Studies on Vacuolar Membrane Microdomains Isolated from Arabidopsis Suspension-Cultured Cells: Local Distribution of Vacuolar Membrane Proteins

Abstract

The local distribution of both the vacuolar-type proton ATPase (V-ATPase) and the vacuolar-type proton pyrophosphatase (V-PPase), the main vacuolar proton pumps, was investigated in intact vacuoles isolated from Arabidopsis suspension-cultured cells. Fluorescent immunostaining showed that V-PPase was distributed evenly on the vacuolar membrane (VM), but V-ATPase localized to specific regions of the VM. We hypothesize that there may be membrane microdomains on the VM. To confirm this hypothesis, we prepared detergent-resistant membranes (DRMs) from the VM in accordance with well established conventional methods. Analyses of fatty acid composition suggested that DRMs had more saturated fatty acids compared with the whole VM in phosphatidylcholine and phosphatidylethanolamine. In the proteomic analyses of both DRMs and detergent-soluble mebranes (DSMs), we confirmed the different local distributions of V-ATPase and V-PPase. The observations of DRMs with an electron microscope supported the existence of different areas on the VM. Moreover, it was observed using total internal reflection fluorescent microscopy (TIRFM) that proton pumps were frequently immobilized at specific sites on the VM. In the proteomic analyses, we also found that many other vacuolar membrane proteins are distributed differently in DRMs and DSMs. Based on the results of this study, we discuss the possibility that VM microdomains might contribute to vacuolar dynamics.

Introduction

The plant vacuole is the largest organelle that is enveloped with a single lipid bilayer, the tonoplast. Vacuoles play important roles in plant cell function, such as space filling, storage of inorganic ions and metabolites, protein degradation, detoxification and control of ionic homeostasis of the cytoplasm. There are many kinds of transmembrane proteins and peripheral proteins involved in vacuolar activity. Proteomic analyses of the vacuolar membrane (VM) (Carter et al. 2004, Shimaoka et al. 2004, Jaquinoid et al. 2007, Schmidt et al. 2007, Whiteman et al. 2008) have revealed that in excess of 100 proteins are likely to be involved in VM activities. However, the functions of most of these proteins are still unknown (Martinoia et al. 2012).

In plant vacuoles, the two enzymes, vacuolar type H⁺-ATPase (V-ATPase) and vacuolar type H⁺-pyrophosphatase (V-PPase), function to generate a proton motive force for secondary transport systems and maintain the acidic conditions in the vacuole (Maeshima 2001, Kluge et al. 2003, Martinoia et al. 2007). V-ATPase is a multisubunit enzyme composed of two complexes (the V_o complex comprising up to six subunits is membrane integral; the V₁ complex consisting of eight subunits is bound to the V_o complex outside the membrane). V-PPase is a highly hydrophobic single subunit protein, which is believed to function as a dimer or oligomer (Maeshima 2001, Mimura et al. 2005). It is known that both the V-ATPase and the V-PPase localize to the same VM in Chara intermodal cells (Shimmen and MacRobbie 1987). It is not known, however, the reason why the vacuole requires two types of proton pump in the same membrane.

In animal cells, the plasma membrane (PM) is composed of liquid-ordered domains enriched in cholesterol/sphingolipids which co-exist with liquid crystalline domains rich in phospholipids containing unsaturated fatty acids. These membrane domains were termed lipid rafts by Simons and Ikonen (1997) and can be isolated as detergent resistant-membranes (DRMs). Recently it has been reported that PM microdomains also exist in the plant PM (Peskin et al. 2000, Mongrand et al. 2004, Bhat and Panstruga 2005, Borner et al. 2005, Laloi et al. 2007, Zappel and Panstruga 2008, Simon-Plas et al. 2011, Cacas et al. 2012). In organelle membranes, it has been reported that V-ATPase locates in DRMs of membrane vesicles of the endocytic pathway in animal cells (Lafourcade et al. 2008). Very recently, Ozolina et al. (2013) described the lipid composition of the DRMs of vacuoles isolated from Beta vulgaris. However, the biological roles of microdomains of both the plant PM and the VM have yet to be established. In the present study, we have attempted to clarify how proton pumps distribute on the VM and have found evidence for the presence of microdomains in the VM.

Results

Distributions of V-ATPase and V-PPase on isolated vacuoles

In order to visualize the distribution of V-ATPases and V-PPases, isolated intact vacuoles were immunostained and observed with a confocal laser microscope (Fig. 1). Counterstaining of the immunostained vacuole with FM 4-64, a lipophilic dye, to visualize the VM (Fig. 1B, F) frequently stained intravacuolar structures (Fig. 1F). Fluorescence from V-ATPase or V-PPase overlapped with that of FM 4-64 (Fig. 1C, G) in the VM, and it appeared that V-ATPase was clustered in small areas on the isolated vacuole, but V-PPase was more evenly distributed (Fig. 1A, E).

Fig. 1

Distribution of proton pumps on an isolated vacuole. The localization of V-ATPase and V-PPase was observed using immunostaining with a laser confocal microscope (A and E, respectively). To visualize the VM, it was co-stained with FM 4-64 (B, F). (C and G) Merged images. DIC images are shown on the right (D, H). (I and J) Simultaneous observations of V-ATPase and V-PPase on an isolated vacuole. (I) Confocal image at the equatorial plane of the isolated vacuole. (J) The surface image of the isolated vacuole. Green (I, J), V-ATPase. Magenta (I, J), V-PPase. Bars = 10 = µm.

The differential distribution of V-ATPase and V-PPase was confirmed by staining them together on the same VM and visualizing them at different wavelengths (Fig. 1I). When focused on the equatorial plane of the vacuole, the fluorescence from the V-ATPase was observed as discrete large dots at only a few locations, whereas the fluorescence from the V-PPase was relatively even. The variation in distribution was more obvious when focusing on the surface of the vacuole (Fig. 1J), which revealed a large number of small fluorescent spots (magenta: V-PPase) and a smaller number of much large spots (green: V-ATPase).

