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Transmembrane Topologies of Ca2�-permeableMechanosensitive Channels MCA1 and MCA2 in Arabidopsisthaliana*

Received for publication, September 15, 2015, and in revised form, November 9, 2015 Published, JBC Papers in Press, November 10, 2015, DOI 10.1074/jbc.M115.692574

Shumpei Kamano‡1,2, Shinichiro Kume‡1,3, Kazuko Iida§1, Kai-Jian Lei‡4, Masataka Nakano‡5, Yoshitaka Nakayama‡6,and Hidetoshi Iida‡7

From the ‡Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei, Tokyo 184-8501, Japan and§Laboratory of Biomembrane, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya,Tokyo 156-8506, Japan

Sensing mechanical stresses, including touch, stretch, com-pression, and gravity, is crucial for growth and development inplants. A good mechanosensor candidate is the Ca2�-permeablemechanosensitive (MS) channel, the pore of which opens to per-meate Ca2� in response to mechanical stresses. However, thestructure-function relationships of plant MS channels arepoorly understood. Arabidopsis MCA1 and MCA2 form ahomotetramer and exhibit Ca2�-permeable MS channel activ-ity; however, their structures have only been partially eluci-dated. The transmembrane topologies of these ion channelsneed to be determined in more detail to elucidate the underlyingregulatory mechanisms. We herein determined the topologiesof MCA1 and MCA2 using two independent methods, theSuc2C reporter and split-ubiquitin yeast two-hybrid methods,and found that both proteins are single-pass type I integralmembrane proteins with extracellular N termini and intracellu-lar C termini. These results imply that an EF hand-like motif,coiled-coil motif, and plac8 motif are all present in the cyto-plasm. Thus, the activities of both channels can be regulated byintracellular Ca2� and protein interactions.

Mechanical stimuli affect plant growth, development, andresistance to herbivores (1– 6). Natural stimuli, such as windand gravity, modify the height and shape of grasses and trees.

Touch retards the elongation of inflorescence and increasesresistance to herbivores in Arabidopsis thaliana. Thus, stimu-lus perception is fundamental to plants, and the elucidation ofmechano-sensors is important for understanding the molecu-lar basis of plant mechanics and morphogenesis.

A Ca2�-permeable mechanosensitive (MS)8 channel hasbeen suggested as a component of mechano-sensors (7–10).Using Nicotiana plumbaginifolia seedlings carrying the Ca2�-dependent photoprotein, aequorin, as an intracellular Ca2�

indicator, a previous study reported that touch elicits animmediate increase in the cytosolic concentration of Ca2�

([Ca2�]cyt), which may act as a Ca2� signal (11). We recentlyidentified novel Ca2�-permeable MS channels in Arabidop-sis (A. thaliana), named MCA1 and MCA2 (mid1-comple-menting activity 1 and 2) (12–15). Both proteins share 74%identity in their amino acid sequences, form a homote-tramer, have no homology to any known ion channels ortransporters, and mediate Ca2� influx upon mechanicalstimulation, such as hypo-osmotic shock and membranestretch. Genes coding for MCA orthologs are found exclu-sively in the plant kingdom (9, 12). Rice and tobacco MCAproteins are also shown to mediate an increase in [Ca2�]cytupon hypo-osmotic shock (16, 17).

An in silico study suggested that MCA1 and MCA2 haveseveral motifs, such as an EF hand-like motif, coiled-coil motif,and plac8 (DUF614) motif as well as a few predicted putativetransmembrane segments (12, 13). To understand the molecu-lar and physiological functions of both proteins and theirmotifs, their transmembrane topologies need to be elucidatedin more detail. In the present study we determined these topolo-gies using two different methods. The results provide evidencethat MCA1 and MCA2 have one transmembrane segment neartheir N termini which is oriented to the outside of the plasmamembrane, in contrast to the prediction with transmembranetopology algorithms available online. Thus, these proteins areidentified as single-pass type I transmembrane proteins. Thisresult suggests that all of the above-mentioned motifs are ori-ented toward the cytosol and provides an insight into the reg-ulatory mechanisms of MCA1 and MCA2. Although other fam-

* This work was supported by Grant-in-aid for Scientific Research on PriorityArea 25120708 (to H. I.) from the Ministry of Education, Culture, Sports,Science, and Technology of Japan and Grant-in-Aid for Scientific ResearchB 26291026 (to H. I.) from the Japan Society for the Promotion of Science.The authors declare that they have no conflicts of interest with the con-tents of this article.

1 These authors contributed equally to this work.2 Present address: Dept. of Molecular Cardiovascular Biology and Pharmacol-

ogy, Ehime University School of Medicine, Shitsukawa, Toon, Ehime, 791-0295, Japan.

3 Present address: Division of Biophysics and Neurobiology, Dept. of Molecu-lar Physiology, National Institute for Physiological Sciences, Myodaiji, Oka-zaki 444-8585, Japan.

