apparatus (see Supplemental
Figures 5A to 5G online). Thus, it seems that both K and D/E in
the KXD/E motif are essential for Golgi localization or retention of
EMP12. In addition, the KXD/E motif must be presented at the
C-terminal end to function as a Golgi retention signal, as the addition
of three Ala residues at the C terminus of the Golgi-localized
Targeting and Subcellular Localization of EMP12
Figure 7. The ER Export Signal at the C Terminus of EMP12 Binding to
COPII Subunit in a Sequence-Specific Manner.
Soluble proteins extracted from Arabidopsis protoplasts expressing
YFP-Sec24 were incubated with Sepharose beads conjugated with
synthetic peptides corresponding to the wild type (WT; lane 3) or mutated (mut; lane 4) C terminus of EMP12 for a pull-down assay, followed
by immunoblot detection of bound proteins using a GFP antibody. M,
molecular weight marker in kilodaltons.
EMP12-GFP-RNIKCD caused its mislocalization to the vacuole
and PVC (Figure 9E; see Supplemental Figures 5H and 5I online).
The PVC-mediated degradation of these mislocalized EMP12GFP fusions was further proved by coexpression with a constitutively active Rab5 mutant, ARA7(Q69L), which can induce PVC
enlargement and trap the degradative cargo proteins within its
lumen (Kotzer et al., 2004). Indeed, both EMP12-GFP and
EMP12-GFP-RNIKCDAAA occurred as large round dots within
the lumen of mRFP-ARA7(Q69L)–labeled ring-like structures,
distinct from the punctate patterns of Golgi-localized GFP-EMP12
and EMP12-GFP-RNIKCD (see Supplemental Figure 6 online).
The detection of GFP signals in the vacuole in cells expressing
GFP-tagged transmembrane proteins usually indicates the occurrence of protein degradation (Kleine-Vehn et al., 2008; Langhans
et al., 2008; Foresti et al., 2010; Cai et al., 2012). To provide further
evidence that these various EMP12-GFP-RNIKCD mutation fusions
were degraded in vacuoles upon their expression in Arabidopsis
protoplasts (e.g., Figure 9), we performed immunoblot analysis using
GFP antibodies. As shown in Figure 10, upon expression in Arabidopsis protoplasts, the Golgi-localized EMP12-GFP-RNIKCD largely
(>90%) remained as the full-length protein in the CM fraction, while
<10% of the signal was detected as the GFP core (Foresti et al.,
2010) in the CS fraction. By contrast, in cells expressing the three
other constructs with a mutation on the KXD/E motif (EMP12-GFPRNIACD and -RNIKCA) or C-terminal addition (–RNIKCDAAA),
>50% (50 to 70%) of the detected signals were found as the GFP
core in the CS fraction (Figure 8C), indicating that their turnover rate
is much higher than that of EMP12-GFP-RNIKCD. Quantification
analysis of these GFP signals in the CM versus CS fractions of
individual proteins further confirmed this conclusion (Figure 10C).
Thus, both confocal data (Figure 9) and immunoblot analysis (Figure
10) point to the same conclusion that the C-terminal KXD/E motif
functions as a Golgi retention signal for EMP12.
The KXD/E Golgi Retention Motif Can Interact with
COPI Vesicles
To further explore the molecular mechanism of KXD/E motif
function in Golgi retention, we next used the synthetic peptides
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corresponding to the EMP12 CT as bait to isolate possible interacting proteins from wild-type Arabidopsis proteins in pulldown experiments, followed by protein elution, separation via
SDS-PAGE, and tandem mass spectrometry (MS/MS) analysis
for protein identification. In duplicate experiments, several proteins that were specifically pulled out by the EMP12 CT peptide
column were identified as the COPI coat subunits (see
Supplemental Figure 7 online). The COPI vesicles are the predominant means by which the membrane proteins recycle back
to the ER or are retained in the Golgi (Cosson and Letourneur,
1994; Tu et al., 2008). We thus further tested whether COPI
coatomers could bind with the KXD/E motif using in vitro peptide
binding experiments as described previously (Contreras et al.,
2004b). The soluble fraction isolated from Arabidopsis protoplasts expressing Sec21-Myc, an Arabidopsis g-COP protein
(Movafeghi et al., 1999), was used as a source of COPI coat
protein in the binding experiment using the synthetic peptide of
EMP12 CT or its mutants of the KXD/E motif as binding baits. As
shown in Figure 11, Sec21-Myc specifically binds to the EMP12
CT as detected by anti-Myc antibody (Figure 11, lane 3), but
such binding was completely abolished when the KXD motif in
the CT was mutated to either ACA or ACD (Figure 11, lanes
4 and 5), while the binding ability was reduced significantly for
the KCA peptide (Figure 11, lane 6), indicating that the Lys in the
KXD motif played a more critical role in COPI binding. These
binding results are consistent with those obtained with confocal
subcellular localization and immunoblot analysis of EMP12GFP-RNIKCA (Figures 9 and 10). Therefore, binding with the
COPI coatomer was a means by which the KXD/E motif achieved
the Golgi retention function.