Behavior of individual molecules in an isolated vacuole

Fig. 2A and B shows the fluorescent images of V-ATPase or V-PPase captured by total internal reflection fluorescent microscopy (TIRFM). Almost all fluorescent signals actively moved on the membrane (Supplementary Videos S1, S2). The behavior of fluorescent spots was traced for 60 frames (2 s) and plotted (Fig. 2C, D). Both V-ATPase and V-PPase often remained still at certain places on the VM for a short time. Diffusion coefficients were calculated from the trace of fluorescence signals for 30 frames (1 s). The coefficients of V-ATPase were calculated from 154 traces, and that of V-PPase from 157 traces. Fig. 2E shows the distribution of all calculated values. The average values were similar (V-ATPase, 0.832 ± 0.442 µm² s⁻¹; V-PPase, 0.879 ± 0.398 µm² s⁻¹), but the medians were clearly different (V-ATPase, 0.735 µm² s⁻¹; V-PPase, 0.810 µm² s⁻¹) (P = 0.0456).

Fig. 2

Single molecule behavior of V-ATPase and V-PPase. V-ATPase and V-PPase were visualized using immunostaining, and the fluorescent images were captured by TIRFM (A, V-ATPase; B, V-PPase). The motion of the fluorescent signals was traced for 2 s at 30 frames s⁻¹ (C, V-ATPase; D, V-PPase). The starting point of the trace represents the origin. Diffusion coefficients were calculated from traces over 1 s (30 frames). The distribution of diffusion coefficients is shown in E (green bar, V-ATPase; magenta bar, V-PPase). Median values are indicated by arrowheads (green, V-ATPase, magenta, V-PPase). Bar = 5 µm (A, B).

Molecular analyses of the VM and vacuolar DRMs

We hypothesized that the different behavior of the two H⁺ pumps on the same vacuole resulted from the existence of membrane microdomains in the VM. Thus, DRMs and DSMs (detergent-soluble membranes) of the isolated intact vacuoles were purified by conventional methods. DRM and DSM proteins (10 µg) were separated on an SDS–polyacrylamide gel (Fig. 3A). The band patterns visualized with silver staining were different between DRMs and DSMs. In order to compare the V-ATPase and V-PPase content of each fraction, we conducted Western blotting using purified specific antibodies against V-ATPase A-subunit (VHA-A), V-ATPase E-subunit (VHA-E) or V-PPase (AVP1). Both VHA-A and VHA-E subunit proteins were more strongly detected in the DRM fraction than in the DSM fraction (Fig. 3B, C). On the other hand, V-PPase (AVP1) was more abundant in the DSM fraction (Fig. 3D).

Fig. 3

Molecular analyses of vacuolar DRMs. (A–D) Western blot analyses. (A) Proteins contained in the vacuolar DRM and DSM fraction (10 mg) were loaded onto a 10% acrylamide gel, separated with SDS–PAGE and silver stained. Western blotting was used to reveal proton pumps: (B) VHA-A. (C) VHA-E. (D) AVP-1. (E–J) Lipid and fatty acid composition of the whole VMs and vacuolar DRMs. Lipid extracts were separated with the positive mode of liquid chromatography by their retention time. (E) VMs, (F) vacuolar DRMs. Mass spectra of PC (G, H) and PE (I, J) were analyzed in the whole VM (G, I) and vacuolar DRM (H, J), respectively. The numbers following the value of m/z indicate the number of carbons and the number of double bonds in two fatty acid chains.

The lipid composition of the whole VM and the vacuolar DRMs were analysed by liquid chromatography–tandem mass spectromety (LC-MS/MS) (Fig. 3E–J). Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylglycerol (PG) and lysophosphatidylcholine (LysoPC) were detected in the VM. Peak 1 and Peak 2 in Fig. 3E show that the VM contains other lipid species that do not correspond to the common phospholipids. The DRM fraction mainly consisted of PE and PC (Fig. 3F). Glycosylceramide (GlcCer) was also detected in both fractions with a retention time around 7.5–8.5 min.

The fatty acid composition was also analyzed by LC-MS/MS. Fig. 3G, H and I, J shows the distribution of the m/z for PC and PE. The number of carbon and double bonds in fatty acids were presented as the sum of the two fatty acids of each lipid molecule. DRMs had more saturated fatty acids (Fig. 3H, J) compared with the whole VM in PC and PE (Fig. 3G, I). As for GlcCers, variation in the saturation ratio was observed in the very long chain fatty acids (Supplementary Fig. S1).

Microstructure of the VM and vacuolar DRM

The microstructures of the VM and vacuolar DRM were also examined with an electron microscope (Fig. 4). The VM was observed as aggregates of small membrane vesicles (Fig. 4A, C), whereas the vacuolar DRM was observed as membrane fragments (Fig. 4B). Each fragment of the vacuolar DRM was very small with a large curvature (Fig. 4D, E). Further, we confirmed the accumulation of V-ATPase on the vacuolar DRM with immunoelectron microscopy (Fig. 4F–I). Fig. 4F and G shows the localization of gold particles indicating V-ATPase or V-PPase on the VM. Both antibodies showed similar distributions. This was also confirmed using the isolated VM fractions (Supplementary Fig. S2). In the vacuolar DRM fraction, gold particles against V-ATPase (Fig. 4H) located much more than those against V-PPase (Fig. 4I).

Fig. 4

Observation of the microstructure of the VM and vacuolar DRM. The pellets of total VM (A, C) and vacuolar DRM (B, D and E) were observed with transmission electron microscopy. Immunoelectron microscopic observation was also carried with whole cells (F, G) and vacuolar DRM (H, I) with antibodies against V-ATPase (F, H) or V-PPase (G, I). Bar = 1 µm (A, B), 200 nm (C, D), 100 nm (E), 500 nm (F, G, H, I).

Proteomic analysis of the vacuolar DRMs and DSMs

The composition of the membrane fractions was examined by proteomic analysis. The analyses were conducted three times and revealed 474 unique proteins. Of these, 382 were found in DSMs and 185 in DRMs. All identified proteins are listed in Supplementary Table S1. In Tables 1–3, proteins which have >5 matched peptides are listed.