4 Present address: Institute of Pharmacy, Henan University, Kaifeng 475004,China.

5 Present address: Dept. of Applied Biological Science, Tokyo University ofScience, Noda, Chiba 278-8510, Japan.

6 A Japan Society for the Promotion of Science Fellow (Grant 10J02008). Pres-ent address: Molecular Cardiology and Biophysics Division, Victor ChangCardiac Research Institute, Darlinghurst, NSW 2010, Australia.

7 To whom correspondence should be addressed. Tel.: 81-42-329-7517; Fax:81-42-329-7517; E-mail: [email protected].

8 The abbreviations used are: MS, mechanosensitive; MCS, multi-cloning site;Endo H, Endoglycosidase H; ER, endoplasmic reticulum; Cub, C-terminalubiquitin; Nub, N-terminal ubiquitin; TF, transcription factor.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 52, pp. 30901–30909, December 25, 2015

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

DECEMBER 25, 2015 • VOLUME 290 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 30901

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ilies of plant MS channels have already been identified andcharacterized, their transmembrane topologies have not yetbeen determined experimentally (18, 19). Therefore, this is thefirst study to experimentally elucidate the transmembranetopologies of plant MS channels.

Experimental Procedures

Yeast Strains and Media—Strain H319 (MATa cch1�::HIS3mid1�::HIS3 his3�-1 leu2-3,112 trp1-289 ura3-52 sst1-2) wasdescribed previously (20), and the two-hybrid reporter strainNMY32 (MATa his3�200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lacZ ade2::(lexAop)8-ADE2GAL4) was obtained from a manufacturer (Dualsystems Bio-tech AG, Zürich, Switzerland). Synthetic SD medium wasdescribed previously (21), to which 20 mg/liter histidine, 30mg/liter leucine, 20 mg/liter tryptophan, 20 mg/liter uracil, and20 mg/liter adenine sulfate were added if necessary.

Escherichia coli Strain and Medium—Strain XL1-Blue(supE44 hsdR17 recA1 endA1 gyrA46 thi relA1 lac� F’ [proAB�

lacIq lacZ �M15 Tn10 (tetr)]; Agilent Technologies, SantaClara, CA) and DH5� (supE44 �lacU169 (�80 lacZ �M15)hsdR17 recA1 endA1 gyrA96 thi-1 relA1; Toyobo Co., Tokyo,Japan) were used for plasmid construction. LB medium wasprepared as described previously (22).

Construction of MCA1-FLAG and MCA2-FLAG—To con-struct the low copy expression plasmids YCpT-MCA1-FLAGand YCpT-MCA2-FLAG, the open reading frames (ORF) ofMCA1-HA and MCA2-HA in the multicopy expression plas-mids YEpT-MCA1-HA4 and YEpT-MCA2-HA4 (14) wereinserted into the multi-cloning site (MCS) of the YCplac111(23)-based, low-copy expression vector YCpTDHxb (LEU2CEN4 ARS1 TDH3p-MCS-ADH1t ampr). The 4�HA tag of theresultant plasmids was replaced by a 5�FLAG tag derived frompFLAG-CMV2 (Sigma).

Construction of MID1-FLAG and MID1(16Q)-FLAG—Toconstruct the low-copy plasmid YCpS-MID1-FLAG, whichexpresses the Mid1 protein with a 5�FLAG tag at its C termi-nus under the control of its own promoter, the 5�FLAG tagwas inserted just ahead of the stop codon of MID1 in YCpS-MID1 (20). To construct YCpS-MID1(16Q)-FLAG, in which allof the Asn residues in the 16 potential N-glycosylation sites(NX(S/T)) of Mid1 were replaced by Gln, YCpS-MID1-FLAGwas mutated with the QuikChange II XL site-directedmutagenesis kit (Stratagene Co., La Jolla, CA) or KOD-Plus-Mutagenesis kit (Toyobo Co.) according to the manufacturers’instructions.

Construction of the Short Suc2 Reporter, Suc2C—To con-struct the short Suc2 reporter, Suc2C, a SUC2 fragment encod-ing a polypeptide corresponding to amino acid residues 350 –

409, which contains four potential N-glycosylation sites NX(S/T), was amplified by PCR with DNA polymerase (KOD-Plus-Neo, Toyobo Co.) using the template plasmid pCS4 –14 (URA32-�m-ori SUC2-HIS4C ampr; Ref. 24) and three sets of primers,S2C-5F-Nco and S2C-3R-Bam for N-terminal fusions and S2C-5F-RI and S2C-3R-Not or S2C-5F-SalI and S2C-3R-Not forC-terminal fusions (Table 1). The PCR products were treatedwith the corresponding restriction enzymes and ligated toYEpTDHXho-MCA1 (12), YEpTDHXho-MCA2 (13), YCpT-MCA1-FLAG, YCpT-MCA2-FLAG, and YCpS-MID1(16Q)-FLAG, which had been linearized with the respective restric-tion enzymes. The resulting plasmids are listed in Table 2.