RNIKCD Functions as a Golgi Retention Motif for Post-Golgi
Membrane Proteins
To find out if the Golgi retention signal RNIKCD identified in the
EMP12 CT would have a similar function in other membrane
proteins, we next used a gain-of-function approach by adding
this motif to the C terminus of GFP fusions of two selective postGolgi membrane proteins: SCAMP1-GFP and TPK1-GFP (Figure
12). GFP fusions with the SCAMP1 (SCAMP1-GFP or GFPSCAMP1), an integral membrane protein with four TMDs, were
localized to both the TGN and PM via an ER-Golgi-TGN-PM
pathway (Lam et al., 2007b; Cai et al., 2011). Indeed, the original
TGN/PM localization of SCAMP1-GFP was altered by the addition of the RNIKCD motif to the C terminus of SCAMP1-GFP
because SCAMP1-GFP-RNIKCD was largely colocalized with
the Golgi marker Man1-mRFP (Figure 12A, panels 1 and 2) in
Arabidopsis protoplasts. A similar result was obtained when the
RNIKCD motif was fused to the C terminus of GFP-SCAMP1
because the resulting GFP-SCAMP1-RNIKCD fusion was also
found to be trapped in the Golgi, colocalizing with the Golgi
marker Man1-mRFP (Figure 12A, panel 3), indicating that the
RNIKCD motif showed similar Golgi retention function even
without direct fusion with GFP.
The Arabidopsis two-pore potassium channel (TPK1) protein
is a tonoplast-localized potassium channel with four TMDs and
a cytoplasmic N terminus and C terminus (Gobert et al., 2007;
Latz et al., 2007; Dunkel et al., 2008; Isayenkov et al., 2011). As
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Figure 8. EMP12-GFP Localized to the TGN, PVC, and Vacuole.
(A) EMP12-GFP construct with GFP fused at the C terminus of EMP12. CT, C terminus; NT, N terminus; T, TMD.
(B) to (D) Subcellular localization of EMP12-GFP upon its coexpression with known organelle markers (as indicated) in
Arabidopsis protoplasts.
(E) Overexpression of Sec12p was used to inhibit the ER export of GFP-EMP12 and Man1-mRFP in cells coexpressing
these two fusion proteins. Confocal images were collected from cells at 12 to14 h after transfection. Bars = 10 mm.
expected, TPK1-GFP showed a typical tonoplast localization
pattern that was distinct from the Golgi marker Man1-mRFP in
Arabidopsis protoplasts (Figure 12B). However, when RNIKCD
was added to its C terminus, the resulting TPK1-GFP- RNIKCD
was found to localize to both Golgi (colocalized with the Golgi
marker Man1-mRFP) and tonoplast (Figure 12B), indicating that
RNIKCD partially trapped the tonoplast-localized TPK1-GFP
within the Golgi. Such dual localization of TPK1-GFP-RNIKCD in
the Golgi and tonoplast may be caused by the competition
between the KXD/E Golgi retention signal and the tonoplast
targeting signals found in TPK1 (Dunkel et al., 2008; Isayenkov
et al., 2011). Thus, it will be interesting to investigate the combinational effect of these distinct targeting signals in determining
targeting of these proteins. Taken together, the C-terminal addition of RNIKCD caused Golgi retention of two post-Golgi
membrane proteins, SCAMP1-GFP and TPK1-GFP, which were
originally localized in the TGN/PM and tonoplast, respectively. In
addition, since most of these targeting studies were performed
using the Arabidopsis protoplast transient expression system, it
will be of interest to generate transgenic tobacco Bright Yellow2 or Arabidopsis cell lines and transgenic Arabidopsis plants
expressing some of the key fusion constructs (e.g., EMP12GFP-CT, SCAMP1-GFP-RNIKCD, and TPK1-GFP-RNIKCD) for
use in future studies.