Table 1

Vacuolar proton pumps identified by proteomic analysis

Locus	Description (TAIR 10)	Footnote	Matched peptides in DSMs	Matched peptides in DRMs	Mol. wt (kDa)	Transmembrane domain
V-ATPase V1 subunits
AT1G78900.1	Vacuolar ATP synthase subunit A	VHA-A	139	1,307	69.1	0
AT1G76030.1	ATPase, V1 complex, subunit B protein	VHA-B1	–	809	54.2	0
AT4G38510.1	ATPase, V1 complex, subunit B protein	VHA-B2	185	752	54.4	0
AT1G20260.1	ATPase, V1 complex, subunit B protein	VHA-B3	198	804	54.4	0
AT1G12840.1	Vacuolar ATP synthase subunit C (VATC)/V-ATPase C subunit/vacuolar proton pump C subunit (DET3)	VHA-C	150	462	42.9	0
AT3G58730.1	Vacuolar ATP synthase subunit D (VATD)/V-ATPase D subunit/vacuolar proton pump D subunit (VATPD)	VHA-D	46	174	29.1	0
AT4G11150.1	Vacuolar ATP synthase subunit E1	VHA-E1	183	520	26.3	0
AT3G08560.1	Vacuolar H+-ATPase subunit E isoform 2	VHA-E2	–	107	27.0	0
AT1G64200.1	Vacuolar H+-ATPase subunit E isoform 3	VHA-E3	–	324	27.4	0
AT3G01390.1	Vacuolar membrane ATPase 10	VHA-G1	–	146	12.4	0
AT3G42050.1	Vacuolar ATP synthase subunit H family protein	VHA-H	127	383	50.7	0
V-ATPase V0 subunits
AT2G21410.1	Vacuolar proton ATPase A2	VHA-a2	144	646	93.6	6
AT4G39080.1	Vacuolar proton ATPase A3	VHA-a3	265	929	93.3	6
AT1G19910.1	ATPase, F0/V0 complex, subunit C protein	VHA-c2	16	114	16.7	4
AT2G25610.1	ATPase, F0/V0 complex, subunit C protein	VHA-c¢¢2	–	26	18.4	4
AT3G28710.1	ATPase, V0/A0 complex, subunit C/D	VHA-d1	71	354	41.3	0
AT3G28715.1	ATPase, V0/A0 complex, subunit C/D	VHA-d2	–	342	41.3	0
V-PPase
AT1G15690.1	Inorganic H pyrophosphatase family protein	AVP1	223	158	81.5	16
V-ATPase related
AT1G16820.1	Vacuolar ATP synthase catalytic subunit-related/V-ATPase-related/vacuolar proton pump-related		8	48	10.5	0

Table 2

Transporters and ion channels identified by proteomic analysis

Locus	Description (TAIR 10)	Footnote	Matched peptides in DSMs	Matched peptides in DRMs	Mol. wt (kDa)	Transmembrane domain
ABC transporters
AT2G36910.1	ATP binding cassette subfamily B1	ABCB1/ATPGP1	–	21	141.3	12
AT4G01820.1	P-glycoprotein 3	ABCB3/MDR3/PGP3	–	14	133.8	11
AT2G47000.1	ATP binding cassette subfamily B4	ABCB4/MDR4/PGP4	–	30	139.4	11
AT3G28860.1	ATP binding cassette subfamily B19	ABCB19/PGP19	–	9	137.2	12
AT5G39040.1	Transporter associated with antigen processing protein 2	ABCB27/ATTAP2	87	55	69.3	5
AT1G30400.1	Multidrug resistance-associated protein 1	ABCC1/ATMRP1	45	18	182.7	15
AT2G34660.1	Multidrug resistance-associated protein 2	ABCC2/ATMRP2	56	–	182.8	14
AT3G13080.1	Multidrug resistance-associated protein 3	ABCC3/ATMRP3	154	56	170.1	16
AT2G47800.1	Multidrug resistance-associated protein 4	ABCC4/ATMRP4	91	71	170.1	16
AT1G04120.1	Multidrug resistance-associated protein 5	ABCC5/ATMRP5	43	–	170.1	14
AT3G13090.1	Multidrug resistance-associated protein 8	ABCC6/ATMRP8	26	16	166.0	15
AT3G13100.1	Multidrug resistance-associated protein 7	ABCC7/ATMRP7	22	16	169.2	16
AT3G21250.1	Multidrug resistance-associated protein 6	ABCC8/ATMRP6	151	79	162.2	10
AT3G60160.1	Multidrug resistance-associated protein 9	ABCC9/ATMRP9	11	–	169.5	16
AT3G62700.1	Multidrug resistance-associated protein 10	ABCC14/ATMRP10	57	44	173.2	16
AT2G07680.1	Multidrug resistance-associated protein 11	ABCC13/ATMRP11	9	7	159.3	9
AT4G15236.1	ABC-2 and plant PDR ABC-type transporter family protein	ABCG43	–	44	158.8	13
AT1G67940.1	Non-intrinsic ABC protein 3	ABCI17/ATNAP3	6	–	28.7	0
AT1G71330.1	Non-intrinsic ABC protein 5	NAP5	15	–	36.1	2
MATE family
AT1G33110.1	MATE efflux family protein		8	–	54.2	12
AT3G21690.1	MATE efflux family protein		68	29	55.4	12
AT3G26590.1	MATE efflux family protein		26	4	54.7	12
AT1G61890.1	MATE efflux family protein		13	–	55.5	12
AT3G23560.1	MATE efflux family protein		13	–	52.4	12
Ion transporter
AT1G53210.1	Sodium/calcium exchanger family protein		85	74	63.9	11
AT1G16010.1	Magnesium transporter 2		5	–	50.6	2
AT1G29820.1	Magnesium transporter CorA-like family protein		16	–	61.2	2
AT2G46800.1	Zinc transporter of Arabidopsis thaliana		8	–	44.1	6
AT5G20650.1	Copper transporter 5		8	13	15.9	3
AT2G23150.1	Natural resistance-associated macrophage protein 3	NRAMP3	22	87	56.4	12
AT2G22950.1	Cation transporter/E1-E2 ATPase family protein		18	–	111.7	10
Sugar transporter
AT2G48020.1	Major facilitator superfamily protein	Sugar transporter	–	20	50.1	12
AT5G14120.1	Major facilitator superfamily protein		12	–	63.6	13
AT5G17010.1	Major facilitator superfamily protein	Sugar transporter	9	–	54.0	12
AT1G75220.1	Major facilitator superfamily protein		14	17	53.2	12
AT5G27360.1	Major facilitator superfamily protein		–	7	52.9	11
AT1G71880.1	Sucrose-proton symporter 1		8	23	55.2	12
AT4G35300.1	Tonoplast monosaccharide transporter2		7	2	80.2	12
AT3G03090.1	Vacuolar glucose transporter 1		9	–	54.0	12
AT3G11320.1	Nucleotide-sugar transporter family protein		–	5	34.1	10
Peptide/amino acid transporter
AT2G02040.1	Peptide transporter 2		85	42	65.1	11
ATPase
AT3G57330.1	Autoinhibited Ca2+-ATPase 11		200	113	112.8	10
AT2G41560.1	Autoinhibited Ca(2+)-ATPase, isoform 4		125	65	113.5	10
AT1G27770.1	Autoinhibited Ca2+-ATPase 1		22	–	112.2	10
AT5G44240.1	Aminophospholipid ATPase 2		–	22	130.2	10
Channels
AT5G12080.1	Mechanosensitive channel of small conductance-like 10	Mechanosensitive ion channel	13	–	83.3	6
AT4G03560.1	Two-pore channel 1	Voltage-gated calcium channel	51	22	85.4	12
AT5G33280.1	Voltage-gated chloride channel family protein		–	12	84.7	11
AT2G16850.1	Plasma membrane intrinsic protein 2;8	Water channel	9	15	29.7	6
AT4G35100.1	Plasma membrane intrinsic protein 3	PIP2;7 water channel	–	26	30.0	6

