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RESEARCH ARTICLE

Arabidopsis ENDOMEMBRANE PROTEIN 12 contributes to the endoplasmic reticulum stress response by regulating K/HDEL receptor traffickingKing Pong Leunga,1, Ming Luoa,b,1,2, Caiji Gaoa,c, Yonglun Zenga, Qiong Zhaoa, Mee-Len Chyed, Xiaoqiang Yaoe, Liwen Jianga,f,2 aCentre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China bGuangdong Provincial Key Laboratory of Applied Botany, Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China cGuangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China dSchool of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China eSchool of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China fCUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen 518057, China 1These authors contributed equally to this work. 2Address correspondence to: Liwen Jiang (Email: ljiang@cuhk.edu.hk) and Ming Luo (Email: luoming852@hotmail.com).

Short Title: EMP12 maintains ERD2a in the Golgi

One-sentence Summary: EMP12 maintains the K/HDEL receptor ERD2a at the Golgi apparatus and thereby prevents its premature retrograde transport.

The authors 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).

ABSTRACT ENDOMEMBRANE PROTEIN 70 (EMP70) proteins constitute a 12-member superfamily in Arabidopsis thaliana, and are the most abundant protein species in plant Golgi proteomes. However, the physiological functions of EMPs in plants remain largely unknown. Here we have demonstrated that two AtEMP12 T-DNA insertion mutants are sensitive to ER (endoplasmic reticulum) stress as induced by tunicamycin and dithiothreitol treatments. The unfolded protein response (UPR) is constitutively activated in the knockout mutant emp12-1 under normal growth conditions, suggesting that the activation is a result of insufficient chaperones in the ER to aid protein folding. Indeed, we have further shown that BiP is secreted into the apoplast in emp12-1, while the K/HDEL receptor ERD2a (ER lumen protein-retaining receptor A), which regulates BiP trafficking, is exclusively localized in the ER in emp12-1, instead of its normal ER-Golgi dual-localization. Given the enhanced retrograde transport of ERD2a, along with the reduction in dimerized receptor formed in the absence of EMP12, ERD2a may be prematurely returned to the ER without its bound ligands. Therefore, we propose that EMP12 may act as a novel regulator of the K/HDEL receptor that ensures an effective retrograde transport of K/HDEL ligands.

Plant Cell Advance Publication. Published on May 23, 2019, doi:10.1105/tpc.18.00913

©2019 American Society of Plant Biologists. All Rights Reserved

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INTRODUCTION

The endomembrane system of eukaryotic cells contains a variety of membrane-bound compartments, where

biomolecules are exchanged between these organelles via transport intermediates. Secretory proteins, for

example, are first translocated into the endoplasmic reticulum (ER) and then trafficked to the Golgi apparatus

for further modifications. Subsequently they are sorted to post-Golgi compartments including the trans-Golgi

network (TGN) or to the extracellular space such as the apoplast (Barlowe et al., 1994). Traffic between the

ER and the Golgi apparatus is mediated by two vesicular carriers with coat complexes, coat protein complex

II (COPII) (Andreeva et al., 2000; Phillipson et al., 2001; Ritzenthaler et al., 2002; daSilva et al., 2004; Yang

et al., 2005) and COPI (Cosson and Letourneur, 1997; Kuehn et al., 1998; Movafeghi et al., 1999; Takeuchi

et al., 2002; Stefano et al., 2006; Di Sansebastiano et al., 2007). Transport mediated by both COPII and COPI

vesicles are energy-dependent processes (Goldberg, 2000; Brandizzi et al., 2002); thus, mechanisms have been

developed that sort proteins effectively to their destinations. For example, specific sorting signals on the cargo

proteins dictate their packaging into COPII vesicles for anterograde transport from the ER to the Golgi, or into

COPI vesicles for retrograde transport from the Golgi to the ER (Barlowe, 2003; Beck et al., 2009). In addition,

the plant COPII machinery may have unique features distinct from those in yeast and mammals (Zeng et al.,

2015; Chung et al., 2016).

Endomembrane Proteins (EMPs), also known as Nonaspanins or Transmembrane Nine Superfamily (TM9SF)

proteins, are a group of superfamily proteins possessing an EMP70 domain (Pfam accession number PF02990).

EMPs contain nine transmembrane domains (TMDs), with a long luminal N-terminus and a signal peptide,

and a short cytosolic C-terminus (Gao et al., 2012). This superfamily is highly conserved among eukaryotes

and can be classified into 5 groups phylogenetically (Supplemental Figure 1; Supplemental Data Set 1).

While there are 3–4 homologues in lower eukaryotes such as the slime mold (Dictyostelium discoideum;

DdPhg1A – DdPhg1C) and yeast (Saccharomyces cerevisiae; ScTMN1 – ScTMN3), as well as in higher

eukaryotes such as the fruit fly (Drosophila melanogaster; DmTM9SF2 – DmTM9SF4) and humans

(HsTM9SF1 – HsTM9SF4), the EMP superfamily is expanded in plants, where 12 and 17 members were

found in Arabidopsis thaliana (termed EMP1 – EMP12 in this study) and rice (Oryza sativa), respectively.

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We previously demonstrated that Arabidopsis EMP12 (AtEMP12) is a cis-Golgi-localized protein containing

both ER export and Golgi retention signals in the cytosolic C-terminus (Gao et al., 2012; Gao et al., 2014).

This novel Golgi retention signal, the KXD/E (Lys-Xaa-Asp/Glu) motif, was shown to interact with the COPI

vesiculation machinery and thereby maintain the cis-Golgi localization of EMPs in Arabidopsis, yeast and

mammalian cells (Woo et al., 2015). In addition, the EMP protein family is the most abundant species in plant

Golgi proteomes (Nikolovski et al., 2014; Ford et al., 2016). Despite knowing its trafficking mechanism, the

function of EMPs in plants remains unknown.

In the secretory pathway of plant cells, the folding of proteins is prone to various stresses, which could lead

to an accumulation of misfolded proteins in the ER and induce ER stress. The unfolded protein response (UPR)

is activated as a protective mechanism in response to stress by increasing the level of protein-folding

chaperones. One of the UPR pathways in plants includes bZIP28 (Basic-leucine Zipper Transcription Factor

28), a membrane-bound transcription factor (Liu and Howell, 2010). The inactive, membrane-bound form of

bZIP28 normally resides in the ER due to binding to the protein-folding chaperone BiP (Srivastava et al.,

2013). Under ER stress, BiP dissociates from bZIP28 and aids protein folding, thus bZIP28 is transported to

the Golgi apparatus via COPII vesicles and cleaved by site-1 and site-2 proteases at the cytoplasmic side to

release the transcription factor (Liu et al., 2007; Sun et al., 2015; Iwata et al., 2017). bZIP28 then migrates

into the nucleus and triggers UPR-specific transcription.

In this study, we characterized EMP12-deficient mutant Arabidopsis plants and found that EMP12 functions

in the ER stress response. We provided evidence that loss of EMP12 leads to a leakage of BiP from the ER.