DISCUSSION
The Position of the GFP Tag Affects the Proper Golgi
Localization of EMP12
Reporter fusion proteins have been a useful tool for studying
the targeting and subcellular localization of integral membrane proteins in plant cells. Because of the complex nature
of membrane protein topology, care must be taken when
Targeting and Subcellular Localization of EMP12
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Figure 9. The C Terminus of EMP12 Contained a Golgi Retention Signal.
(A) EMP12-GFP-CT mutation constructs and their subcellular localization in Arabidopsis protoplasts. CT, C terminus; NT, N terminus; T, TMD.
(B) to (E) Subcellular localization patterns of four selective EMP12-GFP-CT mutation fusions with or without Golgi retention signals at their CT upon their
coexpression with Golgi or PVC marker in Arabidopsis protoplasts. Confocal images were collected from cells at 12 to 14 h after transfection. Bars = 10 mm.
a reporter (e.g., GFP) is fused to an integral membrane protein
to ensure that the fusion protein’s topology remains identical
and that the targeting signals are not affected. For example,
a SP-GFP was used to replace the N terminus of BP-80,
a type I integral membrane protein belonging to the VSR
family, to generate the GFP-BP-80 reporter that was colocalized with the endogenous VSRs to PVC/MVB (Tse et al.,
2004), demonstrating the correct targeting of GFP-BP-80 and
various GFP-VSR fusions in plant cells (Jiang and Rogers,
1998; Tse et al., 2004; Miao et al., 2006). In this study, we
adopted a similar approach and used SP-GFP to replace the
N terminus of EMP12, whereas the generated GFP-EMP12
fusion was shown to colocalize with the endogenous EMP12
proteins to the Golgi apparatus in transgenic Arabidopsis
plants and protoplasts (Figures 2 to 4). Similar Golgi localization
was shown for GFP fusions to three other EMPs (EMP2, EMP3,
and EMP8) in Arabidopsis protoplasts (see Supplemental Figure
8 online). Such Golgi localization of EMPs in Arabidopsis is
consistent with previous results obtained from an organelle
proteome study using Arabidopsis culture cells (Dunkley et al.,
2006). By contrast, N-terminal GFP fusion with the human EMP
protein (i.e., GFP-TM9SF4) was shown to localize to early endosomes in mammalian cells (Lozupone et al., 2009). Such
variation in EMP localization (i.e., Golgi versus endosome localization in plant versus mammalian cells) suggests that EMPs
evolved distinct functions in different organisms.
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Figure 10. Immunoblot Analysis of Protoplasts Expressing the Four EMP12-GFP Fusions with the Golgi Retention Motif or Its Mutation.
(A) Arabidopsis protoplasts expressing the four EMP12-GFP fusions (as described in Figures 7B to 7E) were subjected to protein isolation into CS and
CM fractions, followed by protein separation via SDS-PAGE and protein gel blot analysis using GFP antibody. Arrowhead and arrow indicated the fulllength EMP12-GFP fusion and its degradative GFP core, respectively. M, molecular weight marker in kilodaltons.
(B) Coomassie blue–stained gel was used as control for showing equal protein loading.
(C) The relative grayscale intensity (scale 0 to 1.0) of the two protein bands in the CM (full-length fusion) and in the CS (GFP core) fractions of individual
EMP12-GFP fusions was quantified using ImageJ software, in which the combinational intensity of CM plus CS was expressed as 1.0 (or 100%).