Table 3

Cytoskeleton- and membrane traffic-related proteins identified by proteomic analysis

Locus	Description (TAIR 10)	Footnote	Matched peptides in DSMs	Matched peptides in DRMs	Mol. wt (kDa)	Transmembrane domain
Cytoskeleton
AT5G09810.1	Actin 7		36	–	41.9	0
AT1G64330.1	Myosin heavy chain-related		73	–	64.7	0
AT1G14830.1	DYNAMIN-like 1C		8	11	69.0	0
AT3G60190.1	DYNAMIN-like 1E		7	12	70.1	0
AT1G59610.1	Dynamin-like 3		–	32	100.5	0
AT5G42080.1	Dynamin-like protein		–	59	68.5	0
AT1G10290.1	Dynamin-like protein 6		–	21	99.4	0
AT2G14120.1	Dynamin-related protein		14	–	86.9	0
AT4G33650.1	Dynamin-related protein 3A		8	–	90.0	0
Clathrin and coatmer proteins
AT3G08530.1	Clathrin, heavy chain		129	17	194.4	0
AT3G11130.1	Clathrin, heavy chain		134	–	194.4	0
AT4G34450.1	Coatomer gamma-2 subunit, putative/gamma-2 coat protein, putative/gamma-2 COP, putative		40	–	99.1	0
SNARE related
AT5G39510.1	Vesicle transport v-SNARE family protein	ATVTI11/SGR4	5	11	25.0	1
AT1G04750.1	Vesicle-associated membrane protein 721		5	–	25.1	1
AT3G56190.1	alpha-soluble NSF attachment protein 2		21	15	33.2	0
AT4G20410.1	gamma-soluble NSF attachment protein		15	–	32.7	0
ESCRT-III complex
AT1G73030.1	SNF7 family protein	VPS46.2	–	19	22.7	0
AT2G06530.1	SNF7 family protein	VPS2.1	7	18	25.3	0
AT2G19830.1	SNF7 family protein	SNF7.2	–	25	24.1	0
AT4G29160.1	SNF7 family protein	SNF7.1; AtVPS32.2	–	22	24.4	0
AT5G22950.1	SNF7 family protein	VPS24.1	–	10	26.0	0
AT1G34220.2	Regulator of Vps4 activity in the MVB pathway protein		–	28	69.2	0
AT2G44100.1	Guanosine nucleotide diphosphate dissociation inhibitor 1	ATGDI1	28	–	50.2	0
AT3G52850.1	Vacuolar sorting receptor homolog 1	VSR-1/ATELP1/BP-80	15	–	70.9	2
Small G protein-related proteins
AT5G45750.1	RAB GTPase homolog A1C	AtRABA1c	9	–	24.0	0
AT1G07410.1	RAB GTPase homolog A2B	AtRABA2b	6	–	23.8	0
AT4G17530.1	RAB GTPase homolog 1C	AtRABD2c/RAB1C	6	–	22.5	0
AT1G49300.1	RAB GTPase homolog G3E	AtRABG3e/ATRAB7	7	–	23.3	0
AT1G22740.1	RAB GTPase homolog G3B	AtRABG3b	5	–	23.3	0
AT3G09900.1	RAB GTPase homolog E1E	AtRABE1e	10	–	24.5	0
AT1G02130.1	RAS 5		6	–	22.8	0
SPFH domain-containing proteins
AT5G62740.1	SPFH/Band 7/PHB domain-containing membrane-associated protein family		57	116	31.7	0
AT1G69840.1	SPFH/Band 7/PHB domain-containing membrane-associated protein family		25	75	31.7	0
AT3G01290.1	SPFH/Band 7/PHB domain-containing membrane-associated protein family		32	100	31.5	0
AT5G51570.1	SPFH/Band 7/PHB domain-containing membrane-associated protein family		61	74	32.6	0

Tables 1–3 and Supplementary Table S1 show identified proteins with the locus, annotations by TAIR 10, the number of matched peptides and the number of transmembrane domains. The numbers of matched peptides indicate the total number of peptides detected in one experiment, and are shown for a rough estimation of the total amount of each protein.