Construction of a Series of C-terminally Truncated MCA1-FLAG and MCA2-FLAG Tagged with Suc2C—To synthesize aseries of truncated derivatives of MCA1 and MCA2, PCR wasperformed using the plasmid YCpT-MCA1-FLAG or YCpT-MCA2-FLAG as a template and BamHI site-containing for-ward primers and SalI site-containing appropriate reverseprimers. The resulting products were cut with BamHI and SalIand inserted into the Suc2-tagged MCA1 or MCA2 plasmidsdescribed above in place of the full-length ORF. The resultingplasmids carrying the inserts encoding the appropriately trun-cated forms of MCA1 and MCA2 are listed in Table 2.

Construction of Bait Plasmids—The vectors pNCW, carryingthe LexA-VP16-Cub cassette for N-terminal fusions, andpCCW, carrying the Cub-LexA-VP16 cassette for C-terminalfusions (25), were used to construct the bait plasmids. pCCWwas modified to pCCWc, which contained an additional SacIIrestriction site in the MCS. The ORFs of MCA1, MCA2, andtheir truncated derivatives were inserted in-frame to the MCSof pNCW or pCCWc, respectively. The nucleotide sequences ofthe bait plasmids were confirmed by DNA sequencing. Theresulting plasmids were shown in Table 3. pCCW, pNCW, andthe prey plasmids pAI-Alg5 and pDL2-Alg5 were purchasedfrom a manufacturer (Dualsystems Biotech AG, Zürich,Switzerland).

Endoglycosidase H (Endo H) Treatment and Western BlotAnalysis—Yeast cells were grown to the late log phase at 30 °C,harvested by centrifugation, resuspended in TE buffer (10 mM

Tris-HCl, pH 8.0, 1 mM EDTA) containing 1 mM PMSF, anddisrupted by stirring at 4 °C for 7 min with acid-washed glassbeads (425– 600 �m, Sigma). Whole cell lysates were preparedby centrifuging the extracts for 5 min at 800 � g. The superna-tant received a 1⁄10 volume of the denaturation buffer (5% SDSand 0.4 M DTT), was heat-denatured at 70 °C for 5 min, thenreceived a 1⁄10 volume of the reaction buffer (0.5 M sodium ace-tate, pH 5.2) and divided into two equal parts (29 �l each). One

TABLE 1Primers to construct the short Suc2 reporter, Suc2CUnderlines indicate NcoI, BamHI, EcoRI, SalI, and NotI restriction sites (from the top to the bottom). The initiation and stop codons are boldfaced.

Primer name Nucleotide sequence

S2C-5F-Nco 5�-ccccATGGCCGAACCAATATTGAACATTAGTAA-3’S2C-3R-Bam 5�-ggggatccGGCAAAGACGGATTTGGATATGGT-3’S2C-5F-RI 5�-gggaattcGCCGAACCAATATTGAACATTAGTAA-3’S2C-5F-SalI 5�-tggtcgacGCCGAACCAATATTGAACATTAGTAA-3’S2C-3R-Not 5�-gggcggccgcTTAGGCAAAGACGGATTTGGATATGGT-3’

Transmembrane Topologies of MS Channels, MCA1 and MCA2

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received 5 milliunits (1 �l) of Endo H (Roche Applied Science),and the other received an equal volume of Milli-Q water. Themixtures were incubated at 37 °C for 1 h, received Laemmli SDSsample buffer (26), and were then heated at 70 °C for 5 min.SDS-PAGE and Western blot analyses were performed essen-tially according to the method described by Iida et al. (21).FLAG-tagged proteins were detected with a mouse anti-FLAGM2 monoclonal antibody (Sigma, catalog no. 3165) and anaffinity-purified, peroxidase-conjugated, sheep anti-mouse IgG(GE Healthcare: NXA931). MCA1 and MCA2 without theFLAG tag were detected with the rabbit polyclonal antibodyApep2 that recognizes both the MCA1 and MCA2 proteins (14)and an affinity-purified, peroxidase-conjugated, donkey-antirabbit IgG (GE Healthcare Bio-Science; NA934).

Growth Assay—Cells of the two-hybrid reporter strainNMY32 expressing Cub-TF-tagged MCA1 or MCA2 withAlg5NubI or Alg5-NubG were grown to the mid log phase in SDliquid medium containing adenine and histidine but lackingleucine and tryptophan, harvested by centrifugation, and resus-pended in saline at a density of 4 � 108 cells/ml, after which 5 �lof a 1:10 serial dilution was spotted on SD agar medium lackingleucine and tryptophan with or without adenine and histidineand then incubated at 30 °C for 2 to 4 days.

