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Am J Physiol Cell Physiol 292: C1132-C1136, 2007. First published November 1, 2006; doi:10.1152/ajpcell.00136.2006
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes

Alexandre Leduc-Nadeau,1 Karim Lahjouji,1,2 Pierre Bissonnette,1,2 Jean-Yves Lapointe,1 and Daniel G. Bichet1,2

1Groupe d’Étude des Protéines Membranaires, Département de Physiologie, Université de Montréal, and 2Centre de Recherche, Hôpital du Sacré-Coeur de Montréal, Montreal, Quebec, Canada

Submitted 24 March 2006 ; accepted in final form 29 October 2006


   ABSTRACT
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Over the past two decades, Xenopus laevis oocytes have been widely used as an expression system to investigate both physiological and pathological properties of membrane proteins such as channels and transporters. Past studies have clearly shown the key implications of mistargeting in relation to the pathogenesis of these proteins. To unambiguously determine the plasma membrane targeting of a protein, a thorough purification technique becomes essential. Unfortunately, available techniques are either too cumbersome, technically demanding, or require large amounts of material, all of which are not adequate when using oocytes individually injected with cRNA or DNA. In this article, we present a new technique that permits excellent purification of plasma membranes from X. laevis oocytes. This technique is fast, does not require particular skills such as peeling of vitelline membrane, and permits purification of multiple samples from as few as 10 and up to >100 oocytes. The procedure combines partial digestion of the vitelline membrane, polymerization of the plasma membrane, and low-speed centrifugations. We have validated this technique essentially with Western blot assays on three plasma membrane proteins [aquaporin (AQP)2, Na+-glucose cotransporter (SGLT)1, and transient receptor potential vanilloid (TRPV)5], using both wild-type and mistargeted forms of the proteins. Purified plasma membrane fractions were easily collected, and samples were found to be adequate for Western blot identification.

expression studies; aquaporin 2 mutations

XENOPUS LAEVIS OOCYTES have been widely used as means for the cloning and characterization of heterologous proteins from sources as diverse as viruses, bacteria, plants, and animals (16, 17, 19). More particularly, the use of oocytes has been extremely fruitful in expression cloning of channels and transporters, taking advantage of sensitive techniques such as electrophysiology and radiotracer uptake (6, 12, 16, 20). Rapidly, the use of oocytes has expanded to include physiological and biochemical characterization of heterologous proteins, which often led to the identification of pathological mechanisms associated with naturally occurring mutations. More specifically, the topic of targeting, which turned out to be of highest importance in transporter- and channel-related pathologies, has been addressed with oocytes (7, 15). Adequate plasma membrane targeting is essentially demonstrated by positive functionality and/or by using immunolabeling techniques such as immunofluorescence. Definite characterization is strengthened by studies on purified plasma membrane fractions using Western blots or other biochemical analysis. Unfortunately, existing techniques allow for partial purification of plasma membrane fractions (11) or require laborious steps involving manual removal of vitelline membranes (5, 14), which is cumbersome and severely limits the number of oocytes that can be treated in a reasonable experiment. Recently, a new technique using centrifugation on density gradient has been proposed (13). Unfortunately, this technique was designed for a large number of oocytes (400–1,000) and is therefore not applicable to expression studies requiring individually injected oocytes. It thus became desirable to design a new technique that could generate highly purified plasma membrane fractions from oocytes, one that would be both rapid and easy and could be performed in reasonable batches of injected oocytes.

The present article describes a new technique that was elaborated from the combination of three already existing techniques and allows a high level of purification of plasma membrane fractions from X. laevis oocytes. The technique combines partial digestion of the vitelline membrane, polymerization of the plasma membrane, and low-speed centrifugations of large plasma membrane leaflets. This technique allows for simultaneous processing of multiple samples from 10 to >100 oocytes and can be used for proteins that are more faintly expressed at the plasma membrane. The purified fractions can be efficiently used for biochemical characterizations of proteins using Western blot assays.


