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.

Identification of residues/sequences in the human riboflavin transporter-2 that is important for function and cell biology

“…Many nutrient/substrate transporters contain sequences/regions that are important for their function and targeting to cell membrane that are embedded within their N-terminal [ 24 , 31 ], C-terminal [ 23 , 24 , 32 , 33 ], and/or transmembrane backbone [ 34 , 35 ]. In this study, we show that both the N- (amino acids 1–9) and the C- terminal (amino acids 425–445) sequences of the hRFVT-2 protein are important for its function and cell biology.…”

“…After 24–48 hrs transient transfection cells were used for imaging. For stable transfection, U87 cells were selected using G418 (0.5 mg/ml) (Invitrogen) for 4–6 weeks as described before [ 23 , 24 ].…”

“…U87 cells transiently or stably expressing WT, mutated and truncated constructs were grown in 12 well plates and 3 H-RF uptake assay was performed in Krebs-Ringer (K-R) buffer at 37°C for 3 min (initial linear period; data not shown) following established procedure [ 23 , 24 ]. 3 H-RF (14 nM) was added to the K-R buffer at the time of uptake assay, and after 3 min the reaction was terminated by ice-cold K-R buffer.…”

Alteration of Liver Enzymes Is a Feature of the Myh9-Related Disease Syndrome

“…Myosin II was found to be enriched in the actin microfilament network around the bile canaliculus and was identified as the essential motor for the bile canalicular contraction [25] – [27] . Moreover, myosin II is involved in vesicle trafficking between the cytoplasmic compartment and plasma membrane and regulates the apical membrane expression of several transporters associated with bile secretion, such as the bile salt export pump, whose genetic defects are associated with some forms of familiar intrahepatic cholestasis [28] , [29] . Recent studies identified additional key roles for myosin II in hepatocytes, since it was implicated in postnatal hepatocytes polyploidization [30] spatial reorganization of hepatocytes during development and liver regeneration [31] , and cell cycle progression and motility [32] , [33] .…”

Vitamin C Transporters in Cancer: Current Understanding and Gaps in Knowledge

“…The localization of SVCT2 within the cell may determine its transport activity, although data are conflicting. SVCT2 protein can be retained within intracellular compartments or transport vesicles and may have different kinetic properties depending on its localization ( 63 ), but data in cancer cells are limited. In neurons in culture, SVCT2 translocated to the plasma membrane upon increased extracellular ascorbate concentration ( 64 ).…”