Results

Bioinformatic Predictions Presented Various MembraneTopologies of MCA1 and MCA2—We first predicted the mem-brane topologies of MCA1 and MCA2 with various web-basedprediction servers, including PredictProtein (27), TMpred (28),SOSUI (29), and TOPCONS (30). As shown in Fig. 1, theseservers gave different results for MCA1 and MCA2. In addition,although the amino acid sequence identities and similaritiesbetween MCA1 and MCA2 were 73 and 89%, respectively (12),the predicted topologies were different between the two pro-teins. These inconsistent results prompted us to determinemembrane topologies experimentally.

Construction of a Short Suc2C Reporter to Determine Mem-brane Topology—The membrane topologies of many trans-membrane proteins have been determined by a method using aSUC2-HIS4C dual topology reporter expressed in yeast cells(24, 31). In this reporter, invertase encoded by SUC2 may beN-glycosylated at 14 potential sites when localized in the endo-plasmic reticulum (ER) lumen, whereas the C-terminal domainof the His4 protein harboring histidinol dehydrogenase activitycan rescue the histidine auxotrophy of his4 cells spread on thehistidinol plate when exposed to the cytoplasmic side of the ERmembrane. This reporter is regarded as being very useful

TABLE 2Plasmids carrying MCA1 and MCA2 tagged with SUC2CTDH3p, TDH3 promoter; ADH1t, ADH1 terminator; MID1p, MID1 promoter.

Plasmid name Markers

YEpT-SUC2C-MCA1 LEU2 2-�m-ori TDH3p-SUC2C-MCA1-ADH1t ampr

YEpT-SUC2C-MCA2 LEU2 2-�m-ori TDH3p-SUC2C-MCA2-ADH1t ampr

YEpT-MCA1-SUC2C LEU2 2-�m-ori TDH3p-MCA1-SUC2C-ADH1t ampr

YEpT-MCA2-SUC2C LEU2 2-�m-ori TDH3p-MCA2-SUC2C-ADH1t ampr

YCpT-MCA1-FLAG LEU2 CEN4 ARS1 TDH3p-MCA1–5xFLAG-ADH1t ampr

YCpT-MCA2-FLAG LEU2 CEN4 ARS1 TDH3p-MCA2–5xFLAG-ADH1t ampr

YCpT-SUC2C-MCA1-FLAG LEU2 CEN4 ARS1 TDH3p-SUC2C-MCA1–5xFLAG-ADH1t ampr

YCpT-SUC2C-MCA2-FLAG LEU2 CEN4 ARS1 TDH3p-SUC2C-MCA2–5xFLAG-ADH1t ampr

YCpT-MCA1-FLAG-SUC2C LEU2 CEN4 ARS1 TDH3p-MCA1–5xFLAG-SUC2C-ADH1t ampr

YCpT-MCA2-FLAG-SUC2C LEU2 CEN4 ARS1 TDH3p-MCA2–5xFLAG-SUC2C-ADH1t ampr

YCpT-SUC2C-MCA1-(1–185)-FLAG LEU2 CEN4 ARS1 TDH3p-SUC2C-MCA1 (1–185)-5xFLAG-ADH1t ampr

YCpT-SUC2C-MCA2-(1–186)-FLAG LEU2 CEN4 ARS1 TDH3p-SUC2C-MCA2 (1–186)-5xFLAG-ADH1t ampr

YCpT-MCA1-(1–185)-FLAG-SUC2C LEU2 CEN4 ARS1 TDH3p-MCA1 (1–185)-5xFLAG-SUC2C-ADH1t ampr






YCpS-MID1-FLAG LEU2 CEN4 ARS1 MID1p-MID1–5xFLAG-ADH1t ampr

YCpS-MID1(16Q)-FLAG-SUC2C LEU2 CEN4 ARS1 MID1p-MID1 (16Q)-5xFLAG-SUC2C-ADH1t ampr

pCS4–14a URA3 2-�m-ori SUC2-HIS4C ampr

a A gift from Sengstag (24).

TABLE 3Bait and prey plasmids used in this studyCYC1p, CYC1 promoter; CYC1t, CYC1 terminator; ADH1p, ADH1 promoter.

Plasmid name Markers

pNCW-MCA1 LEU2 CEN/ARS CYC1p-LexA-VP16-Cub-MCA1-CYC1t kanr

pNCW-MCA2 LEU2 CEN/ARS CYC1p-LexA-VP16-Cub-MCA2-CYC1t kanr

pCCW-MCA1 LEU2 CEN/ARS CYC1p-MCA1-Cub-LexA-VP16-CYC1t kanr

pCCW-MCA2 LEU2 CEN/ARS CYC1p-MCA2-Cub-LexA-VP16-CYC1t kanr

pCCW-MCA1-(1–185) LEU2 CEN/ARS CYC1p-MCA1 (1–185)-Cub-LexA-VP16-CYC1t kanr






pAI-Alg5a TRP1 2-mm-ori ADH1p-Alg5-HA-NubI-CYC1t ampr

pDL2-Alg5a TRP1 2-mm-ori ADH1p-Alg5-HA-NubG-CYC1t ampr

a The two prey plasmids were purchased from Dualsystems Biotech AG.