This subsequently caused a mild UPR activation, since there is less BiP present to aid protein folding. We

propose that EMP12 maintains the Golgi localization of the K/HDEL receptor ERD2 and thereby prevents its

premature retrograde transport without capturing K/HDEL ligands, such as BiP, thus guaranteeing an effective

ligand retrieval and ER residence maintenance.

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RESULTS

emp12 mutant lines exhibit an ER stress sensitive phenotype

We isolated two T-DNA insertion mutants for EMP12 from the GABI-Kat collections (Kleinboelting et al.,

2012) obtained from the European Arabidopsis Stock Centre. The T-DNA insertions in emp12-1 (GK_284G01)

and emp12-2 (GK_437A12) were confirmed by Sanger sequencing with chromatograms shown in

Supplemental Figure 2, and the insertion sites of emp12-1 and emp12-2 are at the 1st intron and 5' untranslated

region respectively (Figure 1A).

To examine whether the expression of EMP12 is altered in emp12-1 and emp12-2 homozygous mutant lines,

we characterized the protein expression levels using EMP12-specific antibodies. Using Col-0 as the wild-type

(WT) control, we found that there is no expression in emp12-1, whereas there is about a 70% reduction in

emp12-2 (Figure 1B and 1C). Therefore, emp12-1 and emp12-2 are knockout and knockdown mutants,

respectively.

When grown under standard conditions, both mutant lines grew similar to the WT (Figure 1D). Since the

mutant plants exhibited no abnormal growth defects under normal conditions, we hypothesized that EMP12

should function under certain stresses. We discovered that when subjected to dithiothreitol (DTT) and

tunicamycin (TM) treatments, which induce unfolded protein accumulation in the endoplasmic reticulum (ER)

and hence ER stress, emp12 mutants showed hypersensitivity to these two drugs when compared to WT plants.

As shown in Figure 1D, while WT seedlings still exhibited root growth when grown on Murashige and Skoog

(MS) agar supplemented with 1 mM DTT for 7 days, there was no root growth in both emp12-1 and emp12-

2. Smaller and paler leaves were observed in emp12-1 as compared to emp12-2 and WT seedlings. Under a

50 ng/ml TM treatment for 7 days, WT seedlings showed stunted growth with green cotyledons. Both emp12-

1 and emp12-2 seedlings, on the other hand, have brownish cotyledons and shorter root lengths as compared

to WT.

To examine whether EMP12 can rescue the emp12 mutant phenotype, we transformed emp12-1 with EMP12

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expressed under the UBQ10 promoter. Seeds from WT, emp12-1, and three individual complementation plants

(C1 – C3) were germinated on MS agar with or without 50 ng/ml TM (Supplemental Figure 3). There is no

observable difference in growth phenotypes among WT, the emp12-1 mutant and the complementation lines

grown on MS agar plates. Under TM treatment, WT and the complementation lines showed a similar growth

phenotype, while emp12-1 plants exhibited a hypersensitive phenotype towards ER stress.

We measured several physiological parameters to further characterize the effect of ER stress on WT and emp12

mutants. Fresh weights of both emp12-1 and emp12-2 mutants were 69% lower with DTT treatment and 76%

lower with TM treatment, respectively, in comparison to 50% lower with DTT treatment and 60% lower with

TM treatment in WT plants (Figure 1E). DTT inhibited root growth of WT plants, but the inhibition was much

more severe in emp mutants, which showed little growth (Figure 1F). Tunicamycin also inhibited root growth

of WT plants, but the inhibition was much more severe in emp mutants (50% and 40% reduction in growth

relative to the untreated plants, respectively). It appears that there is a discrepancy between the effect of the

two drugs in root length and fresh weight. It is known that DTT inhibits root growth but not shoot growth

(Hossain et al., 2016), while TM affects the growth of the whole seedling (Nawkar et al., 2017). Therefore,

the fresh weight of TM-treated seedlings would be affected more substantially than that of seedlings treated

with DTT. Chlorophyll a and b contents of emp12 mutants showed no significant difference when compared

to WT plants under normal conditions (Figure 1G). Chlorophyll contents were significantly lower in emp12

mutants under DTT and TM treatments, with emp12-1 plants containing less chlorophyll than emp12-2 plants.

A measurement of ion leakage, which indicates cell death, showed a higher ion leakage in emp12 mutants than

in WT plants after TM treatment (Figure 1H). emp12-1 plants also produced a higher ion leakage than emp12-

2 plants under stress. All these results suggest that a reduction or absence of EMP12 causes the plants to

become more prone to ER stress.

Loss of EMP12 led to a constitutive UPR activation

Since both emp12-1 and emp12-2 showed similar hypersensitivity towards ER stress, we next focused on

characterizing the knockout mutant emp12-1. In response to ER stress due to the accumulation of unfolded

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proteins, the UPR, which increases the transcription of chaperones such as BiP that aid protein folding in the

ER lumen, is triggered (Srivastava et al., 2013). Therefore, we investigated the expression of several UPR-

responsive genes such as BiP1/BiP2 (Luminal Binding Protein 1/2), PDIL1 (Protein Disulfide Isomerase-like

1), SHD (SHEPHERD) and CNX1 (Calnexin 1) in WT and emp12-1 with or without drug treatments using

quantitative PCR (qPCR). As shown in Figure 2A, expression of UPR markers in both WT and emp12-1 was

induced under both DTT and TM treatments. Interestingly, the UPR gene expression levels were higher in

emp12-1 plants than in WT plants grown under normal conditions. Such an increase in basal UPR marker gene

expressions in emp12-1 may suggest that UPR is activated constitutively in the absence of EMP12.

We then observed the subcellular localization of YFP-bZIP28 expressed in the roots of WT and emp12-1 plants

with confocal microscopy in order to determine if UPR is activated constitutively in emp12-1. In WT plants

expressing YFP-bZIP28, fluorescence was localized in the nucleus, along with a cytosolic and punctate pattern

observed when treated with 5 μg/ml TM for 6 h (Figure 2B), indicating an UPR activation. Yet, there was no

observable signal when the plants were mock-treated with DMSO. This observation is in agreement with a

previous report that full-length bZIP28 is unstable (Sun et al., 2013). Nevertheless, we attempted to detect the

possible initial ER localization of YFP-bZIP28 in WT plants under normal conditions by treating them with

proteasomal inhibitor MG132 together with TM, but we were not able to observe the initial ER pattern of

bZIP28. Since we do not have robust antibodies for bZIP28 as described previously (Iwata et al., 2017), we

also tried to use GFP antibodies to observe YFP-bZIP28 cleavage in mock or TM-treated WT or emp12-1

mutant lines, but we failed to clearly differentiate the cleavage of bZIP28. To sum up, there is no UPR

activation in the WT under normal conditions. On the other hand, as expected, YFP-bZIP28 showed a nuclear

localization pattern in emp12-1 with or without TM treatment, suggesting that UPR is indeed activated

constitutively in emp12-1 plants.

BiP, an ER chaperone aiding protein folding, is secreted into the apoplast

Although BiP was overexpressed in emp12-1 under drug treatments (Figure 2A), it seems that such an increase

in chaperone amount is insufficient to reverse the ER stress sensitive phenotype. This observation implies that

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BiP is not functioning normally as a chaperone in the ER. Therefore, we investigated the subcellular

localization of BiP, suspecting that BiP is not maintained in the ER at levels to support a sufficient folding

capacity. We observed that BiP was partially secreted into the apoplast in emp12-1, in contrast to WT plants.