   MATERIALS AND METHODS
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Oocyte Preparation, Injection, and Maintenance

Ovary nodes were surgically removed from gravid X. laevis frogs anesthetized with 2-aminobenzoic acid ethyl ester. Oocytes (stages V–VI) were dissected by hand, and follicular layers were removed with a collagenase treatment (17.5 mg/ml, type 1A; Sigma-Aldrich) for 1 h under mild agitation in a Ca2+-free Barth solution (in mM: 90 NaCl, 3 KCl, 0.82 MgSO4, and 5 HEPES, pH 7.6). Oocytes were allowed to rest overnight at 18°C in normal Barth solution [same as above with 0.4 mM CaCl2 and 0.33 Ca(NO3)2] supplemented with horse serum (5%), sodium pyruvate (2.5 mM), and antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.1 mg/ml kanamycin). On the next day, oocytes were injected with cRNA solutions in water and further incubated for 2 [aquaporin (AQP)2 and transient receptor potential vanilloid (TRPV)5] or 5 [Na+-glucose cotransporter (SGLT)1] days before proceeding to membrane purification. cRNA preparations were diluted to 1 µg/µl [except wild-type AQP2 (wt-AQP2), which was 0.025 µg/µl] and injected at 46 nl/oocyte with a microinjection apparatus (Drummond Scientific, Broomall, PA).

Vectors and cRNA

All constructs used in this study were elaborated with the pT7Ts vector. For AQP2, the wild-type protein (wt-AQP2) construct was kindly provided by Peter M. T. Deen (Nijmegen, The Netherlands). The two point mutations, D150E and G196D, were inserted with site-directed mutagenesis, and validity of constructs was confirmed through sequencing. For SGLT1, a fully functional myc-tagged version of the protein was used (1), along with two nonfunctional SGLT1 mutants, C351A and C361A (10). Vectors for TRPV5, wild type and mutants (N518C and F531C), were kindly provided by L. Parent (University of Montreal) (8). For preparation of cRNAs, all vectors were linearized and cRNAs were synthesized with the mMessage mMachine T7 kit (Ambion, Austin, TX).

Preparation of Total and Plasma Membrane Fractions of Oocytes

Total membranes. Five oocytes were rinsed in Barth solution and homogenized in 1 ml of the same solution supplemented with protease inhibitor cocktail (Sigma-Aldrich) with 10 strokes of a Potter-Elvehjem tissue grinder (Wheaton). Homogenates were centrifuged at 250 g for 10 min at 4°C to discard cell debris, and the supernatant was centrifuged at 16,000 g for 20 min at 4°C to pellet down total membranes. Pellets were resuspended in 10 µl of Barth solution (2 µl solution/oocyte) and frozen until use.

Plasma membranes. Forty oocytes (except in Fig. 1, where 10–120 oocytes were treated) were rinsed in MES-buffered saline solution (MBSS; 80 mM NaCl, 20 mM MES pH 6.0) and incubated for 10 min at room temperature in the same solution with 0.005% subtilisin A (Sigma-Aldrich) under very mild agitation to partially digest the vitelline membranes. From this moment on, oocytes are somewhat sticky although resistant enough to tolerate subsequent treatments. The following polymerizing steps were performed at 4°C under mild agitation. Polymerization was performed by two sequential 60-min incubations in MBSS, first with 1% ludox and then with 0.1% polyacrylic acid (Sigma-Aldrich). Between each step, oocytes were thoroughly rinsed in MBSS. The oocytes were then homogenized in an Eppendorff tube with 0.5 ml of cold HbA (in mM: 5 MgCl2, 5 NaH2PO4, 1 EDTA, 80 sucrose, and 20 Tris pH 7.4). This homogenization was performed by hand with a P200 pipettor until no particles (dark granules) were visible (~15 pipettings). This produces a homogeneous solution containing large leaflets of plasma membranes attached to vitelline membranes, which become visible after subsequent centrifugation steps. The homogenates were raised to 1.5 ml with HbA and centrifuged at 16 g for 30 s at 4°C. The supernatants were removed, leaving the bottom 75–100 µl, which contains the plasma membranes, and diluted again with 1 ml of cold HbA. The tubes were centrifuged again at same speed, and the top 1 ml of supernatant was removed and replaced again with the same amount of fresh HbA. At this moment, large leaflets should become visible when the tube is tipped. With the same procedure, the tubes were centrifuged once at 25 g and then at 35 g, with membranes always visible in each pellet. A final centrifugation at 16,000 g for 20 min pelleted the purified plasma membranes, which were resuspended in 10 µl of HbA and frozen until use.