Interestingly, C-terminal GFP fusion to EMP12 caused mistargeting of EMP12-GFP to post-Golgi compartments and, ultimately, degradation in vacuoles (Figures 8 and 13); a trafficking
pathway previously demonstrated to be mediated by PVCs (Tse
et al., 2004; Miao et al., 2008). Indeed, EMP12-GFP was found
to internalize into the ARA7(Q69L)-induced enlarged PVCs
(Kotzer et al., 2004) prior to reaching the vacuole. The mislocalization of EMP12-GFP is likely caused by masking of the
KCD Golgi retention signal within the short CT of EMP12 because C-terminal fusions of both GFP (Figure 8) and short triple
peptide sequences (AAA) (Figure 9) resulted in its mislocalization
to the post-Golgi compartment. Such mislocalization upon
C-terminal fusion was also true for other tested EMP members,
including EMP2, EMP3, and EMP8 (see Supplemental Figure 8
online), indicating a general targeting mechanism for the EMPs
in Arabidopsis plants. These results support the idea that the
vacuole rather than the PM is the default destination for missorted or misfolded integral membrane proteins in plant cells
(Höfte and Chrispeels, 1992). In addition, our study might explain the different subcellular localizations of two EMPs in yeast:
While the localization of Yer113c to the Golgi (Huh et al., 2003) is
consistent with that of plant EMPs, the localization of a C-terminal
GFP-tagged yeast EMP (TMN2-GFP) to the endosome and vacuole (Aguilar et al., 2010), rather than the Golgi, was likely caused
by the C-terminal GFP fusion masking the Golgi retention signal
as described in this study. It would thus be interesting to find out if
GFP-TMN2 would localize to the Golgi apparatus in yeast in
future experiments.
Multiple Sorting Signals and Proper COPII Vesicle Function
Are Involved in ER Export of EMP12
In the secretory pathway of eukaryotic cells, soluble and membrane cargo proteins are packed into ER-derived COPII vesicles
for Golgi and post-Golgi transport. The COPII coat consists of
Figure 11. The Cytoplasmic C Terminus of EMP12 Binds to COPI
Subunit in a Sequence-Specific Manner.
Soluble proteins extracted from Arabidopsis protoplasts expressing
Sec21-Myc were incubated with Sepharose beads conjugated with
synthetic peptides corresponding to the wild-type (WT) C terminus of
EMP12 (lane 3) or its various mutations (lanes 4 to 6; K587A/D589A,
K587A, and D589A) for a pull-down assay, followed by immunoblot
detection of bound proteins using anti-Myc antibodies. Blank Sepharose
beads without peptide conjugation were used as a control. M, molecular
weight marker in kilodaltons.
Figure 12. The Golgi Retention Motif of EMP12, RNIKCD, Retained Post-Golgi Membrane Proteins within the Golgi.
(A) C-terminal fusion of RNIKCD to SCAMP1-GFP or GFP-SCAMP1 caused their relocalization from their original PM/TGN (panel 1) to the Golgi
apparatus (panels 2 and 3). Bars = 10 mm.
(B) C-terminal fusion of RNIKCD to the tonoplast-localized TPK1-GFP (panel 4) partially trapped TPK1-GFP within the Golgi (panel 5). Bars = 10 mm.
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Figure 13. Working Model of EMP12 Trafficking in Plant Cells.
(A) Structure and topology of EMP12, GFP-EMP12, and EMP12-GFP fusions. Both EMP12 and GFP-EMP12 reached Golgi via ER. The ER export
motifs FV/Y of EMP12 interacted with the COPII subunit for its transport via COPII vesicles to the Golgi apparatus for residency because of the presence
of the Golgi retention motif KXD and its interaction with the COPI subunit.
(B) Pathways and signals. EMP12-GFP reached the Golgi via the ER. Since its Golgi retention motif KXD was masked by C-terminal GFP fusion,
EMP12-GFP reached the vacuole via the TGN and PVC.
[See online article for color version of this figure.]
Sar1, Sec23-Sec24, and Sec13-Sec31 complexes that are sequentially recruited to the ER membrane (Barlowe, 2003).
Transmembrane cargo proteins usually contain ER export signals in their cytoplasmic region that interact with the COPII coat
complex. Typical ER export signals identified in membrane
cargo proteins in yeast, mammalian, and plant cells include the
diacidic motif (DXE), dihydrophobic motif (LL), diaromatic motif
(FF, YY), dibasic motif (RR, RK), and Tyr-based motif YXXF
(where X is any amino acid, and F is a bulky hydrophobic group)
(Sato and Nakano, 2002; Barlowe, 2003; Hanton et al., 2005).
In this study, using both loss-of-function and gain-of-function
approaches in transiently transformed Arabidopsis protoplasts,
we also identified the dihydrophobic and Tyr-based motif, FV/Y,
at the short cytosolic C terminus of EMP12 to be an ER export
signal for EMPs in plants. FV and Y in EMP12 may function
synergically as the ER export signals to facilitate its ER export
and Golgi localization, as triple mutations of FV/Y resulted in
complete ER localization, whereas double mutations of FV or
a single mutation of Y resulted in dual ER and Golgi localization.