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because of this duality. However, this method sometimes givesself-contradictory results; i.e. no N-glycosylation (suggestive ofcytoplasmic), but no rescue on medium containing histidinolinstead of histidine (suggestive of luminal or extracellular) andvice versa (31–33). Furthermore, the molecular mass of a fullymodified form of this reporter protein is �130 kDa, which ismarkedly larger than the estimated molecular mass of full-length MCA1 and MCA2 proteins (�50 kDa). Because we ana-lyzed even smaller, truncated forms of MCA1 and MCA2 in thepresent study, this large reporter may lead to discrepant resultsbeing obtained, similar to those described above.

To overcome this issue, we constructed a new reporter com-posed solely of a Suc2 protein fragment (amino acid residues350 – 409) that only had four potential N-glycosylation sites.This reporter was named Suc2C because it was derived from aregion close to the Suc2 C terminus.

MCA1 and MCA2 Are Not Naturally N-Glycosylated—Before using Suc2C as a probe, we examined the possible N-gly-cosylation of full-length MCA1 and MCA2 because the web-server GlycoEP (34) predicted that MCA1 has two potentialN-glycosylation sites at Asn279 and Asn371, whereas MCA2 hasone site at Asn363. If N-glycosylated, these sites may complicatethe results of the experiments described below, in which EndoH is used to cleave N-linked oligosaccharides. To determinewhether N-glycosylation occurred, MCA1 and MCA2 taggedC-terminally with five consecutive FLAG epitopes (MCA1-

FLAG and MCA2-FLAG) were expressed in yeast cells, andwhole cell extracts were treated with Endo H before SDS-PAGEand a Western blot analysis with the anti-FLAG antibody. Fig.2A shows that the positions of MCA1-FLAG and MCA2-FLAGon the Western blot were not shifted by the Endo H treatment.In contrast, the position of the positive control, the yeast Mid1protein, which is known to be N-glycosylated (21), was moveddownward. These results suggest that MCA1 and MCA2 arenot N-glycosylated in yeast cells.

Determination of Membrane Topology with the Suc2C Re-porter—We fused the Suc2C reporter to the N terminus or Cterminus of MCA1 and MCA2 and determined whether theresulting constructs, Suc2C-MCA1, Suc2C-MCA2, MCA1-Suc2C, and MCA2-Suc2C, were N-glycosylated using the pro-cedures described above. Fig. 2B shows that the upper bands ofSuc2C-MCA1 and Suc2C-MCA2 (an arrow in the figure) dis-appeared after the Endo H treatment. On the other hand, theirlower bands were not shifted by this treatment, and neitherwere the bands of MCA1-Suc2C and MCA2-Suc2C. Theseresults suggest that some of the Suc2C-MCA1 and Suc2C-MCA2 polypeptides are N-glycosylated, whereas the MCA1-Suc2C and MCA2-Suc2C polypeptides are not. The reason why alarger amount of Suc2C-MCA1 and Suc2C-MCA2 polypeptides

FIGURE 1. Hydrophobicity plots and transmembrane topology predic-tions of MCA1 and MCA2. Hydrophobicity plots were made using the Kyteand Doolittle method with a window of 19 amino acids (47). The abscissashows the amino acid number, and the ordinate represents hydrophobicity.Transmembrane topologies were predicted with the four membrane topol-ogy algorithms available online: PredictProtein, TMPred, SOSUI, and TOP-CONS. Green bars represent putative transmembrane segments, namedpTM1, pTM2, pTM3, and pTM4, among which pTM1 was identified as the realtransmembrane segment in the present study.

FIGURE 2. Examination of N-glycosylation of MCA1 and MCA2 and Endo Hsensitivity of MCA1 and MCA2 fused N-terminally with Suc2C. A, MCA1and MCA2 were not N-glycosylated in yeast cells. Whole cell lysates preparedfrom yeast cells expressing MCA1-FLAG and MCA2-FLAG were treated withEndo H or mock-treated and subjected to Western blotting. As a positivecontrol, the yeast Mid1 protein tagged with a 5�FLAG was treated in thesame manner. The proteins were detected by an anti-FLAG antibody. Thearrow indicates an Endo H-sensitive protein. B, Suc2C-MCA1 and Suc2C-MCA2 are sensitive to Endo H. Whole cell extracts prepared from yeast cells(strain H319) expressing MCA1 and MCA2 fused N-terminally or C-terminallywith Suc2C were treated with Endo H or mock-treated and processed asdescribed above. The arrow indicates an Endo H-sensitive protein. Becausethese constructs have no FLAG tag, they were detected with the anti-Apep2antibody that recognizes both MCA1 and MCA2 (14).




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are not N-glycosylated currently remains unknown. The amountsof both proteins may have been too much to be N-glycosylated inthe ER because they are expressed under the control of the strongpromoter, TDH3p, on multicopy plasmids in yeast cells (35). Weused this promoter because it was necessary to make MCA1,MCA2, and their derivatives detectable on Western blots.