Figure 1
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  		Fig. 1. Detection according to sample size. Oocytes were injected with 1 ng cRNA coding for wild-type aquaporin (AQP)2 and processed as described in MATERIALS AND METHODS to purify plasma membranes, using increasing numbers of oocytes (10–120). Samples were loaded on gels, and AQP2 was detected by Western blot.

 
Western Blots

Western blots were performed as described previously (1), using total membranes and purified plasma membranes isolated from X. laevis oocytes. Samples loaded represent either 1 oocyte (total membranes) or 40 oocytes (purified plasma membranes), except for Fig. 1, where the number of oocytes varied from 10 to 120. For TRPV5 and SGLT1, total membrane fractions were solubilized in 1% Triton X-100 to eliminate a major contaminating band of equivalent molecular mass that alters the normal migration of both proteins analyzed. Interestingly, this contaminant is not found in purified plasma membrane. Samples were run on either a 7.5% [TRPVS and SGLT1] or a 12% (AQP2) gel and transferred onto nitrocellulose membranes. The efficiency of the overall procedure was monitored by Ponceau red staining. The membranes were blocked with 5% nonfat milk in Tris-buffered saline (TBS) + Tween 20 (TBS-T, 0.1%) and probed with specific antibodies. For AQP2, we used {alpha}-AQP2 at 1/100 (N-20 from Santa Cruz Biotechnology, Santa Cruz, CA) followed by horseradish peroxidase (HRP)-linked chicken anti-goat at 1/5,000 (Santa Cruz Biotechnology). For SGLT1, we used {alpha}-myc at 1/300 (9E10 clone from Santa Cruz Biotechnology) followed by HRP-linked goat anti-mouse at 1/3,000 (Jackson Immunoresearch). For TRPVS, we used {alpha}-CAT21-A at 1/100 (Jackson Immunoresearch) followed by HRP-linked donkey anti-rabbit at 1/10,000 (Jackson Immunoresearch, PA). For protein disulfide isomerase (PDI), we used {alpha}-PDI at 1/200 (Santa Cruz Biotechnology) followed by HRP-linked donkey anti-rabbit at 1/10,000 (Santa Cruz Biotechnology). All incubations were performed in TBS-T with milk. Membranes were rinsed adequately between every step with TBS-T and revealed with enhanced chemiluminescence detection (Phototope-HRP, New England Biolabs, Pickering, ON, Canada).

Volume Measurements

Functionality of AQP2 was assessed by water flux measurements in noninjected and AQP2-injected oocytes. Briefly, the oocytes were placed in a 0.07-ml bath on the stage of an inverted low-power microscope equipped with a camera and a recording system for analysis of the oocyte cross section. The oocytes were challenged with a hyposmotic solution (–20 mosmol/kgH2O), and the swelling induced was monitored. The variations in volume were used to determine water permeability values (Pf) which are given in micrometers per second. For a more detailed description of this setup and procedure, see Duquette et al. (9) and Charron et al. (4).


