Plant Physiol. (1998) 118: 315-322

Distinct Biochemical and Topological Properties of the 31- and 27-Kilodalton Plasma Membrane Intrinsic Protein Subgroups from Red Beet¹

Lucille M. Barone, Helen He Mu, Connie J. Shih, Kenan B. Kashlan, and Bruce P. Wasserman^*

Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, 65 Dudley Road, New Brunswick, New Jersey 08901-8520

	ABSTRACT

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Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387-392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfide-linked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.

	INTRODUCTION

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The PM of higher plants has been the subject of extensive research focusing on the areas of recognition, water and ion transport, signal transduction, and cell wall polymer biosynthesis (Chrispeels and Maurel, 1994; Sussman, 1994; Delmer and Amor, 1995; Maurel, 1997; Schaffner, 1998). However, the PM remains one of the least-understood membrane systems in higher plants. Although PM vesicles are relatively simple to isolate and characterize, the hydrophobicity and low abundance of plant PM polypeptides have complicated purification and characterization efforts. Thus, only a limited number of plant PM proteins have been definitively identified. These include the H⁺-ATPase (Anthon and Spanswick, 1986; Katz and Sussman, 1987; Schaller and Sussman, 1988), a Ca²⁺-ATPase (Bonza et al., 1998), a Suc-binding polypeptide (Overvoorde and Grimes, 1994), and a group of PMIPs. Many of the PMIPs studied in plants have been shown to function as aquaporins, channels that permit the bidirectional passage of water through cellular membranes (Daniels et al., 1994; Kammerloher et al., 1994; Qi et al., 1995; Johansson et al., 1996; Schaffner, 1998). Molecular models based on deduced amino acid sequences suggest that PMIPs share a common membrane topology, with six hydrophobic membrane-spanning domains, connected by five hydrophilic loops (Chrispeels and Maurel, 1994; Nielsen and Agre, 1995; Lee et al., 1997; Maurel, 1997; Agre et al., 1998; Schaffner, 1998). The molecular structure of the mammalian aquaporin-1 from erythrocyte membranes has been described as a tilted hourglass, with a narrow pathway through the center of oligomerized tetrameric CHIP subunits (Jung et al., 1994; Cheng et al., 1997; Walz et al., 1997; Agre et al., 1998).

PM vesicle fractions isolated from storage tissue of red beet (Beta vulgaris L.) contain two highly abundant bands migrating at 31 and 27 kD. Both bands were identified to contain one or more MIPs by comparison of tryptic peptides with known MIP/aquaporin sequences (Qi et al., 1995). More recently, cDNA clones encoding storage tissue PMIPs were obtained (Qi et al., 1996). Collectively, these data show that the PMIP species of 31 and 27 kD both contain polypeptides sharing a high degree of sequence similarity with known PM aquaporins, such as tomato pTOM75 (Fray et al., 1994), pea clone 7a (Guerrero et al., 1990), and the Arabidopsis PIP proteins (Kammerloher et al., 1994). From a biochemical perspective, our laboratory has shown that the PMIP species of 31 and 27 kD are resistant to extraction with salts or chaotropic agents, largely insoluble in Triton X-100, and partially soluble in the detergents 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, digitonin, and octylglucoside (Wasserman et al., 1992). Each PMIP subgroup is highly prone to aggregation, and reducing agents are required to minimize disulfide-linked aggregation (Qi et al., 1995). In PMIP31 addition of exogenous Hg²⁺ leads to a conformational change characterized by exposure of a previously inaccessible proteolytic site immediately preceding the highly conserved Gly-Gly-Gly-Ala-Asn-X-X-X-X-Gly-Tyr motif. Based on this information, the topological orientation of surface loop C in PMIP31 was directly established (Barone et al., 1997).

The existence of two structurally related PMIPs, PMIP31 and PMIP27, within a single PM vesicle fraction presented a unique opportunity to directly compare their biochemical and topological properties. To explore the comparative properties of PMIP31 and PMIP27, affinity-purified antibodies specifically recognizing each PMIP species were generated. In this study the antibodies were used to assess the differential responses of PMIP31 and PMIP27 to proteolysis, detergent extraction, and exposure to sulfhydryl modification reagents. We also sought to determine if PMIP31 and PMIP27 were able to form mixed disulfide-linked heterodimers within the PM vesicle system. Our results show that PMIP31 and PMIP27 are topologically similar, but each responds differently to various perturbations placed upon the PM.

