Glyoxalate reductase/hydroxypyruvate reductase interacts with the sodium-dependent vitamin C transporter-1 to regulate cellular vitamin C homeostasis

Abstract

The human sodium-dependent vitamin C transporter 1 (hSVCT1) contributes to cellular uptake of ascorbic acid (AA). Although different aspects of hSVCT1 cell biology have been extensively studied, nothing is currently known about the broader hSVCT1 interactome that modulates its role in cellular physiology. Here, we identify the enzyme human glyoxalate reductase/hydroxypyruvate reductase (hGR/HPR) as an hSVCT1 associated protein by yeast two-hybrid (Y2H) screening of a human liver cDNA library. The interaction between hSVCT1 and hGR/HPR was further confirmed by in vitro GST pull-down assay, in vivo coimmunoprecipitation and mammalian two-hybrid firefly luciferase assays. This interaction had functional significance as coexpression of hGR/HPR with hSVCT1 led to an increase in AA uptake. Reciprocally, siRNA-mediated knockdown of endogenous hGR/HPR led to an inhibition of AA uptake. Given that oxalate is a degradation product of vitamin C and hGR/HPR acts to limit cellular oxalate levels, this association physically couples two independent regulators of cellular oxalate production. Furthermore, confocal imaging of human liver HepG2 cells coexpressing GFP-hSVCT1 and hGR/HPR-mCherry demonstrated that these two proteins colocalize within a subpopulation of intracellular organelles. This provides a possible molecular basis for organellar AA transport and regulation of local glyoxylate/glycolate concentration in the vicinity of organelle membranes.

Association of TM4SF4 with the Human Thiamine Transporter-2 in Intestinal Epithelial Cells

“…The proteins bound to beads were then eluted with 10mM glutathione and subjected to western blotting using a rabbit polyclonal anti-hTHTR-2 antibodies (Abcam, MA, USA). The immunoreactive bands were visualized using anti-rabbit IRDye-800 secondary antibodies and an Odyssey infrared imaging system (LI-COR Bioscience, Lincoln, NB, USA) [18]. …”

“…Cellular extracts prepared from control cells and TM4SF4 siRNA pretreated HuTu-80 cells were used to perform western blotting [16, 18]. Total proteins (60 μg) were resolved onto premade 4–12% Bis-Tris minigel (Invitrogen), and subjected to western blotting [16, 18].…”

“…Total proteins (60 μg) were resolved onto premade 4–12% Bis-Tris minigel (Invitrogen), and subjected to western blotting [16, 18]. Proteins were electro-blotted onto immobilon polyvinylidene difluoride membrane (Fisher Scientific, CA, USA).…”

“…Along with blocking buffer (LI-COR Bioscience), the membranes were incubated overnight at 4°C with human TM4SF4 specific rabbit polyclonal antibodies (Sigma) together with the human β-actin monoclonal antibody (Santa Cruz Biotechnology). The immunoreactive bands were detected and the band intensity was quantified using LI-COR software [18]. …”

Extracellular Cl− regulates human SO4 2−/anion exchanger SLC26A1 by altering pH sensitivity of anion transport

“…The widely expressed SLC26A1 is particularly abundant in sinusoidal membranes of hepatocytes and in basolateral membranes of intestinal enterocytes and renal proximal tubular cells, with higher expression in male than female rats [12,37,42,60-62]. The liver is also the main site of oxalate uptake from dietary absorption, oxalate biosynthesis from ascorbate and amino acid metabolism, and oxalate catabolism [53,76]. Hepatic SLC26A1 is the likely sinusoidal exchanger of intracellular oxalate for extracellular SO 4 2− , enabling SO 4 2− uptake and oxalate secretion for eventual excretion by intestine and kidney [11].…”