   RESULTS AND DISCUSSION
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 RESULTS AND DISCUSSION
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Technical Considerations

The need for an improved technique for the purification of plasma membrane of X. laevis oocytes came from the fact that all available techniques report various levels of contaminants originating from different intracellular stores (11, 13, 21), except that of Kamsteeg and Deen (14), which results in a high level of purification without any evidence of contaminants. Unfortunately, this technique requires a certain level of technical skill and turns out to be inappropriate when >40 oocytes are required for a given experiment.

The present technique was elaborated out of preexisting techniques and essentially permits rapid and efficient purification of highly purified plasma membranes from X. laevis oocytes. The overall technique is accessible, does not require the manual removal of the vitelline membranes, and is accomplished in ~3 h. The first step consists of the partial enzymatic digestion of the vitelline membrane with the use of subtilisin A. The purpose of this procedure is to make the vitelline membrane more permeable to the polymerizing agents applied thereafter. In contrast to techniques in which the vitelline membrane is physically removed with the use of microforceps, the partial digestion of this layer does not make the oocytes fragile and sticky, which greatly facilitates subsequent manipulations. In addition, the manual removal of this membrane is cumbersome and thus greatly limits the number of oocytes that can be treated by a single person. This treatment has been proposed to facilitate patch-clamp studies on oocytes (5). The enzymatic permeabilization of the vitelline membrane enables an efficient polymerization of the plasma membrane to the vitelline layer.

The use of polymerization as a strategy for the purification of oocyte plasma membranes was proposed by Chaney and Jacobson (2) and subsequently modified by Kamsteeg and Deen (14). This technique presents rapid and easy purification of plasma membranes without the use of more fastidious techniques such as density-gradient centrifugation (13). Polymerized plasma membranes from the latter technique, although adequate, generate small membrane fragments that are difficult to separate from the remainder of the homogenate. On the other hand, when a similar purification is performed on oocytes in which the plasma membranes have been polymerized to the vitelline layer, large leaflets are generated that are easier to precipitate and also allow for their direct visualization through the successive centrifugation steps. The final pellet consists of highly purified plasma membranes attached to the vitelline fraction.

Limitation of Technique

In a first set of experiments, we aimed at defining the limitations of this technique, delineating the number of oocytes that could be adequately processed in a given sample and also the ability to detect variations in expression levels in plasma membrane of the oocyte. For these purposes, we have chosen to express the AQP2 water channel since its detection in oocytes through Western blots is found to be quite sensitive. In Fig. 1, plasma membranes were purified from oocytes expressing AQP2 (1 ng cRNA per oocyte) in batches of 10–120 oocytes. As seen in Fig. 1, the specific signal found at 29 kDa gradually increases with the increasing number of oocytes used in the purification procedure. Although quite faint, AQP2 can be visualized even with as few as 10 oocytes, thus validating the technique from very low to high sample sizes. This flexibility in sample size makes it convenient to adapt for varying expression levels and Western blot sensitivity. Second, we also wanted to demonstrate the ability to visualize variations in expression levels of a given protein at the plasma membrane. In Fig. 2, plasma membranes were purified with the same sample size (40 oocytes per sample) expressing increasing levels of AQP2 (0–1 ng per oocyte) and visualized through Western blot. As seen in Fig. 2B, specific signals increasing in intensity are found in correlation with increasing activity levels for AQP2 (Fig. 2A) corresponding to injected cRNA. Identification of possible contamination from intracellular stores, as determined by evaluation of PDI in the same blots, failed to show any specific contaminants (data not shown).


Figure 2
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  		Fig. 2. Detection according to expression level. Oocytes were injected with increasing amounts of cRNA (0–1 ng) coding for wild-type AQP2 and tested for both activity and Western blot detection. A: evaluation of water permeability (Pf) as described in MATERIALS AND METHODS. B: Western blot of plasma membranes (40 per condition) purified from same oocytes tested in A showing a direct correlation between AQP2 activity and protein expression at the plasma membrane.