Leaf protoplasts of WT and emp12-1 plants were isolated, whereas secGFP was also transiently expressed in

the protoplasts to serve as a secretion control. The culture medium with protein secreted after overnight

incubation was then collected for immunoblot analysis. As shown in Figure 3A, there is an increased

expression of BiP in emp12-1 relative to WT, which is consistent with the up-regulated BiP expression as

shown in Figure 2A. All 3 isoforms of BiP can be recognized by the BiP antibody (Supplemental Figure 4).

Such an increase was not due to an unequal loading of protein samples because an equal amount of cFBPase

was detected. In addition, when culturing media with secreted proteins from WT and emp12-1 leaf protoplasts

were assayed, BiP was present in emp12-1 samples, in contrast to a barely detectable signal from the WT

counterparts. CRT was also observed to be secreted in a similar fashion. A lack of cFBPase signal from both

samples indicates the lack of intracellular contaminants during the course of sample preparation.

We further verified the subcellular localization of BiP in WT and emp12-1 plants using confocal microscopy.

spRFP-BiP1 was transiently expressed in protoplasts of WT and emp12-1 with CNX-GFP (an ER marker) and

YFP-SYP32 (a cis-Golgi marker) (Supplemental Figure 5). spRFP-BiP1 did not colocalize with YFP-SYP32

and colocalized with CNX-GFP in both WT and emp12-1. To sum up, BiP was present in the ER in both WT

and emp12-1, but BiP is leaked to the apoplast in the absence of EMP12.

Steady-state localization of ERD2a is shifted to the ER in the absence of EMP12

Since BiP is an ER luminal protein, whose trafficking is governed by the K/HDEL receptor ERD2 (Montesinos

et al., 2014; Pastor-Cantizano et al., 2018), we wondered if its partial loss of ER residency might be due to a

change of ERD2 subcellular localization. Therefore, we examined the localization of ERD2a overexpressed

in WT and emp12-1 using confocal microscopy. ERD2a-GFP was largely localized in the ER in emp12-1, with

few punctae that are presumably Golgi (Figure 3B). By contrast, ERD2a-GFP in WT plants showed mainly a

punctate pattern with a minor ER reticular network signal. A similar localization pattern can be observed in

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roots, hypocotyls and leaves of both plants. We also confirmed that the expression level of ERD2a did not

change in the absence of EMP12, although there was a slight increase in the expression in both WT and emp12-

1 plants when under ER stress induction (Supplemental Figure 6).

To confirm that such localization difference of ERD2 between WT and emp12-1 mutant plants is not due to

the positional effect of T-DNA insertion, we obtained five individual transgenic lines expressing ERD2a-GFP

in WT and emp12-1 respectively. ERD2a-GFP is expressed in all lines as a single polypeptide at various

expression levels (Supplemental Figure 7A). We chose WT / ERD2a-GFP #1 and emp12-1 / ERD2a-GFP #1

for the following experiments because of their similar expression levels of ERD2a-GFP. Confocal microscopy

confirmed similar localization patterns of ERD2a-GFP in WT and emp12-1 in other transgenic lines

(Supplemental Figure 7B).

On the other hand, we verified the ER and Golgi localization of ERD2a-GFP in WT and emp12-1 plants by

transiently expressing CNX-RFP (an ER marker) and ManI-RFP (a cis-Golgi marker) via Agrobacterium

transformation (Figure 4A). To quantify the relative abundance of ERD2a in the ER versus the Golgi

apparatus in WT and emp12-1, we quantified the fluorescence intensity of ERD2a-GFP localized on the ER

and the Golgi (Figure 4A). As shown in Figure 4B, about 60 % of the total signal could be detected in the

Golgi of WT plants, while the remaining 40 % of the signal was detected in the ER. We observed that ~97 %

of ERD2a-GFP localized in the ER in the emp12-1 mutant, while only ~3 % was found in the Golgi, both

being significantly different from their WT counterparts. A co-localization test conducted by calculating

Manders’ co-localization coefficient showed that ERD2a-GFP co-localized well with both CNX-RFP and

ManI-RFP in WT plants, implying that ERD2a-GFP was dually localized in the ER and Golgi. As for emp12-

1 plants, only CNX-RFP co-localized well with ERD2a-GFP but not ManI-RFP, indicating that ERD2a-GFP

mainly localized in the ER (Figure 4C).

Given that overexpressing EMP12 could revert the ER stress sensitive phenotype in emp12-1, the Golgi

residence of ERD2a should be restored by EMP12 overexpression in emp12-1. We then transiently expressed

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EMP12 in WT/ERD2a-GFP #1 and emp12-1/ERD2a-GFP #1 via Agrobacterium transformation using ManI

as a negative control (Figure 4D). While the 40/60% ER/Golgi localization ratio of ERD2a was maintained

in WT/ERD2a-GFP #1 co-expressed with both ManI and EMP12 (Figure 4E), the 97/3% ER/Golgi ratio of

ERD2a in emp12-1/ERD2a-GFP #1 was reverted to 40/60% when co-expressed with EMP12 in comparison

to that with ManI (Figure 4F). EMP12 overexpression could indeed revert the Golgi localization of ERD2a

from an exclusive ER localization.

Enhanced retrograde transport of ERD2a with few dimer formations is observed in emp12-1

It was reported that ERD2a cycles between ER and Golgi to carry out its K/HDEL ligand retrieval function

through binding to both COPII and COPI vesicle subunits (Montesinos et al., 2014). Therefore, we examined

the affinity of ERD2a-GFP with COPII and COPI subunits Sec24 and Sec21 respectively via co-

immunoprecipitation (Co-IP) assays using a GFP-Trap. This affinity may reflect the ability of ERD2a-GFP to

be loaded onto COPII and COPI vesicles for anterograde (ER-Golgi) or retrograde (Golgi-ER) transport,

respectively. The amount of ERD2a-GFP loaded into these vesicles is independent of the existing ERD2a

localized in the ER or Golgi. There is no obvious difference in the binding of ERD2a-GFP with Sec24 between

WT and emp12-1 protein samples, implying that the amount of ERD2a-GFP exported from ER to Golgi was

similar in WT and emp12-1. However, more Sec21 was bound to ERD2a-GFP in the emp12-1 sample than in

the WT sample (Figure 5A). We also performed a reciprocal IP by capturing ERD2a-GFP using anti-Sec21,

where less ERD2a-GFP was pulled down by Sec21 in WT plants than in emp12-1 plants (Supplemental

Figure 8). Such a higher affinity of ERD2a towards COPI coatomer may indicate that more ERD2a was loaded

onto COPI vesicles, and hence there is an enhanced retrograde transport of ERD2a from Golgi to ER, thus

explaining the shift of ERD2a localization exclusively to the ER in emp12-1. It would be ideal to observe the

co-localization of Sec24 or Sec21 and ERD2a in WT and emp12-1 plants under confocal microscopy in order

to examine the anterograde/retrograde transport status of ERD2a via COPII and COPI vesicles, but this

experiment would be practically challenging because according to the current model of coatomer disassembly

(Sato and Nakano, 2007; Beck et al., 2009), coatomers tend to disintegrate soon after the formation of

COPII/COPI vesicles, rendering the co-localization between ERD2 and the coatomers on COPII/COPI

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vesicles very difficult to be observed.