Table 1 shows the list of subunits of the proton pumps. Almost all subunits of V-ATPase were identified in DRMs. The numbers of matched peptides of these subunits in DRMs significantly exceeded those in DSMs. The numbers of VHA-E2 and VHA-a1 were small compared with those of other isoforms. AVP1 was slightly more prevalent in DSMs than in DRMs. Many types of transporters and channels were found in both fractions (Table 2; Supplementary Table S1). Nineteen ABC transporters (five ABCB type, 11 ABCC type, one ABCG type, one ABCI type and one other ABC transporter) were identified in the present study. Most of the ABCC-type transporters were mainly present in DSMs, while more ABCB-type and ABCG-type transporters were detected in DRMs than in DSMs. MATE family transporters were mainly identified in DSMs, while ion transporters, sugar transporters, Ca²⁺-ATPase and channels were detected in both DRMs and DSMs. Membrane traffic-related proteins were identified (Table 3). In particular, ESCRT-III complex-related proteins were localized in DRMs, and small G proteins were localized in DSMs. Some dynamin-like proteins were identified in DRMs. Band 7 family proteins were detected in DRMs much more than in DSMs.

Discussion

Isolated vacuoles display proton pumping activity

Intact vacuoles were isolated from Arabidopsis suspension-cultured cells. Naked vacuoles have a spherical shape when the surrounding cytoplasm is removed and replaced by an artificial medium. In the present study, we confirmed that the integrity of the VM remained intact and that both V-ATPase and V-PPase remained active after isolation of vacuoles by monitoring the acidification of the vacuoles with neutral red following the addition of ATP or pyrophosphate (PPi) (Supplementary Fig. S3).

Distribution of proton pumps on the VM

In the Western blot analysis, most V-ATPases were found in the vacuolar DRMs (Fig. 4) which suggests that V-ATPase is mainly located in VM microdomains. On the other hand, V-PPase (AVP1) was mostly found in DSMs rather than in DRMs. Proteomic analysis also showed that subunits of V-ATPase were detected in DRMs much more than in DSMs (Table 1). These differences in distribution of proton pumps seem to indicate physical compartmentation within the VM.

In previous reports, V-ATPase has often been detected in DRMs of membrane fractions in animal cells (Lafourcade et al. 2008). In plant cells, V-ATPase has also been detected in DRMs of the tobacco PM fraction (Mongrand et al. 2004, Morel et al. 2006). Although it is unclear whether the V-ATPase in the tobacco PM DRM is a contaminant or a real PM protein, it does show that plant V-ATPase locates in operationally defined DRMs.

We also tried to visualize the distribution of vacuolar H⁺ pumps in a whole cell. Fluorescence derived from V-PPase was observed more or less evenly around the VM, but that from V-ATPase was not (Supplementary Fig. S3). In isolated maize root cells, V-ATPase was shown to be localized in the VM where its distribution appeared to be non-uniform (Kluge et al. 2004). Observation of the VM in a cell is very difficult because the boundary surface between the VM and cytoplasm is not clear, and also because vacuoles in a cell have complex morphologies. Plant vacuoles can have special features such as a spherical structure known as a ‘bulb’ (Saito et al. 2002), an intravacuolar sheet in the vacuolar lumen (Uemura et al. 2002) and tubular vacuoles (Hicks et al. 2004). Saito et al. (2002) showed that γ-TIP occurred in the ‘bulbs’, but AtRab75c did not. Their results suggested that different proteins associated with the VM can be compartmented into specific membrane regions. The finding that V-ATPase was found in DRMs, whereas V-PPase was found in DSMs, lends support to this hypothesis.

V-ATPase is a multisubunit enzyme composed of two complexes. The V_o complex comprising up to six subunits is membrane integral. The V₁ complex consisting of eight subunits is bound to the V_o complex outside the membrane. Some subunits have 2–5 isoforms. For example, the VHA-a subunit has three isofoms, VHA-a1, VHA-a2 and VHA-a3. Dettmer et al. (2006) reported that VHA-a2 and VHA-a3 are localized on the VM, while VHA-a1 is localized on the trans-Golgi network (TGN). VHA-a1 is specifically involved in TGN functions. The V-ATPase B and C subunits are known to be involved in interaction with the actin cytoskeleton. Ma et al. (2012) reported that Arabidopsis V-ATPase B subunits (VHA-B1, B2 and B3) showed different activities in capping the barbed ends of actin microfilaments. They described different tissue-specific gene expression using the Arabidopsis eFP browser. In almost all tissues, gene expression of VHA-B2 was lower than that of B1 or B3; however, in mature pollen, gene expression of VHA-B2 was higher. Thus, it appears that each subunit of the V-ATPase has unique tissue specificity in terms of gene expression and intracellular localization.

In the present study, VHA-a1 was detected in two of three repetitions, but only in small amounts (data not shown). This indicates that our isolated vacuole and VM specimens contained very little contaminating TGN. Interestingly, the number of matched peptides of VHA-E2 was smaller than that of VHA-E1 or VHA-E3. In Arabidopsis, VHA-G has three isoforms (VHA-G1, VHA-G2 and VHA-G3), but we could detect only VHA-G1. There is a possibility that VHA-E or VHA-G isoforms may share their roles, but further experiments will be necessary to support this speculation.

Behavior of proton pumps

The observation with TIRFM revealed the dynamic nature of membrane proteins on the isolated VM. All fluorescent signals moved rapidly on the surface (Supplementary Videos S1, S2). The coefficients of distribution were very high for both V-ATPase and V-PPase (means >0.83 µm² s⁻¹) compared with other membrane proteins (viral coat protein, 0.48 µm² s⁻¹; bovine rhodopsin, 0.33 µm² s⁻¹) (Almeida and Vaz 1995). The high fluidity is most probably due to the lipid composition and/or protein : lipid ratio of VMs. The coefficient of distribution of V-ATPase was slightly lower than that of V-PPase (Fig. 2), which suggests that V-ATPases were trapped in a region resembling a membrane microdomain more frequently or for longer than V-PPase, by unknown mechanisms. In an intact cell, cytoplasmic cellular components such as the cytoskeleton may interact with the VM proteins and the vacuole may therefore have a more complex structure. Thus, the behavior of organellar membrane proteins may also be more complex in vivo.