 
Validation of Technique

In subsequent experiments, total membranes and purified plasma membrane fractions were tested for the presence of heterologously expressed proteins through Western blot analysis. For the sake of demonstrating the efficacy of the technique, it was applied to proteins normally targeted to the plasma membrane and to mutant forms known to be mistargeted or nonfunctional. Again, we have chosen AQP2 as a model to test this technique since the wild-type form of the protein (wt-AQP2) is strongly expressed at the plasma membrane while most mutant forms are known to be completely retained in intracellular stores. Also, we took advantage of one AQP2 mutant (D150E) that demonstrates predominant endoplasmic reticulum (ER) retention with low expression at the plasma membrane, thus displaying partial targeting. In Fig. 3A, we present water permeabilities of control and AQP2-injected oocytes. As shown in Fig. 3A, oocytes injected with 1 ng of wt-AQP2 cRNA display a net increase in water permeability (93 ± 22 µm/s) while G196D-AQP2-injected oocytes (10 ng) display water permeabilities similar to controls (3.6 ± 1.5 and 4.4 ± 1.4 µm/s, respectively). The oocytes injected with D150E-AQP2 (10 ng) exhibit intermediary water permeability (24.8 ± 6.5 µm/s), consistent with its partial targeting at the plasma membrane. Figure 3B presents Western blot detection of AQP2 in control and AQP2-injected oocytes on both total membranes and purified plasma membrane fractions prepared following the technique described above. As seen in the total membrane lanes, the AQP2 protein is adequately synthesized in both wild type (29-kDa band) and mutants (29- and 31-kDa bands). On the other hand, the signal found in the purified plasma membrane lanes is a clear reflection of the membrane activity found in Fig. 3A. While a strong signal is found in wild-type protein, a fainter signal is seen with D150E-AQP2 and no signal is found for the nonfunctional G196D-AQP2 mutant. It should be noted that only the mature band of D150E-AQP2 (29 kDa) is found in the purified plasma membrane fraction, as for wt-AQP2. Once again, PDI was used as marker for intracellular contaminants. Even in overexposed blots, no trace of PDI could be seen in purified plasma membrane fractions.


Figure 3
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  		Fig. 3. Western blot of total membranes and purified plasma membranes from AQP2-injected oocytes. Oocytes were injected with wild-type (wt-AQP2, 1 ng) or mutant (D150E and G196D, 10 ng) AQP2 and tested for both activity and Western blot detection. Functionality was assessed by water permeability analysis (A) for controls (Ctl; Pf = 3.6 ± 1.5 µm/s), wild-type (Pf = 93 ± 22 µm/s), D150E (Pf = 25 ± 6.5 µm/s), and G196D (Pf = 4.4 ± 1.4 µm/s) AQP2s. B: Western blot performed on total membranes (5 oocytes) and purified plasma membranes (40 oocytes) from same samples. In total membranes, mature AQP2 is shown at 29 kDa while high-mannose form is seen at 31 kDa for both mutants. In purified plasma membrane, only the mature form of the protein is found. Note the strong signal for wild type compared with the fainter D150E. G196D is not detected in this purified fraction. Bottom: protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) marker, was used to show the absence of this major contaminant in the purified membrane fraction.

 
To further substantiate the efficacy of this technique, we have performed similar studies on two other proteins: the Na+-glucose cotransporter SGLT1 and the TRPV5 Ca2+-selective channel, testing both wild-type proteins and mutants for which a lack of function has been established. SGLT1 has been extensively studied in X. laevis oocytes. Functional studies as well as immunofluorescence labeling have shown adequate targeting to the plasma membrane (1). In Western blots, both mature (glycosylated) and immature forms of the protein are found (18). In Fig. 4, we compare Western blots for SGLT1 of total membrane and purified plasma membranes for both wild type and mutants (C351A and C361A). Both of these mutants have been previously reported to be nonfunctional in oocytes, but even though mistargeting was suspected their actual processing has not been determined (10). The Western blot of total membranes confirms that both mutant SGLT1 proteins are being synthesized, although the mature glycosylated form is lacking, which is found with the wild-type form of the protein (69- and 61-kDa forms, also see Ref. 18). In purified plasma membranes, only the mature form of the wild-type protein is found. Immature forms of the protein, be it of the wild type or the mutated proteins, are not evidenced. It thus seems that both mutants of SGLT1 are sequestered in intracellular stores, most probably the ER. In Fig. 4, bottom, evaluation of PDI indicates that no intracellular contaminants are found in the purified plasma membrane fractions.