To determine the membrane topologies of MCA1 andMCA2 with respect to putative transmembrane segments, theSuc2C reporter was fused to downstream positions of each ofthe four putative transmembrane segments (named pTM1 topTM4) common to MCA1 and MCA2 (Fig. 1). The fused posi-tions are shown in Fig. 3A. The resulting constructs wereexpressed in yeast cells, and whole cell extracts were treatedwith Endo H and then analyzed as described above. Fig. 3Bshows that only the MCA1 (1–185)-FLAG protein N-termi-nally fused with Suc2C [i.e. Suc2C-MCA1 (1–185)-FLAG] wassensitive to Endo H. The same result was obtained with Suc2C-intact MCA1 (Fig. 2B). In contrast, MCA1-FLAG proteins(including truncated forms) that fused C-terminally withSuc2C, such as MCA1 (1–185)-FLAG-Suc2C, MCA1 (1–338)-FLAG-Suc2C, MCA1 (1–373)-FLAG-Suc2C, and intact MCA1(1– 421)-FLAG-Suc2C, were all insensitive to Endo H.

The intact and truncated forms of MCA2-FLAG gave similarresults (Fig. 3C). Namely, only Suc2C-MCA2 (1–186)-FLAGwas sensitive to Endo H, whereas MCA2 (1–186)-FLAG-Suc2C, MCA2 (1–323)-FLAG-Suc2C, MCA2 (1–363)-FLAG-Suc2C, and intact MCA2 (1– 416)-FLAG-Suc2C were insensi-tive to this enzyme. These results suggest that the N termini ofMCA1 and MCA2 are extracellular and that only pTM1 is anactual transmembrane segment.

In addition to the suggestion about membrane topology, thepresence of the N-glycosylated forms of Suc2C-MCA1-(1–185)-FLAG and Suc2C-MCA2-(1–186)-FLAG suggest thatneither MCA1 nor MCA2 has a signal peptidase-cleavable sig-nal sequence at their N-terminal region (Fig. 3, B and C).

Determination of Membrane Topology by the Yeast Two-hy-brid Method—To confirm the above suggestion, we employedthe split-ubiquitin membrane-based yeast two-hybrid systemas the second method that functions with a different operationprinciple to that of the Suc2C method (25, 36). In this systemthe MCA proteins and their derivatives were fused with theLexA-VP16-Cub cassette at the N terminus or Cub-LexA-VP16cassette at the C terminus, in which Cub is C-terminal ubiqui-tin, and LexA-VP16 is an artificial transcription factor that canbe released by ubiquitin. These fusions were used as bait. Onthe other hand, as prey, the yeast ER membrane protein Alg5was fused with two kinds of Nub (N-terminal ubiquitin), NubIand NubG. The former possessed wild-type Ile13, whereas thelatter carries the Ile13 to Gly mutation. If the LexA-VP16-Cubor Cub-LexA-VP16 moiety of the bait protein is localized on thecytoplasmic side of the ER and plasma membranes, the co-ex-pressed Alg5-NubI forms split-ubiquitin (the native fold ofubiquitin), which cleaves off LexA-VP16 to activate thereporter genes ADE2 and HIS3, the expression of which rendersthe yeast ade2 his3 auxotroph to grow on agar medium lackingadenine and histidine. On the other hand, if the moiety islocated on the extracellular side of the plasma membrane orluminal side of the ER membrane, the co-expressed Alg5-NubI

cannot form split-ubiquitin. The co-expressed Alg5-NubG isused as a negative control, in which NubG cannot form split-ubiquitin even when the Cub-LexA-VP16 moiety is localizedon the cytoplasmic side of the membranes.

Fig. 4A schematically summarizes fusions between MCAproteins (including their truncated derivatives) and the LexA-VP16-Cub or Cub-LexA-VP16 cassette. The fused positionswere the same as those between the MCA proteins and Suc2C

FIGURE 3. Endo H sensitivity of C-terminally truncated forms of MCA1 andMCA2 fused with Suc2C. A, schematic diagram of the truncated derivativesof MCA1 and MCA2 fused with Suc2C. The Suc2C reporter was fused at the Nterminus or downstream of the four putative transmembrane segments(pTM1– 4). MCA1, MCA2, and their truncated derivatives were represented bylight brown-colored boxes. The size of each protein was shown in amino acidnumbers. pTM1– 4 were denoted by brown-colored bars. Suc2C was indicatedby gray-colored boxes. N and C represent the intrinsic N and C termini, respec-tively. The sizes of the proteins and the reporter are arbitrary. The FLAG tagfused to the C terminus of each construct of MCA1 and MCA2 was not drawnin this figure for simplicity. B and C, MCA1, MCA2, and their truncated deriva-tives fused C-terminally with Suc2C are all insensitive to Endo H. The wholecell lysate preparation, Endo H treatment, and Western blotting were per-formed as described under “Experimental Procedures.” Mid1(16Q)-FLAG-Suc2C, a positive control, contains the Asn to Gln mutation at all of the 16putative N-glycosylation sites in Mid1 so that only the Suc2C reporter can beN-glycosylated. Arrows indicate Endo H-sensitive proteins.