Analysis of the vacuolar DRM

Since the microstructure of the DRM is important in relation to its functions, we investigated the microstructure of the VM and vacuolar DRM with an electron microscope (Fig. 4). Several previous studies have examined the ultrastructure of the plant PM DRM (Mongrand et al. 2004, Lefebvre et al. 2007), which revealed the PM DRMs as parallel membrane sheets. In our study, VM was observed as aggregates of small membrane vesicles, whereas vacuolar DRM was observed as membrane fragments. Each fragment of the vacuolar DRM was very small with a high degree of curvature (Fig. 4). Such curved membrane structures have not been reported in previous studies on PM DRMs (Mongrand et al. 2004, Lefebvre et al. 2007). This morphological difference in DRMs might arise due to differences in protein or lipid composition, but these curved structures were not observed in the whole VM.

Previous studies have reported that VMs were mainly composed of PC and PE and that other phospholipids were only minor components (Yoshida and Uemura 1986, Brown and DuPont 1989, Tavernier et al. 1993). In this study, PC and PE were also found to be the main phospholipids in the Arabidopsis VM.

Phospholipids in the PM DRM have more highly saturated fatty acids (Mongrand et al. 2004, Borner et al. 2005) than those in the total PM. Similarly in the vacuolar DRM, most of the identified fatty acids had higher degrees of saturation than fatty acids for the whole VM (Fig. 3G–J; Supplementary Fig. S1). The fact that the vacuolar DRMs had highly saturated fatty acids does not conflict with the general concept of membrane microdomains.

Cholesterol is thought to contribute to the tight packing of liquid-ordered domains by filling interstitial space between lipid molecules (Brown 1998). Recent studies have shown that the sterol composition of plant VM varies between different plant species (Marty and Branton 1980, Yoshida and Uemura 1986, Tavernier et al. 1993), and these sterols might contribute to the formation of the VM microdomains. However, we could not measure sterols in our samples. Very recently, Ozolina et al. (2013) reported the presence of DRMs in the VM of B. vulgaris and showed that the tonoplast microdomains are rich in sphingolipids, free sterols and saturated fatty acids.

The putative roles of VM microdomains

The proteomic analysis provided some novel insight into the composition of the VM and differences between the DRMs and DSMs. Solute transporters and sugar transporters were identified in DRMs, and we speculate that the V-ATPase and these transporters were co-localized in the VM microdomains so as to utilize the proton gradient generated by V-ATPase most effectively. On the other hand, ABC transporters which transport a solute with energy from ATP were localized in DSMs. Domain-dependent localization of different kinds of proteins may be relevant to the observation that some channels and transporters oligomerize to function (Maeshima 2001) and the VM microdomains might therefore provide an appropriate environment for dimerization or oligomerization of those membrane proteins.

In this study, some PM proteins appeared to be contaminants in the VM fraction. However, it is known that PM proteins are recycled by the endocytic pathway (Kleine-Vehn and Friml 2008), and it is possible that we detected proteins which were on this recycling pathway as contaminant proteins. Shimaoka et al. (2004) showed that VMs prepared with this method did not contain PM and endoplasmic reticulum membrane, but it is possible that the isolated vacuoles contained other endomembranes (Golgi apparatus, endosome or pre-vacuolar compartment). Some proteins identified in this study have previously been reported to be associated with other membranes. For example, proteins belonging to the Rab family of small GTPases were identified in the DSM (Table 2; Supplementary Table S1). It has been proposed that Rab family proteins are involved in the regulation of endomembrane trafficking, and that different Rab GTPases regulate trafficking between different membrane compartments (Lycett 2008). The existence of Rab GTPases suggests that the VMs prepared in this study have small components of other endomembranes.

Interestingly, some SNF7 family proteins, which are part of the endosomal sorting complex required for the transport (ESCRT)-III complex, were clearly localized in DRM fractions (Table 3). It is thought that the ESCRT complex is involved in sorting of transmembrane proteins into the vacuole. ESCRT-0, I and II complexes recruit the ubiquitinated cargos on endosomal membranes, and ESCRT-III complex sorts the cargos into the intraluminal vesicles of the multivesicular bodies (MVBs) by exerting membrane bending, scission and fusion. The plant ESCRT system has not been fully explored. Recently, in Arabidopsis, Katsiarimpa et al. (2013) showed that ESCRT-III subunit VPS2.1 plays an important role in the autophagic degradation and autophagy-mediated physiological processes with AMSH3 (Associated Molecule with the SH3 domain of STAM3). Using observation of green fluorescent protein (GFP)-fused protein analysis, Katsiarimpa et al. (2011) found that subunits of ESCRT-III localized on MVBs not on the VM. Further, Ibl et al. (2012) showed interactions of the ESCRT-III subunit and VPS2.2 proteins. Proteins interacting with VPS2.2 included SNF7.1, VPS2.1, VPS46.1 and VPS60.1. Some dynamin-related proteins, DRP1A, 1C, 2A and 2B, were also detected by immunopreciptation with VPS2.2. Their results partially overlap with our proteomic analysis of vacuolar DRMs. These results suggest that VM microdomains may strongly interact with MVBs and be involved in autophagy-mediated physiological processes.

Band 7 family proteins were identified in both DRMs and DSMs (Table 3). It is thought that SPFH domain-containing proteins play an important role in forming membrane microdomains on the PM (Browman et al. 2007); however, their roles have not yet been clarified in plant cells. Jaquinod et al. (2007) indicated that one of the band 7 family proteins, At1g69840, is localized in the VM and may therefore be involved in the formation of VM microdomains.