Furthermore, an in vitro peptide binding assay demonstrated
that triple mutations of FV/Y in the C terminus of EMP12 caused
a dramatic reduction in the original interaction between the
COPII coat protein Sec24 and EMP12 CT. Since the FV/Y motif
is highly conserved in the C terminus of all plant EMPs, including
Arabidopsis, rice, and maize, this motif may represent a general
mechanism that regulates the ER export of EMPs in plants. In
addition, when all EMP12 fusions and their mutants were expressed alone, they showed identical patterns as those coexpressed with other markers (see Supplemental Figure 9 online),
thus verifying that coexpression with the organelle markers did
not affect the localization of EMP12 fusions and its mutants in
this study.
The KXD/E Motif and COPI Vesicle Mediated Golgi
Localization of EMP12
In eukaryotic cells, the best-known signal-mediated Golgi retention mechanism for membrane proteins is related to COPI
function. In yeast, the Golgi-localized glycosyltransferases contain the semiconserved motif (F/L)-(L/V)-(S/T) that directly interacts with the Golgi resident protein Vps74p (Schmitz et al., 2008;
Tu et al., 2008). Vps74 was shown to interact with multiple
subunits of the COPI coat, and Vps74 thus likely served as an
adapter in mediating cargo packaging into the retrograde COPI
Targeting and Subcellular Localization of EMP12
vesicles for retrograde transport in maintaining the steady state
Golgi localization of glycosyltransferases (Tu et al., 2008). Similarly, the localization of Emp46p family proteins to the Golgi in
yeast was achieved via direct interaction of their dilysine signals
with the COPI coat (Sato and Nakano, 2002). Indeed, the
dilysine motifs (KKXX or KXKXX), which are usually located in
the cytoplasmic C terminus of membrane proteins, have been
shown to bind with the COPI subunit, especially the g-subunit of
coatomer, in yeast, mammalian, and plant cells (Cosson and
Letourneur, 1994; Harter and Wieland, 1998; Contreras et al.,
2004a).
In this study, we demonstrated that the KXD/E motif, which
occurs within the last six amino acids of the C terminus of
EMP12, functioned as a Golgi retention signal that regulates the
Golgi localization of EMP12 via its interaction with the COPI
vesicle. This conclusion is supported by several lines of experimental evidence. First, mutation of the KCD residues caused
mistargeting of EMP12. Second, blockage of this cytosolic
signal by C-terminal GFP or triple peptide AAA fusion resulted in
mislocalization of the fusion protein. Third, this motif interacted
specifically with the COPI subunit. Last, when attached to other
post-Golgi membrane proteins, the motif functioned as a Golgi
retention signal that retained these proteins largely within the
Golgi. Similar to the ER export signal, this KXD/E Golgi retention
motif is also highly conserved within the cytosolic C terminus of
all plant EMPs, indicating a general mechanism of Golgi retention for EMPs in plants. Thus, this KXD/E motif represents
a newly identified Golgi retention signal for multiple TMD proteins in eukaryotes.
Interestingly, although the KXD/E motif in EMPs is different
from the dilysine motif KKXX or KXKXX identified in single TMD
proteins, the Lys in the –3 position of these motifs is highly
conserved, indicating the COPI binding ability of the KXD/E
motif because the Lys in the –3 position of KKXX or KXKXX has
been shown to play a more vital role in COPI binding (Harter and
Wieland, 1998; Hardt and Bause, 2002). Indeed, results from the
in vitro peptide binding assay and confocal study demonstrated
that the Lys in the –3 position of the KXD/E motif played a more
critical role in COPI binding and Golgi retention of EMP12,
whereas the last acidic residue played a minor role, indicating
that the Lys-based motif can also function in Golgi retention of
multiple TMD proteins in plant cells.