We were also interested in knowing whether ERD2a could still transport its ligands in emp12-1, given that

there is an enhanced retrograde transport and exclusive ER localization. In mammalian cells, binding of

ligands would induce oligomerization of ERD2 receptor for retrograde transport via COPI vesicles (Majoul et

al., 2001). Therefore, we conducted a crosslinking experiment by adding 1 % paraformaldehyde as a

crosslinker when homogenizing WT and emp12-1 plants. It is expected that after cross-linking, ERD2a-GFP

oligomers would be stabilized, resulting in a band with higher molecular weight from its monomeric form. In

the WT sample, there was a prominent band sized ~100 kDa, presumably a dimer band, apart from the

monomeric band of ERD2a-GFP, which is consistent with our expectation (Figure 5B). Interestingly, the

dimer band was barely detectable in the emp12-1 sample. These observations were further supported by the

higher dimer-to-monomer ratio in the WT sample (0.341) when compared to the emp12-1 sample (0.018),

where a higher dimer-to-monomer ratio indicates more dimer formations. This result indicates that ERD2a

may not be dimerized by an induction of its ligands for retrieval without EMP12, at least not as efficient as

that in WT plants. We also attempted to use DTT/TM treatments to induce dimer formation but failed to detect

an obvious increase. To verify that the dimers formed are not protein aggregates, a crosslinking reversal was

conducted by heating the sample at 100 °C, which produces the monomeric band only.

EMP12 interacts with ERD2a and the interaction is disrupted by the presence of HDEL ligands

As both EMP12 and ERD2a are localized in the Golgi (Gao et al., 2012; Pastor-Cantizano et al., 2018), it is

possible that EMP12 directly interacts with ERD2a for regulation. We transiently co-expressed EMP12-CFP-

RNIKCD and ERD2a-YFP in Arabidopsis protoplasts. Confocal microscopy analysis confirmed that both

fusions co-localized in the Golgi (Figure 6A). The sequence RNIKCD was added after the CFP-fused EMP12

protein to maintain the correct Golgi localization (Gao et al., 2012). We then performed a fluorescence

resonance energy transfer-acceptor photobleaching (FRET-AB) assay to test for a potential protein–protein

interaction between EMP12-CFP-RNIKCD and ERD2a-YFP, using EMP12-CFP-RNIKCD and YFP-SYP32

as a negative control. The assay suggested that there is an in vivo interaction between EMP12-CFP-RNIKCD

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and ERD2a-YFP, as the FRET efficiency was significantly higher than the negative control (Figure 6B). A

Co-IP assay using a GFP-Trap was also performed to demonstrate an interaction between ERD2a-YFP and

spRFP-EMP12, where spRFP-EMP12 was pulled down by ERD2a-YFP, but not YFP-SYP32 (Figure 6C).

To show that there is no functional redundancy between EMP12 and other EMP members, we conducted a

FRET-AB assay to test possible interaction between ERD2a and EMP8 or EMP9 (Figure 6B). The results

suggested that there is no interaction between either pair of proteins, as there is no significant difference in

FRET efficiency when compared to the negative control. This notion is further supported by the observation

that an emp9 knockout mutant (Supplemental Figure 9A-B) did not show ER stress sensitive phenotype

under TM treatment (Supplemental Figure 9C).

To investigate if HDEL ligands could affect the interaction between EMP12 and ERD2a, we also conducted a

FRET-AB assay on EMP12-CFP-RNIKCD and ERD2a-YFP co-expressed with the HDEL ligand spRFP-BiP3,

using CNX-RFP as a K/HDEL receptor-independent control. Examples of triple co-expressed proteins for

FRET-AB assays are shown in Figure 6D, where both CNX-RFP and spRFP-BiP3 remained in the ER

reticular network pattern. The FRET efficiency dropped significantly when the EMP12-ERD2a FRET pair

was co-expressed with spRFP-BiP3, indicating that addition of excess HDEL ligands could disrupt the

interaction between EMP12 and ERD2a (Figure 6E). However, the interaction between EMP12-ERD2a

FRET pair remained unchanged when co-expressed with CNX-RFP as a negative control. Since CNX-RFP is

an ER protein without a HDEL signal, the result indicated that the observed effect is specific for HDEL ligands.

A co-IP assay using a GFP-Trap was also performed to demonstrate that the interaction between spGFP-

EMP12 and ERD2a-RFP was disrupted by co-expression of spRFP-BiP3, but not CNX-RFP (Figure 6F).

DISCUSSION

Diverging roles of EMPs among eukaryotes

Even though EMPs are evolutionarily conserved among eukaryotes, different phylogenetic groups of EMPs

seem to perform different biological functions. For example, a previous study revealed that a TM9SF4

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knockout mutant in fruit fly does not induce UPR (Perrin et al., 2015b). Yet, according to the phylogenetic

analysis of amino acid sequences of EMPs from different organisms as shown in Supplemental Figure 1,

EMP12 is a close relative of TM9SF3, where a loss of EMP12 causes ER stress hypersensitivity in Arabidopsis.

These conflicting observations may suggest that different EMP homologues could have non-overlapping and

unique biological functions, as suggested in previous reports that TM9SF2 and TM9SF4 in Drosophila may

play non-redundant roles in phagocytosis (Bergeret et al., 2008; Perrin et al., 2015a). The functional

specificities among EMP homologues might be due to the highly variable N-terminus sequences in different

EMP members. For example, the N-terminal sequences of AtEMP8 and AtEMP9 (which belong to the same

phylogenetic group) are 90 % identical (95% similar); however, the N-terminus of AtEMP8 and AtEMP12

(which belong to different phylogenetic groups) is only 34 % identical (53 % similar). This may explain the

lack of functional redundancy between AtEMP12 and the other two EMP members, AtEMP8 and AtEMP9, in

this study.

Another possible explanation for the involvement of EMP in functionally diverging processes is that EMPs

have been reported to localize to different organelles other than the Golgi. For example, TM9SF4 in human

metastatic melanoma cells was found to be localized in early endosomes (Lozupone et al., 2009) and appears

to co-localize with V-ATPase for its aberrant constitutive activation and result in a malignant phenotype

(Lozupone et al., 2015).

Reports on fruit fly and slime mold demonstrated that EMP proteins participate in regulating protein

trafficking. In Drosophila, TM9SF2 and TM9SF4 interact with a peptidoglycan recognition protein (PGRP)-

LC that triggers the immune response to bacterial infection (Perrin et al., 2015a). Deletion of TM9SF4 caused

a loss of plasma membrane localization of PGRP-LC. As for slime mold, plasma membrane localization of

SibA, a cell adhesion protein, is found to be regulated by Phg1A (Perrin et al., 2015b). Loss of Phg1A led to

its accumulation in the ER, while overexpressing the EMP homologue caused an enhanced surface localization.