	MATERIALS AND METHODS

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RNA Gel-Blot Analysis

Membrane Isolation

Carboxymethylated microsomal membranes were prepared by substitution of 5 mM I-Ac for DTT in homogenization buffers as described previously (Umbach and Siedow, 1996). All other steps remained unchanged.

Electrophoresis and Immunoblotting

Affinity-purified antibodies to PMIP31 and PMIP27 were prepared as described previously (Barone and Wasserman, 1996; Barone et al., 1997). Immunoblotting with development by enhanced chemiluminescence was conducted as described previously (Wasserman et al., 1996; Barone et al., 1997). Mouse monoclonal antibodies that recognized the 60-kD subunit B of the vacuolar H⁺-ATPase (Ward et al., 1992) were provided by Heven Sze (University of Maryland, College Park). Unless otherwise indicated, each antibody was used at a dilution of 1:2000.

Suc Gradient Centrifugation

Protease Treatment of PM Vesicles

1

1

Sequence Analysis of Proteolytic Fragments

	RESULTS

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PMIP27 Is the Gene Product of BPM3

Tissue Distribution of the BPM Transcripts

View larger version (42K): [in this window] [in a new window]   Figure 1. Tissue distribution of BPM2 and BPM3 by RNA gel-blot analysis.

Membrane Distribution of PMIP31 and PMIP27

Microsomal membranes were applied to a continuous gradient of 15% to 45% (w/w) Suc. Fractions were collected and analyzed for PM and tonoplast marker enzymes. The PM marker callose synthase (Fig. 2, top) was localized as a well-defined peak at 35% to 42% Suc. However, the majority of the 60-kD subunit of the tonoplast H⁺-ATPase was located in lower-density fractions (Fig. 2, middle). These results are consistent with previous work, in which PM vesicles partitioned at 38% to 40% Suc, whereas tonoplast vesicles are found at 25% to 28% Suc (Poole et al., 1984; Wasserman et al., 1985).

View larger version (52K): [in this window] [in a new window]   Figure 2. Suc-gradient centrifugation of a microsomal vesicle fraction. A microsomal membrane fraction (3.4 mg of protein) was centrifuged overnight at 80,000g on a continuous gradient of Suc (15%-45%, w/w). Top, Distribution of the PM marker callose synthase and Suc. Middle, Immunoblot probed with a monoclonal antibody to the 60-kD vacuolar H+-ATPase (1:1,000 dilution). Bottom, Immunoblot dually probed with affinity-purified polyclonal antibodies specific for the PMIP31 and PMIP27 species (1:2,000 dilution). Each lane contained 20 µL from each gradient fraction and was prepared for SDS-PAGE in 100 mM DTT.

To demonstrate the association of PMIP31 and PMIP27 with the PM, gradient fractions were probed with each respective affinity-purified antibody. Both PMIP species distributed with the PM marker (Fig. 2, bottom) and neither correlated with the distribution of the tonoplast H⁺-ATPase subunit. Moreover, PMIP31 and PMIP27 were distributed similarly within the gradient, suggesting that the two PMIP subgroups are associated with the same population of membrane vesicles.

Effects of Sulfhydryl-Modification Reagents on PMIP Aggregation State

To determine the importance of sulfhydryl-modification agents during membrane preparation with respect to PMIP aggregation states, PM vesicles were prepared in tissue homogenization buffer containing I-Ac or NEM, with control membranes prepared in the absence of these reducing agents. The effect of diamide, which promotes sulfhydryl-group oxidation, was also examined (Umbach and Siedow, 1996).

PMIP31 and PMIP27 were differently affected by each of these treatments. With PMIP27, the disulfide-linked 43-kD species was dominant; however, the monomeric form of PMIP27 was clearly present (Fig. 3A, lanes 1, 3, and 5). Consistent with the formation of disulfide-linked species, addition of diamide promoted the oxidation of the monomeric form of PMIP27 to the 43-kD disulfide-linked species (Fig. 3A, lanes 2, 4, and 6). In contrast to PMIP27, the monomeric form of PMIP31 was markedly less visible when membranes were prepared in the absence of reducing agent. In the absence of DTT, only the anomalously migrating 45-kD aggregated species was observed (Fig. 3B).