Both p24 (Contreras et al., 2004b) and EMP (this study) are
shown to interact with the COPI vesicle. However, p24 is recycled back to the ER (Langhans et al., 2008), whereas EMP is
trapped in the Golgi, as demonstrated by both confocal immunofluorescence and immunogold EM in this study, in which both
endogenous EMP12 and the GFP-EMP12 fusion were found to
localize mainly to the medial- and cis-Golgi cisternae. A previous
EM tomography study (Donohoe et al., 2007) suggests the existence of distinct COPI populations in plant cells: The COPIa
vesicles that bud exclusively from cis-Golgi cisternae may mediate the recycling transport to the ER, whereas the COPIb
vesicles that bud exclusively from medial- and trans-Golgi cisternae may mediate the retrograde transport of Golgi resident
proteins (Donohoe et al., 2007). Since EMP12 proteins show
mainly medial- and cis-Golgi localization in this study, we are not
sure if EMP12 would achieve its Golgi localization by interacting
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with COPIb vesicles in Arabidopsis cells. It would therefore be
interesting to test the possible distinct functions of KKXX and
KXD/E motifs in Golgi-ER recycling (for p24) or Golgi retention (for
EMP12) by swapping the two COPI interaction motifs of p24 and
EMP for subsequent dynamics and subcellular localization studies.
METHODS
Plasmid Construction
The full-length cDNA of EMP12 was amplified and cloned into the pBI121
backbone for construction of the GFP fusion and generation of transgenic
plants using floral dip (Clough and Bent, 1998). All EMP12 mutant constructs
used for transient expression in protoplasts were PCR amplified and cloned
into the pBI221 backbone containing the cauliflower mosaic virus 35S
promoter, GFP coding sequence, and the nopaline synthase terminator
(Miao et al., 2008). The SP sequence for making the SP-GFP-EMP12–linked
constructs was derived from barley (Hordeum vulgare) proaleurain (Jiang
and Rogers, 1998) or from the native signal sequence of EMP12 based on
the SignalP 3.0 Server prediction (http://www.cbs.dtu.dk/services/SignalP/).
The protein topology was predicted using TMHMM Server 2.0 (http://www.
cbs.dtu.dk/services/TMHMM/). The primers used to generate various
EMP12 constructs are listed in Supplemental Table 1 online. All constructs
were confirmed by restriction mapping and DNA sequencing.
Plant Materials and Transient Expression in Protoplasts
Procedures for generating transgenic Arabidopsis thaliana plants expressing XFP fusion proteins and maintaining Arabidopsis suspension
cultured cells were described previously (Clough and Bent, 1998; Jiang
and Rogers, 1998; Tse et al., 2004; H. Wang et al., 2010; J. Wang et al.,
2010). Transient expression using protoplasts derived from Arabidopsis suspension culture cells was essentially performed according to our established
protocol (Miao and Jiang, 2007; Lam et al., 2009; Wang and Jiang, 2011).
Immunofluorescence Study and Confocal Microscopy
Fixation and preparation of Arabidopsis roots for immunofluorescent
labeling and confocal analysis were performed as described (Sauer et al.,
2006). Fixed roots were incubated with anti-EMP12 antibody at 4 mg/mL
overnight at 4°C for immunolabeling and were then probed with Alexa 568
goat anti-rabbit IgG (Invitrogen) secondary antibody for confocal observation. Expression of the fluorescent fusion proteins and observation of their
subcellular localization in protoplasts were usually performed at 12 to 14 h
after transient expression. For each experiment or construct, more than 50
individual cells were observed for confocal imaging that represented >80%
of the cells showing similar expression levels and patterns. Images collected
from 10 individual cells were used for the data process and analysis. All
confocal images were captured using the Olympus FV1000 laser scanning
confocal system (http://www.olympusconfocal.com/). The GFP signal was
visualized with excitation at 488 nm and emission at 500 to 550 nm (using
a band-pass filter). mRFP or Alexa 568 signals were visualized in another
detection channel with excitation at 559 nm and emission at 570 to 630 nm
(band-pass filter). The line sequential scanning mode was always selected
in dual-channel observations to avoid possible crosstalk between two
fluorophores. All images were prepared using Adobe Photoshop as described previously (Jiang and Rogers, 1998; Tse et al., 2004; H. Wang et al.,
2010; J. Wang et al., 2010).
EM Study
The general procedures for transmission EM sample preparation, thin
sectioning, and immunogold labeling were performed essentially as
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described previously (Tse et al., 2004; Lam et al., 2007b; Miao et al., 2011;
Shen et al., 2011; Wang et al., 2011; Wang et al., 2012). Root tips of 7d-old wild-type and transgenic GFP-EMP12 Arabidopsis plants were cut
and immediately frozen in a high-pressure freezer (EM PACT2; Leica),
followed by subsequent freeze substitution in dry acetone containing
0.1% uranyl acetate at –85°C in an AFS freeze substitution unit (Leica).
