Molecular determinants dictating cell surface expression of the human sodium-dependent vitamin C transporter-2 in human liver cells

Abstract

The human sodium-dependent vitamin C transporter-2 (hSVCT2) plays an important role in cellular accumulation of ascorbic acid in liver cells. However, little is known about the molecular determinants that direct hSVCT2 to the cell surface in hepatocytes. We addressed this issue using live cell imaging methods to resolve the distribution and trafficking of truncated or mutated hSVCT2 constructs in a cellular model of human hepatocytes, HepG2 cells. Whereas a full-length hSVCT2-yellow fluorescent protein (YFP) fusion protein was functionally expressed at the cell surface in HepG2 cells, serial truncation and mutation analysis demonstrated an essential role for both NH(2)- and COOH-terminal sequence(s) for cell surface expression and function. Video-rate confocal imaging showed evidence of dynamic hSVCT2-YFP containing intracellular trafficking vesicles, the motility of which was impaired following disruption of microtubules using nocodazole. However, in a HepG2 cell line stably expressing hSVCT2-YFP at the cell surface, plasma membrane levels of hSVCT2 were unaffected by inhibition of microtubule-associated motor proteins; rather, surface expression of hSVCT2-YFP was increased following treatment with myosin inhibitors. Together, these results show that 1) both NH(2)- and COOH-terminal sequences are essential for proper localization of hSVCT2, 2) cell surface delivery is dependent on intact microtubules, and 3) peripheral microfilaments regulate insertion and retrieval of hSVCT2 into the plasma membrane.

Fig. 1.

Membrane targeting of human sodium-dependent vitamin C transporter-2 (hSVCT2)-yellow fluorescent protein (YFP) in HepG2 cells. A: schematic representation of the full-length hSVCT2 protein (1–650 amino acids) with YFP fused to the COOH terminus (hSVCT2-YFP). B: lateral (x-y) confocal images of a stable HepG2 cell line expressing hSVCT2-YFP (top) and YFP (bottom). Scale bar = 10 μm. C: [¹⁴C]ascorbic acid uptake (32 μM) assays in a stable hSVCT2-YFP expressing HepG2 cell line. Results represent means ± SE (n ≥ 3, *P < 0.01).

Fig. 2.

COOH-terminal truncations disrupt the cell surface targeting of hSVCT2. A: distribution of hSVCT2 truncation constructs in stably expressing HepG2 cells in lateral (x-y) section. B: uptake of [¹⁴C]ascorbic acid in control HepG2 cells and cells expressing indicated truncation constructs. Shaded columns are data from Fig. 1C for comparison. C: flow cytometry analysis of the mean fluorescence intensity of populations of HepG2 cells stably expressing indicated constructs different from hSVCT2-YFP. Values are means ± SE (*P < 0.01 and **P < 0.05).

Fig. 3.

COOH-terminal mutations disrupt the cell surface targeting of hSVCT2 in HepG2 cells. A: distribution of indicated hSVCT2 mutant constructs in stably expressing HepG2 cells in lateral (x-y) section. Images show examples of differing expression profiles observed with indicated constructs. B: uptake of [¹⁴C]ascorbic acid in control and stable HepG2 cells. Shaded column data are from Fig. 1C for comparison. C: flow cytometry analysis of the mean fluorescence intensity of population of HepG2 cells stably expressing indicated mutant constructs. Values are means ± SE (*P < 0.01).

Fig. 4.

NH₂-terminal sequence is important for cell surface targeting of hSVCT2 in HepG2 cells. A and B: distribution of indicated hSVCT2 truncation and mutant constructs transiently (A) and stably expressing HepG2 cell lines (B), in confocal lateral (x-y) section. Images show examples of differing expression profiles observed with indicated constructs. C: [¹⁴C]ascorbic acid uptake in control stable HepG2 cell lines. Values are means ± SE; *significantly (P < 0.01) increased compared with control, **significantly (P < 0.01) decreased compared with hSVCT2-YFP.

Fig. 5.

Dynamics of hSVCT2-containing trafficking vesicles. A: lateral (x–y) confocal image of a single hSVCT2-expressing HepG2 cell. B: video still of HepG2 cell treated with nocodazole (10 μM, 15 min). Boxed areas (A and B) are enlarged in C and D. Scale bars = 10 μm. C: image stills exemplifying vesicular dynamics in control (green, top) or nocodazole-treated cells. Tracked vesicles are shown at unequal intervals during a period of 3 s. D: examples of individual tracks representing vesicle motility in control cells (green, left) or cells treated with nocodazole (red, right). E: collated data showing peak velocities (μm/s) of individual vesicles (n > 27 individual puncta) in control (black) or nocodazole-treated (red) cells. Vertical line represents population average.

Fig. 6.

Effect of motor protein inhibitors on hSVCT2 trafficking. A: stable hSVCT2-YFP expressing HepG2 cells were treated with DMSO (control), a cytoplasmic dynein inhibitor (vanadate, 100 μM, 24 h), a kinesin inhibitor (monastrol, 100 μM, 24 h), myosin inhibitors (BDM, 20 mM, 24 h) or blebbistatin (100 μM, 24 h). B: uptake of [¹⁴C]ascorbic acid in drug-treated stable hSVCT2-YFP expressing HepG2 cells. C: flow cytometry analysis of the mean fluorescence intensity of populations of hSVCT2-YFP expressing HepG2 cells treated with indicated motor protein inhibitors. Values are means ± SE. (*P < 0.01).