It is worth noting that we could not detect γ-TIP, which is widely believed to be one of major proteins in the VM. In a previous analysis of the VM proteome (Shimaoka et al. 2004) we were also unable to detect this protein. This result suggests that aquaporins are not strongly expressed in liquid suspension-cultured cells.

PM microdomains are believed to be involved in signal transduction. This leads us to propose that VM microdomains may be involved in the regulation of membrane transport or signal transduction across the VM.

Materials and Methods

Plant material

Arabidopsis thaliana suspension-cultured cells (Deep) (Arabidopsis Col-0 cell suspension) were kindly supplied by Dr. Umeda (NAIST) (Mathur et al. 1998) and were cultured in modified Murashige and Skoog medium supplemented with 4.5 µM 2,4-D and 3% sucrose with rotation at 125 r.p.m. at 23°C in the dark (TAITEC, BioShaker BR-43FL).

Isolation of intact vacuoles and preparation of vacuolar DRMs

Vacuoles were isolated from suspension-cultured cells (7 d old) according to Shimaoka et al. (2004). The protoplasts were prepared by enzymatic treatment and treated with hypotonic buffer to burst only the PM. The intact vacuoles released from protoplasts were collected by Percoll density gradient centrifugation. Purity of the vacuole fraction has been confirmed by the measurements of marker enzymes and Western blotting (Shimaoka et al. 2004).

The VM microdomains were collected as the DRM fraction from isolated vacuoles prepared from >40 g (FW) of suspension-cultured cells. Isolated vacuoles were lysed by diluting with 4 vols. of buffer [30 mM HEPES adjusted to pH 7.2 with Tris, 30 mM potassium gluconate, 2 mM MgCl₂, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.5 mg ml⁻¹ leupeptin]. VMs were collected by centrifugation at 120,000×g for 75 min. The pellet was resuspended with TNE buffer (25 mM Tris adjusted to pH 7.5 with HCl, 150 mM NaCl and 5 mM EDTA), and recollected by centrifugation at 120,000×g for 75 min. The washed VMs were resuspended in TNE buffer, and the quantity of protein was determined. This suspended membrane was treated with Triton X-100 (1% final concentration, detergent : protein = 5 : 1) for 30 min at 4°C. Solubilized VMs were placed at the bottom of a centrifuge tube and mixed with 60% sucrose (w/w, dissolved in TNE buffer) to reach a final concentration of 45–50%. A discontinuous sucrose gradient [35, 30, 5% sucrose (w/w), dissolved in TNE buffer] was overlaid. After centrifugation for 16 h at 150,000×g, DRMs were recovered at the boundary between 30% and 35%. The bottom layer was collected as the DSMs.

Preparation and purification of antibodies

Rabbit polyclonal antibodies against peptides in V-ATPase A subunit (VHA-A) (Cys-TKAREVLQREDDLNEI), V-PPase (AVP1) (Cys-DLVGKIERNIPEDDPRN) and V-ATPase E subunit (VHA-E) (Cys-SVSAEEEFNIEKLQLVEAEKKKIRQ) were prepared (SIGMA-ALDRICH JAPAN). These antibodies were purified using an antigenic peptide-conjugated column to remove non-specific antibodies. The specificities of purified antibodies against VM proteins were confirmed by Western blot analysis of purified VMs (Supplementary Fig. S4).

Immunostaining

Isolated vacuoles were stained with purified specific antibodies against V-ATPase or V-PPase. Fluorescently labeled F(ab)₂ fragment [Zenon Alexa Fluor 488 (or 594) Rabbit IgG Labeling Kit, Molecular Probes Inc.] was used as a secondary antibody. The purified antibody was incubated with the fluorescently labeled F(ab)₂ fragment for 10 min at room temperature. Isolated vacuoles were incubated with the labeled specific antibody for 30 min at 4°C. The stained isolated vacuoles were collected by Percoll density gradient centrifugation.

Observation and measurement of molecular behavior

TIRFM was used to confirm the behavior of proton pumps in isolated vacuoles. The images were captured with a C9100 EM-CCD camera (Hamamatsu Co.) controlled by MetaMorph software (Molecular Devices) on a Nikon TE2000-U microscope at an excitation wavelength of 690 nm. Fluorescently labeled F(ab)₂ fragment (Zenon Alexa Fluor 700 Rabbit IgG Labeling Kit, Molecular Probes Inc.) was used as a secondary antibody. Fluorescent labeling and immunostaining were conducted as described above. Movies were captured at 33 ms per frame for 10 s, and smoothed with spatial smoothing filtering. Movement of the object and its behavior were quantified with MetaMorph software. Experiments were repeated twice, and >150 fluorescent spots were measured from >15 randomly selected isolated vacuoles.

For the analysis, it was hypothesized that a membrane protein performs a random walk in a two-dimensional lattice according to the equation <r²> = 4Dt, where <r²> represents the mean square displacement of the trace, D the diffusion coefficient and t the time (Almeida and Vaz 1995). <r²> vs. t plots were generated and the diffusion coefficient was determined from the slope. Median tests were carried out with JMP software (SAS Institute).

Electron microscopy

The VM and vacuolar DRMs were pelleted at 120,000×g for 60 min. Chemical fixation and embedding were carried out according to Mongrand et al. (2004). The agarose blocks were dehydrated with an ethanol series and propylene oxide and then embedded in Spurr’s resin. Ultra-thin sections, 60 nm thick, were stained with uranyl acetate and lead citrate, and observed with a transmission electron microscope (JEM-1200EXα; JEOL Ltd.).