Since we demonstrated that both EMP12 and ERD2a co-localized in the Golgi, where EMP12 may regulate

ERD2a by maintaining its Golgi localization for its ligand capturing and recycling activity, the present study

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adds another clue supporting that one of the functions of EMP is to regulate intracellular trafficking of certain

membrane proteins.

EMP12 and the unfolded protein response

It appears that the ER stress sensitivity due to the loss of EMP12 in emp12 mutants is caused by BiP leakage.

A working model is depicted in Figure 7. Under normal growth conditions, there is no observable growth

retardation in emp12-1 and emp12-2 mutants relative to WT plants. From the subcellular fractionation

experiment, we observed that although BiP has partially lost its ER residence, ~92 % of expressed BiP is still

localized in the ER, which provides a sufficient amount of chaperone in the ER to maintain a basal level of

folding activity. However, when presented with a higher protein folding demand, e.g. under DTT and TM

treatments, we postulate that the basal folding capacity can no longer cater for the surging demand due to a

constant loss of BiP chaperone to post-ER compartments. As the remaining BiP would bind to the unfolded

proteins in the ER, it then dissociates from bZIP28, leading to its nuclear translocation to activate UPR

responsive genes. BiP is then up-regulated. The IRE1/bZIP60 pathway should be activated accordingly as it

is proposed to adopt a similar mode of action by sensing BiP availability in the ER in plants (Ruberti and

Brandizzi, 2014; Ruberti et al., 2015). A positive feedback loop is set up: BiP expression is up-regulated in an

attempt to compensate for the loss, yet the leakage causes an insufficient amount of BiP to be retained in the

ER to perform a chaperoning function. This explanation aligns with our observations that YFP-bZIP28 in

emp12-1 produced a nuclear localization with cytosolic and punctate patterns under normal conditions, and

BiP was found to have partially lost its ER residence and became secreted into the apoplast. Therefore, UPR

is activated constitutively and it eventually leads to a hypersensitivity phenotype in emp12 mutants upon ER

stress induction. In future studies, additional experiments in which EMP12 or ERD2a is overexpressed in

emp12-1 mutants could be conducted to see whether the BiP secretion could be reversed.

It was reported that plants overexpressing the cytoplasmic domain of bZIP28 showed a delayed growth

phenotype when compared to WT plants under normal conditions (Liu et al., 2007). Such overexpression of

the truncated bZIP28 form should cause a full-scale UPR activation similar to plants under ER stress-inducing

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drug treatments. By contrast, UPR was constitutively, but mildly activated in emp12-1 mutants, hence the

effect is not strong enough to cause a visible growth defect. This hypothesis is supported by the observation

that the UPR marker genes were only mildly up-regulated under normal conditions, which were highly up-

regulated upon ER stress induction. Alternatively, we speculate that such mild UPR activation is intended for

compensating the loss of folding capacity due to BiP leakage, and maintaining ER homeostasis. Therefore,

there is no observable phenotype in emp12 mutants under normal conditions, as the constitutive UPR

activation may have a protective effect against ER stress due to a loss of BiP in the ER. In the presence of

DTT and TM, UPR is activated to a much higher extent, but this happens both in WT and emp12-1 mutants.

Indeed, the mutants do not show a higher UPR activation, which together with partial BiP secretion may

explain why they are hypersensitive to both DTT and TM.

EMP12 may act on ERD2 in a p24δ-independent manner

The K/HDEL receptor ERD2 has been extensively studied in yeast, human, and more recently in plants. ERD2

binds to K/HDEL ligands in cis-Golgi at an acidic pH (Wilson et al., 1993), which induces ERD2

oligomerization (Majoul et al., 2001). Overexpression of K/HDEL ligands can enhance the retrograde

transport of ERD2 in both animals (Lewis and Pelham, 1992) and plants (Montesinos et al., 2014), which

appears not to be the case in emp12-1: BiP, one of the K/HDEL ligands, was up-regulated following UPR

activation, compensating for the leakage, yet few ERD2a dimers formed in the cross-linking experiment.

It is also known that ERD2 trafficking is governed by p24 proteins, which facilitate the sorting of ERD2 into

COPI vesicles for its Golgi-to-ER retrograde transport (Majoul et al., 1998; Majoul et al., 2001). In

Arabidopsis, p24δ5 and p24δ9 are the key players in this retrograde transport for the retrieval of K/HDEL

ligands by ERD2 (Montesinos et al., 2014). Interestingly, a recent study using a quadruple knockout mutant

of all p24δ-1 subclass proteins p24δ3δ4δ5δ6 in Arabidopsis showed similar results as emp12-1: the

p24δ3δ4δ5δ6 mutant grew normally under standard conditions, but UPR is activated constantly with BiP

secreted into the apoplast (Pastor-Cantizano et al., 2018). Although the constitutive UPR activation stems from

ERD2 malfunction in both mutants, it appears that the underlying mechanism is not the same. In emp12-1, the

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steady state of ERD2a shifted exclusively to the ER in contrast to being dually localized in ER and Golgi in

WT plants. However, ERD2a was trapped in the Golgi in p24δ3δ4δ5δ6, indicating that the shift of steady-

state localization of ERD2a is not due to a decreased function of p24δ proteins in the absence of EMP12. On

the other hand, one might argue that an increased function of p24δ5 might have occurred in emp12-1, as its

overexpression would lead to a dominant ER localization of ERD2a (Montesinos et al., 2014). However, it

was also shown in the same study that p24δ5 overexpression prevents HDEL ligand secretion in Arabidopsis,

which contradicts our observation that BiP was secreted into the apoplast. Hence, a loss of EMP12 does not

lead to p24δ protein malfunction / gain-of-function that in turn affects ERD2 trafficking. Another line of

evidence is that both anterograde and retrograde transport of ERD2a via COPII and COPI, respectively, are

normal in emp12-1 judging by the binding of COPII and COPI coatomers with ERD2a in the mutant. Thus,

p24δ protein is expected to function normally in emp12-1. Taken together, EMP12 may regulate ERD2 in a

p24δ-independent manner. Analysis of the relationship between EMP12 and p24 proteins could be conducted

in the future.

EMP12 maintains the Golgi localization of ERD2 for ligand retrieval

In this study, we adopted a loss-of-function approach to investigate the function of EMP12. Given that in the

absence of EMP12, the steady-state localization of ERD2a is shifted to the ER, which could be the result of

its enhanced retrograde transport, EMP12 should function to maintain ERD2a at the Golgi in a WT plant.

Together with the observation that EMP12 and ERD2a interact in the Golgi, as shown by co-IP and FRET-AB

assays, EMP12 may maintain ERD2 localization at the Golgi by directly binding to it.