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described above (Fig. 3A). The bait and prey plasmids wereco-introduced into the two-hybrid reporter strain NMY32 andthe sequentially diluted cultures of the resulting three indepen-dent transformants were spotted onto synthetic agar mediumwith or without adenine and histidine. As shown in Fig. 4B,upper panels, transformants expressing the MCA1 proteinfused N-terminally with the LexA-VP16-Cub cassette (i.e.LexA-VP16-Cub-MCA1), and NubI was unable to grow onselection medium lacking adenine and histidine. This was alsothe case with those expressing NubG as negative control prey.

In contrast, transformants expressing MCA1 proteins (includ-ing truncated derivatives) fused C-terminally with the Cub-LexA-VP16 cassette (such as MCA1 (1–185)-Cub-LexA-VP16,MCA1 (1–338)-Cub-LexA-VP16, MCA1 (1–373)-Cub-LexA-VP16, and intact MCA1 (1– 421)-Cub-LexA-VP16) were ableto grow on the selection medium when co-expressed with NubIbut not with NubG (Fig. 4, C–F, upper panels). The same resultswere obtained with the MCA2 proteins (Fig. 4B-F, lower pan-els). These results suggest that the N termini of MCA1 andMCA2 are located extracellularly or in the ER lumen, and the C

FIGURE 4. Split-ubiquitin analysis between MCA proteins fused with TF-Cub or Cub-TF and Alg5-NubI. A, schematic diagram of the truncated derivativesof MCA1 and MCA2 fused with TF-Cub or Cub-TF. TF-Cub and Cub-TF represent the LexA-VP16-Cub and Cub-LexA-VP16 cassettes, respectively. TF-Cub wasfused at the N terminus, whereas Cub-TF was fused downstream of pTM1– 4. The cassettes were indicated by gray-colored boxes. Other boxes, bars, and symbolswere the same as those described in the legend to Fig. 3. The sizes of the proteins and cassettes are arbitrary. B–F, growth of yeast cells (strain NMY32)expressing bait (MCA1, MCA2, or their truncated derivatives fused with the cassettes) together with prey (Alg5-NubI or Alg5-NubG (negative control)) on agarmedium lacking adenine and histidine. Exponentially growing cells were harvested and resuspended at 4 � 108 cells/ml, and 5 �l of a 1:10 serial dilution wasspotted on SD agar medium lacking leucine and tryptophan (�LT) with or without adenine and histidine (�AH or �AH) and incubated at 30 °C. The incubationperiod was 2–3 days for control plates (left panels, SD-LT, �AH) and 3– 4 days for test plates (right panels, SD-LT, �AH). In each experiment three independenttransformants with the same combination of bait and prey were examined.




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termini of the intact and truncated forms of both proteins arelocated intracellularly, again suggestive of only pTM1 being theactual transmembrane segment.

Discussion

In the present study we demonstrated that the N termini ofMCA1 and MCA2 are oriented extracellularly, whereas their Ctermini are located in the cytosol, using two different methods,the Suc2C reporter and split-ubiquitin methods. Our resultsalso suggest that only pTM1 near the N terminus is the actualtransmembrane segment among the four putative transmem-brane segments (pTM1– 4) commonly predicted for both pro-teins. Moreover, the presence of N-glycosylation on the Suc2Creporter fused N-terminally to MCA1 and MCA2 suggest thatneither protein has a signal peptidase-cleavable signal sequencenear their N termini. Therefore, we concluded that both pro-teins are single-pass type I transmembrane proteins without acleavable signal sequence (Fig. 5).

This conclusion is consistent with a finding obtained in ourprevious study on the structure-function relationships ofMCA1 and MCA2. In that study we showed that the C-termi-nally truncated forms of MCA1 and MCA2, namely MCA11–

173, MCA21–173, MCA11–185, and MCA21–186, all of which havethe N-terminal pTM1 as the only putative transmembrane seg-

ment, exhibits Ca2� uptake activity (14). Hence, based on thepresent results and previous findings, we hypothesize thatpTM1 is necessary and sufficient for Ca2� permeation. Thishypothesis is supported by the finding that the sole negativelycharged amino acid residue Asp21 located in the pTM1 ofMCA1 and MCA2 is highly conserved during plant evolutionand required for Ca2� permeation (14); deletion of the pTM1segment results in the complete loss of Ca2� permeation activ-ity in MCA1 and MCA2, whereas the Asp21 to Asn substitu-tion leads to the complete loss of activity in MCA1 and apartial loss in MCA2, suggesting that Asp21 is involved inCa2� coordination.