View larger version (59K): [in this window] [in a new window]   Figure 3. Effects of Cys modification reagents. PM vesicles were isolated in the presence (+) and absence () of I-Ac (5 mM) and NEM (5 mM). Samples were treated with (+) or without () diamide (3 mM) before SDS-PAGE. Immunoblotting was performed using anti-PMIP27 (1:2500, A) and anti-PMIP31 (1:2000, B).

To determine if disulfide-linked aggregation results from oxidation reactions during homogenization and subsequent centrifugation steps, free sulfhydryl groups were blocked immediately upon tissue disruption by inclusion of I-Ac (Fig. 3, A and B, lanes 3 and 4) or NEM (Fig. 3, A and B, lanes 5 and 6) in homogenization buffers. Neither sulfhydryl reagent significantly enhanced monomer formation. Assuming that the reaction of free thiol groups with I-Ac or NEM is rapid, these results imply that the 27- and 31-kD monomers are already linked by disulfide bonds before tissue disruption.

Homodimeric Nature of the Aggregated Species

Proteolytic Fragmentation Patterns

Figure 4 shows that each PMIP species responded uniquely to proteolysis. In the absence of added detergent, PMIP31 was completely refractory to proteolysis by either trypsin or Pronase E (Fig. 4, lanes 2 and 5). Addition of 0.01% digitonin caused a dose-dependent decrease of PMIP31, with its complete disappearance at 40 µg mL¹ Pronase E (Fig. 4, lane 3) and partial disappearance with 112 µg mL¹ trypsin (Fig. 4, lane 6). No distinct intermediary proteolytic fragments were produced when digitonin was present.

View larger version (27K): [in this window] [in a new window]   Figure 4. Different fragmentation patterns of PMIP31 and PMIP27. Limited proteolysis was conducted with PM vesicles treated with 0.01% digitonin. Proteases used were Pronase E (left) and trypsin (right). Membrane-bound proteolytic products were extracted, electrophoresed, and blotted with anti-PMIP27 and anti-PMIP31, as indicated.

In contrast to PMIP31, PMIP27 yielded two proteolytic fragments in the absence of added detergent. Immunoreactive fragments of 25 and 21 kD were generated by Pronase E, and to a greater degree by trypsin (Fig. 4, lanes 5 and 6). The addition of digitonin did not greatly enhance proteolytic degradation of PMIP27. Thus, PMIP31 and PMIP27 each responded uniquely to limited proteolysis of PM vesicles.

Effect of Hg²⁺ and Placement of the Proteolytic Sites

View larger version (67K): [in this window] [in a new window]   Figure 5. Occlusion of tryptic site II in the presence of HgCl2. A, PM vesicles were incubated with increasing levels of HgCl2 and 0.01% digitonin before treatment with trypsin (40 µg mL1). B, Right-side-out vesicles were treated with trypsin in the absence () and presence (+) of 3 mM HgCl2 and digitonin, as indicated. The immunoblots were probed with anti-PMIP27.

A topological model based on the hydropathy plot of BPM3 is depicted in Figure 6. Tryptic sites I and II are defined as the cleavage sites giving rise to p25 and p21, respectively. The location of tryptic site I within the BPM3 gene product was directly established by N-terminal sequence analysis of p25 obtained from a carboxymethylated membrane preparation (Barone et al., 1997). The amount of protein recovered was low, but p25 yielded the N-terminal sequence Asp-Tyr-Val-Glu-Pro-Val-Gly-Ala-Asp-Leu-Phe. Although not a perfect match, this sequence generally corresponds to the predicted BPM3 sequence of Asp-Tyr-Lys-Glu-Glu-Pro-Pro-Pro-Ala-Pro-Leu-Phe beginning at residue 29. This sequence immediately follows an Arg residue, a well-known recognition site for trypsin, and demonstrates that tryptic cleavage site I is located within the N-terminal domain of PMIP27.

View larger version (33K): [in this window] [in a new window]   Figure 6. Topological model of a PMIP27. This model is based on a hydropathy plot (Kyte and Doolittle, 1982) of BPM3 (Qi et al., 1996) and the topological data provided here. The conserved domains located at surface loops C and E are shown. The locations of tryptic cleavage sites I and II are indicated. Conserved Cys residues and the two NPA domains of BPM3 are also shown.