ERD2a can self-interact in plants (Xu and Liu, 2012) and mammalian ERD2 oligomerizes when bound to

K/HDEL ligands (Majoul et al., 2001). In this study, results from crosslinking experiments (Figure 5B)

showed that ERD2a did not form dimers in emp12-1 when compared to WT. The dimeric form of ERD2a

would exist in Golgi in WT plants, as it is the site where ERD2a captures its ligands. Once ERD2a reaches

the ER via COPI vesicles, the ligands would be released and ERD2a would become monomeric. The few

dimeric ERD2a formed in the absence of EMP12 could thus be a result of a more frequent ERD2a recycling

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to the ER in monomeric form without binding to ligands in the Golgi, together with a few ERD2a dimers

bound to its ligands transported to the ER via COPI vesicles. In addition to biochemical assays, future

experiments using FRET assays on transgenic plants co-expressing ERD2a-CFP and ERD2a-YFP in WT or

emp12-1 plants will further demonstrate the oligomerization status in a spatiotemporal manner. Since EMP12-

ERD2a interaction is disrupted when excess HDEL ligands exist (as shown in the FRET-AB assay in Figure

6E), we propose that EMP12 may serve as an anchor that prevents ERD2a from self-oligomerizing and from

returning to the ER prematurely before it can capture its ligands. ERD2a-ligand binding would result in a

conformational change in ERD2a, which would result in its dissociation from EMP12, interaction with p24

proteins, and return to the ER for ligand retrieval.

METHODS

Plasmid Construction

Full-length cDNAs of ERD2a and bZIP28 were amplified and cloned into the pBI121 backbone containing

either the UBQ10 (Ubiqutin-10) promoter (Grefen et al., 2010) or cauliflower mosaic virus 35S promoter, the

GFP or YFP coding sequence and the nopaline synthase terminator for generation of UBQ10pro-ERD2a-GFP

and 35Spro-YFP-bZIP28 transgenic plants. For making constructs for transient expression in protoplasts, full-

length cDNAs of EMP12, ERD2a and BiP3 were amplified and cloned into pBI221 vectors containing the

cauliflower mosaic virus 35S promoter, the CFP, YFP or spRFP coding sequence, and the nopaline synthase

terminator. The signal peptide (sp) sequence for making the spRFP-EMP12 and spRFP-BiP3 constructs was

derived from barley (Hordeum vulgare) proaleurain (Jiang and Rogers, 1998). Primers used for plasmid

construction are listed in Supplemental Table 2.

Plant Materials and Growth and Treatment Conditions

Arabidopsis thaliana T-DNA insertion lines were obtained from the European Arabidopsis Stock Centre

(GK_284G01 and GK_437A12). Genotyping was conducted by PCR amplification using gene-specific and

T-DNA-specific primers, which are listed in Supplemental Table 2. The T-DNA insertions were confirmed

by next generation sequencing. To generate transgenic plants, all constructs were transformed into

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Agrobacterium tumefaciens and transformed into wild-type Arabidopsis thaliana plants by the floral dip

method (Clough and Bent, 1998). For emp12-1/YFP-bZIP28 and emp12-1/ERD2a-GFP transgenic lines, YFP-

bZIP28 or ERD2a-GFP transgenic plants were crossed with emp12-1 homozygous plants, and the T1

generation was selected with kanamycin, followed by screening for GFP/YFP signal by fluorescence

microscopy. Seeds were surface sterilized and plated on standard Murashige and Skoog (MS) growth medium

supplemented with 1% sucrose and 0.8% agar. Plates were kept at 4 °C in darkness for 2 d before being

transferred to growth chambers at 22 °C under 150 μmol m-2 s-1 of cool-white fluorescent light for a long-day

(16 h light / 8 h dark) photoperiod. Plants were transferred to soil after 2 weeks. For ER stress induction, seeds

were directly germinated on MS agar plates supplemented with or without 1 mM DTT or 50 ng/ml TM. For

confocal study of bZIP28 localization, 5-day-old seedlings were transferred into MS medium supplemented

with DMSO or 5 μg/ml TM for 6 h. For transient protein expression in Arabidopsis seedlings with

Agrobacterium, the assays were performed using the AGROBEST method as described (Wu et al., 2014).

Phylogenetic Analysis

Amino acid sequences of EMPs were retrieved from the UniProt database (https://www.uniprot.org/).

Sequences were aligned using multiple sequence alignment algorithm MUSCLE (http://www.phylogeny.fr/;

Supplemental Data Set 1). Alignment was curated with Gblocks. The phylogenetic tree was constructed

using PhyML (http://www.phylogeny.fr/) by the maximum likelihood method.

Chlorophyll Content and Ion Leakage Measurements

Total chlorophylls were extracted from a pool of 20 seedlings with 80% (v/v) acetone at 4 °C overnight. Fresh

weight was measured prior to the acetone extraction. Absorbance at 663 nm and 646 nm was measured, and

chlorophyll content was determined as described previously (Lichtenthaler, 1987). Ion leakage was measured

with a conductivity meter from 10-day-old seedlings treated with 5 µg/ml TM for 6 h as previously described

(Mishiba et al., 2013). Total amount of ions in the cells was determined after autoclaving the samples for 15

min. Ion leakage was expressed as the percentage of solution conductivity before and after autoclave.

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Confocal Microscopy Analysis

All images were captured using a Leica SP8 confocal microscope equipped with a 63× water lens. Fluorescent

signals were detected with the following excitation and emission wavelengths: CFP (405 / 450 - 500 nm), GFP

(488 nm / 495 - 545 nm), YFP (514 nm / 525 - 588 nm) and RFP (552 nm / 600 - 682 nm). Sequential scanning

was used to avoid possible crosstalk between fluorescence channels. Images were processed using Adobe

Photoshop software as previously described (Jiang and Rogers, 1998). For the quantification of fluorescent

intensity of ERD2a-GFP from ER and Golgi, the fluorescence intensity of ERD2a-GFP from a whole cell was

first measured using ImageJ, and the measured value was set as 100% total intensity. Fluorescence intensity

of punctae co-localized with ManI was defined as the intensity from the Golgi and expressed as percentage of

the total intensity. The remaining intensity was set to be the ER intensity and expressed as percentage of the

total intensity. For the co-localization experiments, the Manders’ coefficient was calculated using the ImageJ

Coloc2 tool.

Secretion Assay

Secretion assays were performed using protoplasts derived from WT and emp12-1 plants as described

previously (Chung et al., 2018). SecGFP was expressed as a secretion control.

Formaldehyde Crosslinking

Crosslinking buffer was obtained by preparing 1 % paraformaldehyde (Sigma-Aldrich) in 100 mM HEPES

and 150 mM NaCl with pH adjusted to 7.5. For cross-linking, the seedlings were weighed and transferred to

a mortar. To 100 mg of plant material, 100 μL of crosslinking buffer was added. Homogenization was

immediately started at room temperature. After 5 min, the reaction was quenched by 1.25 M Tris, pH 8.0 with

one-tenth of buffer volume. Lysates were transferred to a 1.5-mL tube and spun for 10 min at 20000 g at room

temperature to remove debris. SDS sample buffer was added and heated at 37 °C for 10 min. For crosslinking

reversal, the sample was further boiled at 100 °C for 10 min.

Transient Expression in Protoplasts

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Transient expression of XFP fusion proteins using protoplasts derived from Arabidopsis suspension culture

cell line PSB-D was performed as described in our established protocol (Miao and Jiang, 2007; Lam et al.,

2009; Wang and Jiang, 2011).

