Figure 1.
Expression and function of hSVCT1 in Xenopus oocytes.
(A) Western blot analysis of isolated membranes from three oocytes expressing hSVCT1. Western blotting was performed using a 6% SDS/polyacrylamide gel and an anti-HA antibody. The prominent band below the 75 kDa marker corresponds to recombinant hSVCT1 (calculated molecular mass based on the amino acid sequence: ∼ 71 kDa). A second, less prominent band between the 100 kDa and 150 kDa markers was assigned to dimeric hSVCT1. (B) Oocytes injected with hSVCT1 cRNA mediate uptake of [14C]ascorbate in contrast to control oocytes injected with water. Mean ± SEM from 20 oocytes are indicated (two independent experiments).
Figure 2.
Purification and biochemical characterization of recombinant hSVCT1.
Silver-stained SDS/polyacrylamide gels of detergent-solubilized membranes prior binding to cobalt resin (A) and purified hSVCT1 after metal affinity chromatography (B). A prominent hSVCT1 band is discerned between the 50 and 75 kDa markers. (C) Western blot analysis of PNGase F untreated (−) and treated (+) purified hSVCT1. A clear shift towards lower molecular mass and a more compact appearance of the hSVCT1 monomer and dimer bands is observed after PNGase F treatment of purified protein. This indicates N-linked glycosylation of hSVCT1 when expressed in Xenopus laevis oocytes. In (A)-(C) samples were run on 6% SDS/polyacrylamide gels, and in the Western blot displayed in (C) an anti-HA antibody was used.
Figure 3.
Negative stain-TEM and SPA of purified hSVCT1.
(A) Overview electron micrograph of purified hSVCT1 adsorbed on a parlodion carbon-coated surface. Projections of differently oriented hSVCT1 proteins are discerned. Large, elliptical (hSVCT1 dimers) and small, round-shaped (hSVCT1 monomers) particles are marked by black and white circles, respectively. Small protein aggregates are also observed (arrowheads). Well-preserved top views of dimers (B) and monomers (C) were magnified and are displayed in the gallery. The rightmost frames in (B) and (C) display averages of dimer (n = 387) and monomer (n = 316) top views, respectively. In hSVCT1 dimers (B) two stain accumulations are discerned, while in monomers (C) only one. Averages are low-pass filtered to 2 nm resolution. The scale bar in (A) is 100 nm. The frame sizes in (B) and (C) are 18.5 nm.
Figure 4.
Chemical crosslinking of hSVCT1 in isolated oocyte membranes.
Western blot analysis of isolated oocyte membranes containing hSVCT1 incubated at different DST concentrations, i.e., 0, 100, 250, 500 and 1000 µM. hSVCT1 monomer (M), dimer (D) and aggregate (A) bands are indicated. At ≥250 µM DST increasing smearing (S) and aggregation, and decreasing amounts of monomers are observed indicating unspecific crosslinking. Western blotting was performed using a 6% SDS/polyacrylamide gel and an anti-HA antibody. The interface between stacking and separating gels is indicated (dotted, white line).