Despite several attempts, it was not possible to obtain the N-terminal sequence from p21; therefore it was not possible to directly ascertain the location. However, if we make the assumption that the proteolytic cleavage site on PMIP27 giving rise to p21 is located at a similar position to the Hg²⁺-induced cleavage site on PMIP31, which generates a 22-kD fragment (Barone et al., 1997), this would place tryptic cleavage site II of PMIP27 near the interface of transmembrane segment 3 and surface loop C (Fig. 6). The assignment of this location is supported by the fact that occlusion of tryptic cleavage site II is observed when added Hg²⁺ binds in close proximity to the four conserved Cys residues.

To establish the orientation of the tryptic cleavage sites we probed the effects of Hg²⁺ and digitonin on a highly latent (94%) right-side-out vesicle fraction (Fig. 5). In the absence of Hg²⁺ and digitonin, p21 was the favored product, with lesser amounts of p25 formed (Figs. 4 and 5B, lane 7).

Hg²⁺ suppressed formation of p21, but simultaneously enhanced the formation of p25 in this vesicle fraction consisting of primarily right-side-out vesicles (Fig. 5). This experiment suggests that the formation of p25 and p21 are conformationally linked. In the absence of Hg²⁺, the buildup of p21 with increasing levels of digitonin (Fig. 5B, lane 4) indicates that tryptic site II is the preferred site of attack. When Hg²⁺ was present, however, there was a marked buildup of p25 at 0.1% digitonin (Fig. 5B, lane 7). Suppression of p21 formation is consistent with occlusion of tryptic site II attributable to the binding of Hg²⁺ near the four conserved Cys residues. Simultaneously, tryptic site I showed increased susceptibility to tryptic attack in the presence of Hg²⁺. This would suggest a Hg²⁺-induced conformational change that renders tryptic site I more accessible to proteolytic attack. Because the formation of p21 was significantly enhanced by the addition of digitonin (Figs. 4 and 5), this indicates that tryptic cleavage site II may lie at the interface between surface loop C and transmembrane segment 3.

In summary, tryptic site II of the PMIP27 subgroup exhibits greater sensitivity to proteolytic attack than tryptic site I in a predominantly right-side-out vesicle system when detergent is not present. The ready formation of p21 in the absence of detergent is indicative of an apoplastic orientation for tryptic site II. Conversely, the relatively low levels of p25 formed in the absence of detergent is consistent with a cytosolic orientation for tryptic site I. The formation of p25 could reflect a limited population of unsealed or inside-out vesicles. It is interesting that the PMIP27 and PMIP31 subgroups exhibited very different responses when exogenous proteases were applied. The resultant topological findings for both PMIP27 and PMIP31, however, are generally consistent with the models predicted by hydropathy analysis for plant PMIPs.

	DISCUSSION

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Previous studies have unequivocally demonstrated that the broad protein bands of 31 and 27 kD from PM vesicles of red beet storage tissue contain MIPs with extensive sequence homologies to known PM aquaporins (Wu and Wasserman, 1993; Qi et al., 1995; Barone et al., 1997). Other than the fact that antibodies prepared against the two species did not cross-react, the degree of similarity between the two PMIP species was unclear. One possibility was that PMIP27 was merely a proteolytic fragment derived from PMIP31. If this were true, one would expect to observe similar proteolytic fragmentation patterns. However, the current evidence indicates that PMIP31 and PMIP27 are topologically similar but contain subtle differences in their structure and biochemistry. These differences are noted by (a) the non-cross-reactivity of the polyclonal antibodies, (b) the distinct proteolytic fragmentation patterns in the absence and presence of Hg²⁺, (c) the different responses to sulfhydryl modification reagents, (d) the inability to form heterodimeric species, (e) the refractory nature of PMIP27 to proteolysis in the presence of detergent, and (d) the presence of a unique tryptic cleavage site in PMIP27 located in proximity to transmembrane segment 1. PMIP31 does not contain an analogous tryptic cleavage site at this position (Barone et al., 1997).