Co-IP Assay and FRET-AB Analysis

Co-IP assays were performed using proteins extracted from protoplasts expressing corresponding constructs

as stated. Extracted proteins were incubated with GFP-TRAP magnetic beads as described previously (Gao et

al., 2015; Shen et al., 2018). After the washing steps, proteins were eluted and boiled in SDS sample buffer,

followed by immunoblot analysis. FRET-AB analysis was performed using a Leica SP8 confocal microscope

as described previously (Gao et al., 2015). Target proteins were transiently expressed in Arabidopsis

protoplasts. After two rounds of washing with PBS, fixed samples were subjected to FRET-AB analysis.

Defined region of interest was selected for photobleaching using a high intensity laser (514 nm). The signal

intensity of the donor and acceptor proteins before and after photobleaching were measured for calculating

FRET efficiency through the built-in SP8 algorithm. For each testing FRET protein pair, 20 individual cells

expressing the target proteins were used for the analysis.

Antibodies

EMP12, VSR, CNX and ManI antibodies were produced in-house (Li et al., 2002; Tse et al., 2004; Pimpl et

al., 2006; Gao et al., 2012; Shen et al., 2014). BiP antibody was a kind gift from Professor Jurgen Denecke

(University of Leeds, UK). Anti-Sec21 antibody (1:1000 dilution; cat. no. AS08327) and cFBPase antibody

(1:2000 dilution; cat. no. AS04043) was purchased from Agrisera. GFP antibody (1:1000 dilution) was

purchased from Clontech (mouse monoclonal JL-8). RFP antibody (1:1000 dilution) was purchased from

Chromotek (rat monoclonal 5F8). For Sec24 antibody, a synthetic peptide (GenScript)

QRFPSPPFPTTQNPPQGPPPP corresponding to the N-terminal of Sec24 were conjugated with keyhole

limpet hemocyanin and used to immunize rats. Antibodies were affinity-purified using cyanogen bromide-

activated Sepharose 4B (Sigma-Aldrich) conjugated with the peptides.

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Statistical Analysis

Sample numbers and the number of biological replicates for each experiment are indicated in figure legends.

Where appropriate, two-tailed Student’s t-test and one-way ANOVA with post tests were performed

(Supplemental Table 3-4).

Accession Numbers

The Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this article are EMP12

(At1g10950), ERD2a (At1g29330), bZIP28 (At3g10800), and BiP3 (At1g09080). Additional identifiers are

listed in Supplemental Table 1.

Supplemental Data

Supplemental Figure 1. Phylogenetic tree of EMP superfamily in eukaryotes. (Supports Figure 1)

Supplemental Figure 2. Sanger sequencing of emp12-1 and emp12-2 mutant plants. (Supports Figure 1)

Supplemental Figure 3. Complementation of emp12-1 mutant plants. (Supports Figure 1)

Supplemental Figure 4. All three BiP isoforms can be recognized by the BiP antibody. (Supports Figure 3)

Supplemental Figure 5. Subcellular localization of BiP1 in WT and emp12-1 protoplasts. (Supports Figure

3)

Supplemental Figure 6. Transcription level of ERD2A is induced by DTT and TM treatments, but no change

is observed in the absence of EMP12. (Supports Figure 2)

Supplemental Figure 7. Expression and subcellular localization of ERD2a-GFP in different individual

transformed lines in WT and emp12-1 plants. (Supports Figure 3)

Supplemental Figure 8. Reciprocal immunoprecipitation assay analysis showing enhanced interaction

between Sec21 and ERD2a in the absence of EMP12. (Supports Figure 5)

Supplemental Figure 9. Other EMP members do not have functional redundancy with EMP12. (Supports

Figure 6)

Supplemental Table 1. Homologues of EMPs in eukaryotes.

Supplemental Table 2. Primers used in this study.

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Supplemental Table 3. Statistical test results for ANOVA.

Supplemental Table 4. Statistical test results for t-test.

Supplemental Data Set 1. Alignments used to generate the phylogeny in Supplemental Figure 1.

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ACKNOWLEDGEMENTS

We thank Professor Jurgen Denecke (University of Leeds, UK) for providing BiP antibodies. This work was

partially supported by grants from the Research Grants Council of Hong Kong (CUHK14130716, 14102417,

14100818, C4011-14R, C4012-16E, C4002-17G, R4005-18F and AoE/M-05/12) and the National Natural

Science Foundation of China (31670179 and 91854201) as well as Research Committee of CUHK to L.J., the

Hong Kong Scholars Program (XJ2013011) and Youth Innovation Promotion Association, Chinese Academy

of Sciences (2017399) to M.L.

Author Contributions

K.P.L., M.L., C.G., Y.Z., and Q.Z. designed and performed the research; K.P.L. and M.L. analyzed the data

and wrote the manuscript; M.L.C. and X.Y. revised the manuscript; L.J. supervised the research and revised

the manuscript.

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Figure 1. Two identified EMP12 T-DNA insertion mutants are sensitive to the ER stress-inducing agents

dithiothreitol (DTT) and tunicamycin (TM).

(A) An illustration of the EMP12 genomic region showing the T-DNA insertions in emp12-1 and emp12-2 mutants.

(B) Representative immunoblots of wild-type Col-0 (WT), emp12-1 and emp12-2 probed with EMP12 antibodies. Equal

loading was confirmed by probing with VSR antibodies.

(C) Quantification of the band optical density corresponding to EMP12 in (B). Values represent mean ± SEM (n = 3).

(D) Phenotypes of WT and emp12 mutant seedlings under DTT and TM treatments. Seeds were germinated on MS

agar plates with or without the indicated drugs for 7 days. Bar = 10 mm.

(E) Fresh weight of seedlings with or without drug treatments. Twenty seedlings were pooled for measurement.

Significant differences as compared to control are denoted by * (p ≤ 0.05) and ** (p ≤ 0.01) respectively, derived from

one-way ANOVA with Dunnett’s test, n = 3. Values represent mean ± SEM.

(F) Root length of seedlings with or without drug treatments. Significant differences as compared to the control are

denoted by *** (p ≤ 0.001), derived from one-way ANOVA with Dunnett’s test, n = 20. Values represent mean ± SEM.

(G) Chlorophyll a and b contents of seedlings with or without drug treatments. Twenty seedlings were pooled for

measurement. Significant differences as compared to control are denoted by * (p ≤ 0.05), ** (p ≤ 0.01) and *** (p ≤ 0.001)

respectively, derived from one-way ANOVA with Dunnett’s test, n = 3. Values represent mean ± SEM.

(H) Ion leakage of seedlings with or without TM treatment. Twenty seedlings were pooled for measurement. Significant

differences as compared to control are denoted by *** (p ≤ 0.001), derived from one-way ANOVA with Dunnett’s test, n

= 3. Values represent mean ± SEM.

RETRACTED

Figure 2. The unfolded protein response is constitutively activated in emp12-1.

(A) qPCR analysis of (i) BiP1 / BiP2, (ii) PDIL1, (iii) SHD, (iv) CNX1, and (v) ACTIN8 transcript levels in WT and emp12-

1 under DTT and TM treatments. Significant differences as compared to untreated WT are denoted by * (p ≤ 0.05), ** (p

≤ 0.01) and *** (p ≤ 0.001) respectively, derived from unpaired t-test, n = 3. Values represent mean ± SEM.