Figure 4
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  		Fig. 4. Western blot of total membranes and purified plasma membranes from Na+-glucose cotransporter (SGLT)1-injected oocytes. Total membranes (5 oocytes) and purified plasma membranes (40 oocytes) from oocytes injected with 46 ng of wild-type or mutant (C356A and C361A) SGLT1, along with controls, were tested in Western blot. As shown in total membranes, products are generated for all SGLT1 variants, although the mature protein (69 kDa) is only found in the wild type. The 2 mutants only display low-molecular-mass products. In purified plasma membranes, only the mature SGLT1 protein is detected in the wild type while immature forms are never found. Bottom: detection of PDI (ER marker) indicates the absence of contamination in purified membrane fractions.

 
The TRPV5 is also well expressed in X. laevis oocytes (3, 8). The protein is adequately targeted at the plasma membrane and exhibits normal functionality. In Western blots from oocytes, TRPV5 is expressed as a doublet that represents glycosylation variants (79 and 88 kDa; also see Ref. 3). We have expressed in oocytes the wild-type form as well as two nonfunctional mutants (N518C and F531C) of TRPV5 (8) and assessed their plasma membrane targeting with Western blots. As shown in Fig. 5, in total membrane samples all three forms of TRPV5 are well expressed, essentially as unglycosylated forms, although glycosylated variants are present for all three proteins. On the other hand, in purified plasma membranes only the wild-type protein is detected, with the mature glycosylated form being predominant. In contrast, the dominant form of the wild-type TRPV5 in the total membrane preparation resides in the lower-molecular-mass form (79 kDa). It may thus be assumed that the lack of function of the two TRPV5 mutants originates from entrapment of the proteins in intracellular stores. Once again, as shown in Fig. 5, bottom, evaluation of PDI shows the absence of intracellular contaminants in the purified plasma membrane fractions.


Figure 5
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  		Fig. 5. Western blot of total membranes and purified plasma membranes from transient receptor potential vanilloid (TRPV)5-injected oocytes. Total membranes (5 oocytes) and purified plasma membranes (40 oocytes) from oocytes injected with 46 ng of wild-type or mutant (N518C and F531C) TRPV5, along with controls, were tested by Western blot. As shown in total membranes, products are generated for all TRPV5 variants, showing essentially low-molecular-mass forms with mature glycosylated forms. In purified plasma membrane fractions, only wild-type proteins are detected, with glycosylated forms being predominant this time. Bottom: PDI detection indicates the absence of contamination in purified membrane fractions.

 
In conclusion, we have elaborated a technique that permits rapid and efficient purification of plasma membranes from X. laevis oocytes that combines partial digestion of the vitelline layer with polymerization of the plasma membrane. High-level purification is thus achieved without fastidious technical steps, such as manual vitelline removal or centrifugation on density gradients. This improved technique provides a new means of investigation for the study of plasma membrane protein targeting, a key feature of both physiological and pathological interest.


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This work was supported by the Canadian Institutes of Health Research grant no. MOP-10580, Chair in Genetics of Renal Disease (D. G. Bichet) and by the Fonds de la Recherche en Santé du Québec infrastructure program no. 5252.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Bissonnette, Dép. Physiologie, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Québec, Canada, H3C 3J7 (e-mail: pierre.bissonnette{at}umontreal.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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 REFERENCES
 
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