Infiltration with HM20, embedding, and UV polymerization were performed stepwise at –35°C. Immunogold labeling was performed with GFP
or EMP12 antibodies at 40 mg/mL and gold-coupled secondary antibody
at a 1:50 dilution. Transmission EM examination was done with a Hitachi
H-7650 transmission electron microscope with a charge-coupled device
camera (Hitachi High-Technologies) operating at 80 kV.
Antibodies, Protein Preparation, and Immunoblot Analysis
Synthetic peptide (GeneScript), CLRNDYAKYAREDDD, corresponding to
the cytosolic region between the TMD1 and TMD2 of EMP12 was conjugated with keyhole limpet hemocyanin and used to immunize rabbits at
the Laboratory Animal Services Center of the Chinese University of Hong
Kong. Antibodies were affinity purified using cyanogen bromide-activated
Sepharose 4B column (Sigma-Aldrich) conjugated with the peptides. Myc
antibody was purchased from Santa Cruz. Alexa 568 goat anti-rabbit IgG
was purchased from Invitrogen.
For the subcellular fractionation study, microsomal membrane fraction
was first isolated from the wild-type Arabidopsis suspension culture cells
via fractionation on a 25 to 50% continuous Suc gradient according to the
published procedure (Tse et al., 2004). These fractions were then probed
with anti-VSR, anti-Man1, and anti-EMP12 antibodies. To prepare cell
extracts from protoplasts, transformed protoplasts were first diluted
threefold with 250 mM NaCl and then harvested by centrifugation at 100g
for 10 min, followed by resuspension in lysis buffer containing 25 mM TrisHCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulphonyl
fluoride, and 25 mg/mL leupeptin. The protoplasts were further lysed by
passing through a 1-mL syringe with needle and then spun at 600g for
3 min to remove intact cells and large cellular debris. The supernatant total
cell extracts were then centrifuged at 100,000g for 30 min at 4°C; the
supernatant and pellet were assigned as soluble and membrane fractions,
respectively. Proteins were separated by SDS-PAGE and analyzed by
immunoblotting. Quantification of the relative gray-scale intensity in
immunoblot analysis was done with ImageJ software v1.45 according to
the procedure (http://lukemiller.org/index.php/2010/11/analyzing-gelsand-western-blots-with-image-j/).
Sample preparation and MS were performed as previously described
(Wu et al., 2009). Briefly, the gel was silver stained after SDS-PAGE separation, and the interesting protein bands were cut out for in-gel trypsin
digestion. The peptides were extracted from the trypsin-digested gel for MS
analysis with a matrix-assisted laser desorption ionization-time of flight/time
of flight tandem mass spectrometer ABI 4700 proteomics analyzer (Applied
Biosystems). Mass data acquisitions were piloted by 4000 Series Explorer
Software v3.0. The MS and MS/MS data were loaded into the GPS Explorer
software v3.5 (Applied Biosystems) and searched against the National
Center for Biotechnology Information nonredundant database (released on
March 5, 2010) with species restriction to Arabidopsis by Mascot search
engine v2.1 (Matrix Science).
Topology Analysis and Protease Protection Assay
Microsome isolation and protein topology analysis were performed as
described with some modifications (Abas and Luschnig, 2010; Cai et al.,
2011). Basically, the protoplasts expressing GFP-EMP12 or GFP-VSR2
were diluted with 250 mM NaCl and harvested by centrifugation. The
harvested protoplasts were then lysed in the extraction buffer (40 mM
HEPES-KOH, pH 7.5, 1 mM EDTA, 10 mM KCl, and 0.4 M Suc) by passing
through a 1-mL syringe with a needle several times. After centrifugation at
600g for 3 min to remove intact cells and large cellular debris, the supernatants were further centrifuged at 100,000g for 30 min at 4°C. The
pellet was assigned as the microsomal-enriched membranes and was
resuspended in the extraction buffer to be divided into three parts with
equal volume for protease protection assay with three distinct conditions:
without trypsin, 100 mg/mL trypsin and 100 mg/mL trypsin with 1% Triton
X-100. After incubation at 37°C for 30 min, the samples were pelleted for
protein extraction and immunoblot analysis using GFP antibody.