(B) Confocal microscopy images of YFP-bZIP28 expressed in the roots of WT and emp12-1 mutant plants with or without

6 h of 5 μg/ml TM treatment. Bar = 10 μm for all images shown. RETRACTED

Figure 3. In emp12-1, BiP was secreted into the apoplast, and a shift in steady-state distribution of the K/HDEL

receptor ERD2a to the ER exclusively was observed.

(A) Protoplast secretion assay for examining BiP secretion. Protoplasts were isolated from 4-week-old WT and emp12-

1 plants. After isolation, protoplasts were transformed with secGFP as a secretion control. After 16 h, incubation medium

was collected. Proteins from remaining protoplasts and medium concentrated by ultrafiltration were subjected to

immunoblot analysis with anti-BiP, anti-CRT, anti-GFP, and anti-cFBPase (a cytosolic marker). Results are

representative of three independent experiments.

(B) Confocal microscopy images of ERD2a-GFP expressed in the leaves, hypocotyls, and roots of WT and emp12-1

mutant plants. Bar = 10 μm.

RETRACTED

Figure 4. ERD2a-GFP localized in the ER exclusively in emp12-1 mutant plants, whereas EMP12 overexpression

restored the Golgi localization of ERD2a-GFP.

(A) Confocal microscopy images of CNX-RFP (ER marker) and ManI-RFP (Golgi marker) transiently expressed via

Agrobacterium transformation in the leaves of WT and emp12-1 mutant plants expressing ERD2a-GFP. Bar = 10 μm.

(B) Quantification of ERD2a fluorescence intensity colocalized with ER or Golgi marker. Significant differences as

compared to WT are denoted by *** (p ≤ 0.001), derived from unpaired t-test, n = 10. Values represent mean ± SEM.

(C) Quantification analysis of colocalization as shown in (A) by Manders' colocalization coefficient. Significant

differences as compared to WT are denoted by ** (p ≤ 0.01), derived from unpaired t-test, n = 10. Values represent

mean ± SEM.

(D) Confocal microscopy images of ManI-RFP and spRFP-EMP12 transiently expressed via Agrobacterium

transformation in the leaves of WT and emp12-1 mutant plants expressing ERD2a-GFP. Bar = 10 μm.

(E-F) Quantification of ERD2a fluorescence intensity colocalized with ManI-RFP or spRFP-EMP12 in (E) WT and (F)

emp12-1 transgenic plants. Significant differences as compared to WT are denoted by *** (p ≤ 0.001), derived from

unpaired t-test, n = 10. Values represent mean ± SEM.

RETRACTED

Figure 5. Enhanced retrograde transport of ERD2a with a reduction in dimer formations in the absence of EMP12.

(A) ERD2a exhibited a higher affinity towards COPI coatomer in emp12-1 than in WT plants. WT and emp12-1 seedlings

expressing ERD2a-GFP were subjected to protein extraction and IP with GFP-Trap followed by immunoblotting with

GFP, Sec21 and Sec24 antibodies.

(B) ERD2a formed fewer dimers in emp12-1 than in WT plants. WT and emp12-1 seedlings were crosslinked with 1 %

paraformaldehyde (PFA) during homogenization. Total protein extracted with or without PFA reversal was immunoblotted

with GFP antibodies. The intensities of monomer and dimer bands were quantified by densitometry, and the

dimer:monomer ratio was calculated from WT and emp12-1 respectively as shown below the image.

RETRACTED

Figure 6. EMP12 interacts with ERD2a and this interaction is disrupted in the presence of excess HDEL ligands.

(A) Colocalizations of EMP12-CFP-RNIKCD with YFP-SYP32 or ERD2a-YFP expressed in Arabidopsis protoplasts. Bar

= 10 μm.

(B) FRET analysis of EMP12-CFP-RNIKCD, EMP8-CFP-RSIKCE or EMP9-CFP-RSIKCE, co-expressed with YFP-

SYP32 or ERD2a-YFP. FRET efficiency was quantified using the acceptor photobleaching (AB) approach. At least 10

individual protoplasts were used for FRET efficiency quantification. Significant difference as compared to the

corresponding YFP-SYP32 control is denoted by ** (p ≤ 0.01), derived from unpaired t-test, n = 10 – 17. Values represent

mean ± SEM.

(C) Co-immunoprecipitation (Co-IP) assay shows association between EMP12 and ERD2a. Arabidopsis protoplasts

expressing YFP-SYP32 or ERD2a-YFP with spRFP-EMP12 were subjected to protein extraction and IP with GFP-Trap

followed by immunoblotting with GFP and RFP antibodies.

(D) Confocal images of EMP12-CFP-RNIKCD and ERD2a-YFP co-expressed with CNX-RFP or spRFP-BiP3 in

Arabidopsis protoplasts. Bar = 10 μm.

(E) FRET-AB analysis of the colocalized puncta between EMP12-CFP-RNIKCD and ERD2a-YFP co-expressed with

CNX-RFP or spRFP-BiP3 as shown in (D). At least 10 individual protoplasts were used for FRET efficiency quantification.

Significant difference as compared to control expressing RFP-CNX is denoted by *** (p ≤ 0.001), derived from unpaired

t-test, n = 10–16. Values represent mean ± SEM.

(F) Co-IP assay shows association between EMP12 and ERD2a with or without HDEL ligand overexpression.

Arabidopsis protoplasts expressing CNX-RFP or spRFP-BiP3 together with spGFP-EMP12 and ERD2a-RFP were

subjected to protein extraction and IP with GFP-Trap followed by immunoblotting with GFP and RFP antibodies.

RETRACTED

Figure 7. Working model of ERD2a trafficking regulation by EMP12.

(1) In the wild-type plant, EMP12 binds to ERD2a and thereby prevents premature retrograde transport without ligand.

(2) When ERD2a binds a ligand such as BiP that escaped from the ER, it dissociates from EMP12 and forms an ERD2a-

ligand complex. The complex is transported to the ER via COPI vesicles.

(3) ER residence of BiP maintains the bZIP28 inactive form. UPR is not activated.

(4) In the absence of EMP12, ERD2a is transported to the ER without capturing ligands.

(5) BiP is leaked to post-ER compartments such as Golgi and eventually secreted into the apoplast. Insufficient BiP is

presented to keep bZIP28 in the ER.

(6) bZIP28 is activated by protease processing in the Golgi. UPR is activated. More BiP is produced to compensate the

leakage. RETRACTED

DOI 10.1105/tpc.18.00913; originally published online May 23, 2019;Plant Cell

and Liwen JiangKing Pong Leung, Ming Luo, Caiji Gao, Yonglun Zeng, Qiong Zhao, Mee-Len Chye, Xiaoqiang Yao

response by regulating K/HDEL receptor traffickingArabidopsis ENDOMEMBRANE PROTEIN 12 contributes to the endoplasmic reticulum stress

 This information is current as of May 22, 2021

 

Supplemental Data /content/suppl/2019/05/22/tpc.18.00913.DC1.html

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