Our conclusion that MCA1 and MCA2 are single-pass typeI transmembrane proteins with a transmembrane segmentlocated near the extracellular N terminus is helpful for specu-lating about the regulatory mechanisms underlying their activ-ities. According to this conclusion, it has become clear that anEF hand-like motif, coiled-coil motif, and plac8 motif are allpresent in the cytosol. The EF hand-like motif can sense[Ca2�]cyt needed to regulate the Ca2� permeation activities ofMCA1 and MCA2. We previously reported that MCA11–135

lacking this motif loses this activity, whereas MCA21–135 doesnot, suggesting that the motif regulates the two proteins differ-

FIGURE 5. A model for the transmembrane topology of MCA1 and MCA2 based on the present study. This figure was drawn using the TOPO2 program (48).MCA1 and MCA2 both have one transmembrane segment, with the N termini being located extracellularly, and C termini located intracellularly. In thetransmembrane segment Asp was highlighted by red, Gln and Asn were in magenta, and Lys was in purple. Note that these hydrophilic amino acid residues arevertically disposed in an array in the transmembrane helix. This feature may enable the hydrophilic residues to face each other and the hydrophobic residuesto face out toward the lipid bilayer in order to form the pore of a channel composed of four subunits (14). In the cytoplasm amino acid residues in the EFhand-like motif are blue, those in the coiled-coil motif are green, and those in the plac8 motif are brown.




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entially (14). Coiled-coil motifs have been shown to mediateprotein-protein interactions. Our previous findings suggestedthat this motif is not required for making a Ca2�-permeablechannel because MCA1 and MCA2 lacking the coiled-coil motif (i.e. MCA11–173, MCA21–173, MCA11–185, andMCA21–186) maintained Ca2� permeation activity. Thecoiled-coil motif appears to regulate the two proteins differ-entially because MCA11–237 carrying the motif does not havethis activity, whereas MCA21–237 does (14).

In some proteins, the plac8 motif has been suggested to forma pore-forming channel (37, 38). However, as discussed in ourprevious study, the plac8 motif of MCA1 and MCA2 is unlikelyto participate in forming a channel (14). We herein only showthat the C-terminally truncated MCA1 and MCA2 lacking themotif, such as MCA11–173 and MCA21–173, exhibit Ca2� per-meation activities. Therefore, this motif may be unnecessary forforming a channel.

The results of the present study suggest that MCA1 andMCA2 are structurally unique MS channels in terms of thenumber of transmembrane segments. To date, the polypeptidesof all MS channels studied structurally have multiple trans-membrane segments. For example, the polypeptides of bacte-rial MscS and MscL have three and two transmembranesegments, respectively (39, 40). Moreover, seven MscS poly-peptides and five MscL polypeptides assemble as subunits toform a pore-forming channel. Therefore, the MS channelsMscS and MscL are composed of a large number of transmem-brane segments (i.e. 21 and 10, respectively). In addition, thepolypeptides of plant MS channels MSL (for MscS-like) andOSCA1 have been predicted to have five to six and nine trans-membrane segments, respectively; however, their transmem-brane topologies have not yet been determined (19, 41). In con-trast, the polypeptides of MCA1 and MCA2 have a singletransmembrane segment, as shown in this study, and assembleas a homotetramer to form a Ca2�-permeable channel (14, 42).Chloride intracellular channels (CLICs) in mammals may havea single N-terminal transmembrane segment, but the numberof subunits has not yet been established (43– 46). In this con-text, MCA1 and MCA2 are structurally unique among MSchannels, and thus, elucidation of the molecular mechanisms ofMCA1 and MCA2 involved in sensing mechanical stresses,including hypo-osmotic stress and membrane stretch, and inpermeating Ca2� is important.

Author Contributions—S. Kamano performed the experiments bythe Suc2C method and revised the paper. S. Kume performed theexperiments using the yeast two-hybrid system and revised thepaper. K. I. designed the Suc2C method, constructed most plas-mids for Suc2C fusions and the yeast two-hybrid system, andrevised the paper. K.-J. L., M. N., and Y. N. helped with the exper-iments and revised the paper. H. I. designed and coordinated thestudy and wrote and revised the paper. All authors reviewed theresults and approved the final version of the manuscript.

Acknowledgments—We thank Chikara Tanaka in our laboratory forhis technical advice on the yeast two-hybrid system, Dr. DominiqueSanglard of University Hospital Lausanne for the plasmid pCS4 –14[SUC2-HIS4C URA3], and Yumiko Higashi for secretarial assistance.

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Yoshitaka Nakayama and Hidetoshi IidaShumpei Kamano, Shinichiro Kume, Kazuko Iida, Kai-Jian Lei, Masataka Nakano,

Arabidopsis thalianaMCA1 and MCA2 in -permeable Mechanosensitive Channels2+Transmembrane Topologies of Ca

doi: 10.1074/jbc.M115.692574 originally published online November 10, 20152015, 290:30901-30909.J. Biol. Chem.

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