Bergeron MJ et al. (2011), Frog oocytes to unveil the structure and supram...

XB-ART-43551

PLoS One 2011 Jan 01;67:e21901. doi: 10.1371/journal.pone.0021901.

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Frog oocytes to unveil the structure and supramolecular organization of human transport proteins.

Bergeron MJ , Boggavarapu R , Meury M , Ucurum Z , Caron L , Isenring P , Hediger MA , Fotiadis D .

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Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

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Species referenced: Xenopus laevis
Genes referenced: aqp1 myc slc15a1 slc1a1 slc5a1.2

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	Figure 1. Schematic representation of the expression vector pMJB08, and the workflow for the expression in X. laevis oocytes and purification of mammalian transport proteins.(A) Three tags, i.e. 10x-His, FLAG and HA, and one proteolytic site for the HRV3C protease were translated in-frame at the N-terminus of our transport proteins. The HRV3C protease cleaves between the amino acids QG (in red). The vector is setup in a modular manner: i.) the FLAG tag (yellow) can be swapped by a different tag X (e.g. c-myc, HA, etc.) using the unique restriction sites SalI and NotI; ii.) the HRV3C protease cleavage site (green) can be replaced by a different proteolytic site Y (e.g. for TEV protease, thrombin, etc.) using the unique restriction sites NotI and NcoI; iii.) the HA tag (red) can be exchanged by a different tag Z (e.g. c-myc, FLAG, etc.) using the unique restriction sites NcoI and all enzymes in the multi cloning site (MCS) except XmaI; iv.) a XmaI digestion removes all tags and epitopes to obtain the original Pol1 vector. (B) Schematic representation of the workflow for the expression and purification of transport proteins in X. laevis oocytes. See Methods section for a detailed description of the different steps.
	Figure 2. Expression, localization and function of recombinant AQP1 and KCC4 in X. laevis oocytes.(A) Cell surface biotinylation experiments and subsequent Western blot analyses using anti-HA antibodies indicated plasma membrane expression of multi-AQP1 and multi-KCC4 (i.e. AQP1 and KCC4 containing a long N-terminal extension as described in Figure 1A): see multi-AQP1 monomer band below the 37 kDa marker, and multi-KCC4 monomer and dimer bands below the 150 kDa and above the 250 kDa markers, respectively. (B) Confocal immunofluorescence microscopy using anti-HA antibodies localized multi-AQP1 and multi-KCC4 in the plasma membrane. These representative images were recorded at 10x magnification. Scale bar: 200 µm. (C) Functional characterization of different AQP1 constructs. Swelling in water of non-injected oocytes (Control) and oocytes injected with cRNA of wt-AQP1 (no N-terminal extension), HA-AQP1 (only N-terminal HA epitope) and multi-AQP1. Multi-AQP1 was not functional while wt-AQP1 and HA-AQP1 had comparable activities. Data correspond to oocyte volume variation measurements in water at different time points. They are shown as averages (± S.E.) of 4-6 experiments. (D) Functional characterization of different KCC4 constructs. Similar levels of Rb+ influx into wt-KCC4 and multi-KCC4 expressing oocytes indicated full activity of the recombinant transporter. Data correspond to Rb+ influxes and are shown as averages (± S.E.) of 4 experiments (9-15 oocytes/experiment). Oocytes were non-injected (Controls), or injected with 20 ng of AQP1 and KCC4 cRNAs for expression and localization studies (A and B), and injected with 5 ng of AQP1 and 20 ng of KCC4 cRNAs for functional studies (C and D).
	Figure 3. SDS-PAGE, silver-staining and Western blot analyses of purified, recombinant channel and SLC transporters expressed in X. laevis oocytes.Silver-stained SDS/polyacrylamide gels (A,D,F,H and J), and Western blots (using anti-HA: B,E,G,I and K and anti-pentahistidine antibodies: C) from representative purifications. (A) and (B) The recombinant, nonglycosylated human AQP1 monomer runs at ∼25 kDa similar to native AQP1 isolated from human erythrocytes [6]. Additional higher molecular mass bands are discerned corresponding to glycosylated and dimeric AQP1 forms. (C) No His-tagged proteins were immunodetected in purified AQP1 preparations by immunoblotting (lane: AQP1). In contrast, strong signals were obtained for the His-tagged protein markers (lane: M; positive control). (D) and (E) Monomer (below 150 kDa marker) and dimer (∼250 kDa) bands of non- and glycosylated, recombinant mouse KCC4 [12], [13]. (F) and (G) Monomeric (∼75 kDa [16]), dimeric (∼150 kDa) and oligomeric glycosylated, recombinant human EAAC1. (H) and (I) Monomeric (∼70 kDa [17]), dimeric (∼140 kDa) and oligomeric non- and glycosylated, recombinant human PEPT1. (J) and (K) Monomeric non- (∼55 kDa [18]) and glycosylated (∼70 kDa), and oligomeric non- and glycosylated, recombinant human SGLT1. All bands on silver-stained SDS/polyacrylamide gels can be assigned to bands observed on corresponding Western blots.
	Figure 4. Negative stain TEM of purified recombinant AQP1 and KCC4.The homogeneity of the purified HA-AQP1 protein is reflected in the electron micrograph. Numerous particles exhibit a square shape (arrowheads), which is typical for AQP1 top views [5]. The gallery in (B) displays well-preserved top views of HA-AQP1. The last particle in this gallery (rightmost) is an average calculated from 270 top views. No four-fold symmetry was imposed. In the raw images and average, four densities are clearly visible. The average was low-pass filtered to 15 Å resolution. (C) Overview micrograph of purified HA-KCC4. Two populations of particles are distinct: smaller (minority; arrows to the left) and larger particles (majority; arrows to the right). The former and latter particles were magnified and are displayed in (D) and (E), respectively. The scale bars represent 500 Å (A and C). The frame sizes of the magnified particles are 138 Å (B) and 204 Å (D and E).
	Figure 5. Tubular crystals of recombinant human AQP1.(A) Coomassie Blue-stained SDS/polyacrylamide gel of the purified HA-AQP1 used for 2D crystallization. Nonglycosylated HA-AQP1 migrates at ∼25 kDa while glycosylated proteins migrate above. (B) Negatively stained tubular crystals of HA-AQP1. The area marked by the white box was magnified and is displayed as inset: tubular crystals with a typical width of ∼0.11 µm are seen. (C) The power spectrum calculated from a flattened tubular HA-AQP1 crystal indicates regular order. Strong and weaker diffraction spots are marked by a circle and arrowheads, respectively. The scale bars represent 0.5 µm (B) and (50 Å)−1 (C). The frame size of the inset in (B) is 0.503×0.362 µm.

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