Immunoelectron microscopy

For post-embedding immunoelectron microscopic observation, we used Arabidopsis suspension-cultured cells (Deep) cultured for 7 d. Cells were pre-fixed with a fixative containing 4% paraformaldehyde, 100 mM HEPES and 0.4% glutaraldehyde in distilled water for 30 min at room temperature, washed three times with 50 mM HEPES and dehydrated with ethanol (50%, 70%) for 10 min each at 0°C and in a series of ethanol (90, 95, 99%, and twice with 100%) for 10 min each at −20°C. After dehydration, samples were infiltrated with LR-White resin (London Resin Co.) at −20°C as follows: ethanol : resin (3 : 1) for 2 h, ethanol : resin (1 : 1) for 2 h, ethanol : resin (1 : 3) for 2 h, and then two changes of pure resin for 2 h each. Cells were then embedded and polymerized with UV light at −20°C for 48 h. Samples were sectioned with a diamond knife on an ultramicrotome (EM UC7, Leica Microsystems). Thin sections were blocked with 1% BSA (bovine serum albumin) in PBS (phosphate-buffered saline) for 1 h at room temperature, incubated for 1 h at room temperature with the rabbit anti-isolated protein polyclonal antibody diluted 1 : 500 in PBS, followed by a 1 h incubation at room temperature with the anti-rabbit IgG conjugated with 10 nm gold particles (EM GAR10, BB International) diluted 1 : 20 in PBS. Thin sections were then washed with PBS, followed by post-fixation with 1% glutaraldehyde in distilled water for 10 min at room temperature, and finally washed with distilled water. After staining with 3% aqueous uranyl acetate for 15 min and lead citrate stain for 5 min at room temperature, specimens were observed with a transmission electron microscope (H-7100, Hitachi).

LC-MS analysis of lipid extracts

VM and vacuolar DRM fractions were washed with TNE buffer to remove Percoll and detergent. Washed membranes were pelleted at 120,000×g for 60 min, re-dissolved in distilled water and lyophilized. Total lipids were extracted from dried VM and DRM according to Bligh and Dyer (1959), and then subjected to LC-MS analysis using a Shimadzu IT-TOF mass spectrometer combined with a Shimadzu LC-20AD HPLC system as reported previously (Okazaki et al. 2009).

Peptide preparation for proteomic analysis

After vacuolar DRM and DSM fractions were collected, equal volumes of 20% trichloroacetic acid were added. Proteins were collected by centrifugation at 120,000×g for 15 min, and washed three times with acetone. These specimens were diluted in the sample buffer [3.4% SDS, 30% glycerol, 2% 2-mercaptoethanol, 0.25 M Tris (pH 6.8) and 0.012% Bromophenol blue], incubated at 55°C for 5 min and applied to a pre-cast acrylamide gel (DRC, Perfect NT gel 10% A.A.). The separated proteins were stained with Flamingo (BIO-RAD). Gel lanes were sliced into 15 bands. Each slice was washed twice with HPLC-grade water containing 60% (v/v) acetonitrile (Kanto 17 Chemical) then alkylated with 5 mM ditiothreitol (for 1 h at 37°C) and 25 mM iodoacetamide (in the dark for 1 h at room temperature). After washing twice with HPLC-grade water containing 60% (v/v) acetonitrile, specimens were digested with 0.25 µg of trypsin (Promega) in 25 mM ammonium bicarbonate for 16 h at 37°C. Digested peptide fragments were extracted from the cut gels by 5% (v/v) formic acid and 50% (v/v) acetonitrile for 15 min. The solutions containing the digested peptides were dried in a vacuum concentrator, and the dried samples were dissolved in 0.1% (v/v) formic acid and 5% (v/v) acetonitrile.

Mass spectrometric analysis and database searching for proteome

Proteomic analysis was performed by LC-MS/MS using an LTQ-Orbitrap-HTC-PAL system (Thermo Electron) according to Fujiwara et al. (2009). The range of the MS scan was m/z 200–2,000 and the top three peaks were subjected to MS/MS analysis. Spectra were compared with a protein database from TAIR10 using the MASCOT server. The mascot search parameters were as follows: set off the threshold at 0.05 in the ion-score cut off, peptide tolerance at 10 p.p.m., MS/MS tolerance at ±0.8 Da, peptide charge of 2+ or 3+, trypsin as enzyme allowing up to one missed cleavage, carbamidomethylation on cysteines as a fixed modification and oxidation on methionine as a variable modification. The ARAMEMNON database (release 7, http://aramemnon.botanik.uni-koeln.de/) was used to predict transmembrane domains in identified proteins. Independent analyses were carried out three times, and the common proteins identified in all analyses were listed. The number of matched peptides can be used as a semi-quantitative reference. It should be noted, however, that the number does not indicate the actual quantity of the original protein, because the number is influenced by the size (molecular weight) of the original protein and/or quantity of peptides from other proteins in the same specimen, etc.

Funding

This work was supported by the Japanese Ministry of Education, Sports, Culture, Science, and Technology [a Grant-in-Aid for Scientific Research of Priority Areas on ‘Organelle Differentiation as the Strategy for Environmental Adaptation in Plants’ (No. 1685101), and a Grant-in-Aid for Scientific Research on Innovative Areas on plant environmental sensing' (No. 22120006)]; Japan Science and Technology Corporation (JST) [Grant-in-Aid for Exploratory Research by CREST].

Acknowledgments

We greatly appreciate Dr. Rob Reid (University of Adelaide, Adelaide, Australia) for his kind discussion and correction of this manuscript. We also thank Dr. Csaba Koncz (Max-Planck-Institut für Züchtungsforschung) and Dr. Masaaki Umeda (The University of Tokyo) for their kind supply of Arabidopsis suspension culture cells.

Disclosures

The authors have no conflicts of interest to declare.

Abbreviations

Abbreviations
 
• DRM

  detergent-resistant membrane

 
• DSM

  detergent-soluble membrane

 
• GlcCer

  glycosylceramide

 
• LC-MS/MS

  liquid chromatography–tandem mass spectrometry

 
• MVB

  multivesicular body

 
• PBS

  phosphate-buffered saline

 
• PC

  phosphatidylcholine, PE

 
• phosphatidylethanolamine

  PG, phosphatidylglycerol

 
• PI

  phosphatidylinositol

 
• PM

  plasma membrane

 
• PPi

  pyrophosphate

 
• TGN

  trans-Golgi network

 
• TIRFM

  total internal reflection fluorescent microscopy

 
• V-ATPase

  vacuolar-type proton ATPase

 
• VM

  vacuolar membrane

 
• V-PPase

  vacuolar-type proton pyrophosphatase