Accession Numbers
The Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this article are EMP2 (At5g25100), EMP3 (At2g24170), EMP8
(At1g14670), EMP12 (At1g10950), Sec21 (At4g34450), and Sec24
(At3g07100).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Plant EMP Proteins.
In Vitro Binding Assay of COPI and COPII Coat Proteins to
Sorting Motifs
In vitro pulldown and peptide binding assay were performed according to
previously published procedures (Contreras et al., 2004b; Suen et al., 2010).
Synthetic peptides (GeneScript) corresponding to the C-terminal cytosolic
tail of EMP12 were coupled, via their N-terminal NH2 group, to cyanogen
bromide-activated Sepharose 4B (3 mg peptides per mL of beads) according to the standard procedure. The coupling reaction was quenched by
incubation with 1 M Gly, pH 8.0, at room temperature for 2 h. Peptide
coupling efficiency was monitored by measuring the absorbance at 280 nm.
For the peptide binding assay, the soluble fractions, which were
extracted from the wild-type protoplasts, the protoplasts transfected with
the COPI coat-Myc fusion (Sec21-Myc), or the COPII coat-GFP fusion
(Sec24-GFP), were diluted with 23 lysis buffer (50 mM Tris-HCl, pH 7.5,
300 mM NaCl, 0.5% Triton X-100, 2 mM phenylmethylsulphonyl fluoride,
and 50 mg/mL leupeptin) and were then incubated with Sepharose beads
coupled with the peptides for 4 h at 4°C in a top to end rotator. After
incubation, the beads were washed five times with 13 lysis buffer and
then eluted by boiling in reducing SDS sample buffer. Proteins were
separated by SDS-PAGE and subjected to liquid chromatography-MS/
MS analysis or immunoblotting using appropriate antibodies.
Supplemental Figure 2. The C Terminus of EMP12 Was Essential for
ER Export.
Supplemental Figure 3. Subcellular Localization of GFP-EMP12 with
Triple Mutations at Its C Terminus.
Supplemental Figure 4. Subcellular Localization of EMP12-GFP with
the Golgi Retention Motif Fused to the C-Terminal End.
Supplemental Figure 5. Subcellular Localization of EMP12-GFP with
C-Terminal Fusion of Mutated Golgi Retention Motif.
Supplemental Figure 6. EMP12-GFP Reached the Vacuole via the
PVC for Degradation.
Supplemental Figure 7. EMP12 C Terminus Interacted with COPI
Subunits.
Supplemental Figure 8. Subcellular Localization of N-Terminal or
C-Terminal GFP Fusions with EMP2, EMP3, and EMP8 in Arabidopsis
Protoplasts.
Supplemental Figure 9. Localization Patterns of Singly Expressed
Various EMP12 Fusions and Their Mutants in Arabidopsis Protoplasts.
Supplemental Table 1. Primers Used in This Study.
Targeting and Subcellular Localization of EMP12
ACKNOWLEDGMENTS
We thank Akihiko Nakano and Takashi Ueda (University of Tokyo, Japan)
for providing the cDNA encoding ARA7(Q69L). This work was supported
by grants from the Research Grants Council of Hong Kong (CUHK466309,
CUHK466610, CUHK466011, CUHK2/CRF/11G, and HKBU1/CRF/10)
and Chinese University of Hong Kong Schemes A/B/C to L.J.
AUTHOR CONTRIBUTIONS
L.J. supervised the project. C.G. and L.J. designed the experiments.
C.G., C.K.Y.Y., S.Q., M.W.Y.S., K.Y.L., and S.W.L. performed specific
experiments and analyzed data. C.G. wrote the article draft, and L.J.
revised the article.
Received January 19, 2012; revised March 12, 2012; accepted April 18,
2012; published May 8, 2012.
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The Golgi-Localized Arabidopsis Endomembrane Protein12 Contains Both Endoplasmic
Reticulum Export and Golgi Retention Signals at Its C Terminus
Caiji Gao, Christine K.Y. Yu, Song Qu, Melody Wan Yan San, Kwun Yee Li, Sze Wan Lo and Liwen
Jiang
Plant Cell; originally published online May 8, 2012;
DOI 10.1105/tpc.112.096057
This information is current as of June 18, 2017
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