Although PMIP31 and PMIP27 partition similarly in Suc gradients (Fig. 2), the inability of these PMIPs to form heterodimeric species could imply that each PMIP species is associated with a distinct subdomain of the PM. The existence of microdomains within the plant PM provides a possible explanation for the distinct topological properties of PMIP31 and PMIP27. In plants PIP1 from Arabidopsis was found to be localized within the plasmalemmasome (Robinson et al., 1996). These structures protrude into the vacuole and may provide a means for rapid exchange of water with the apoplast. Two kinds of vacuolar subcompartments have been revealed by immunofluorescence microscopy using antibodies raised against two sources of tonoplast intrinsic protein (Paris et al., 1996; Swanson et al., 1998). Small invaginations of the PM known as caveolae exist in mammalian cells (Parton and Simons, 1995; Kurzchalia and Parton, 1996). Caveolin, an integral membrane protein, co-purifies in low-density, detergent-insoluble fractions with proteins involved in signal transduction. It was recently suggested that detergent-insoluble complexes can exist in the absence of caveolae, leading to the idea of the existence of distinct microdomains in the PM. A putative role for these microdomains is to integrate signaling with membrane transport (Schnitzer et al., 1995). The yeast PM also contains low-density Triton X-100-insoluble domains (Kubler et al., 1996) containing proteins involved in signal transduction. However, the PM marker enzyme H⁺-ATPase was excluded from low-density Triton X-100-insoluble domains (Kubler et al., 1996), indicating the presence of multiple PM subdomains. To our knowledge, the existence of microdomains in the plant PM has not yet been documented. The distinct topological characteristics and different responses to sulfhydryl-modifying reagents by PMIP31 and PMIP27 could signify the presence of distinct microcompartments within the plant PM.

It is generally accepted that MIPs are strongly hydrophobic, and it is now becoming apparent that the outer surface loops of plant MIPs contain a limited number of exposed proteolytic sites. Assuming that none of the proteolytic fragments escape detection, PMIP31 contains one tryptic site that is accessible only in the presence of Hg²⁺ (Barone et al., 1997). However, PMIP27 yields two tryptic sites. Based mainly on the relative abundance of p25 and p21, we believe that tryptic cleavage site II is partially exposed to the apoplastic face of the PM, and that tryptic site I lies at the cytosolic face (Fig. 6). This would be consistent with predicted orientations. These results could also be reconciled if segment 1 does not fully traverse the PM. Instead, it may form a loop that causes the N-terminal domain to emerge from the PM at the apoplastic face. However, this would run counter to recent crystallographic studies with aquaporin-1 (Cheng et al., 1997; Walz et al., 1997).

There appears to be a conformational linkage between proteolytic sites I and II in PMIP27. The addition of Hg²⁺ led to a concentration-dependent decline of trypsin-generated p21, which was accompanied by the concomitant accumulation of a proteolytic intermediate of 25 kD (Fig. 5). Our interpretation of this result is that the putative Hg²⁺-binding site is located proximal to the Cys residues in transmembrane segments 2 and 3 (Fig. 6). Increasing levels of Hg²⁺ would result in the occlusion of tryptic site II. The concomitant conformational change would then lead to greater tryptic accessibility of site I, resulting in elevated levels of p25.

With at least 23 MIP isoforms occurring in Arabidopsis (Weig et al., 1997), the complexities of biochemical analysis of plant MIPs becomes apparent. The occurrence of two MIP subgroups in beet storage tissue and the availability of the corresponding antibodies have provided a first step toward understanding the membrane topology of this complex group of proteins. This study demonstrates that the PMIP31 and PMIP27 subgroups both display distinct membrane topologies, and do not form mixed heterodimeric species. Moreover, the possibility that each PMIP class is distributed within distinct microdomains of the PM must be further investigated. Specific physiological functions of the PMIP31 and PMIP27 subgroups, as well as regulatory factors, remain to be determined.

	FOOTNOTES

   Received April 9, 1998; accepted June 19, 1998.

	ABBREVIATIONS

Abbreviations: I-Ac, iodoacetic acid. MIP, major intrinsic protein. NEM, N-ethylmaleimide. PM, plasma membrane. PMIP, PM intrinsic protein.

	ACKNOWLEDGMENT

We thank Dr. Heven Sze for the kind gift of monoclonal antibodies recognizing the 60-kD vacuolar H⁺-ATPase subunit.

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