Cys(294) is essential for the function of the human sodium-dependent multivitamin transporter

The sodium-dependent multivitamin transporter (SMVT) plays an important role in biotin uptake in the intestine and other cell types. While significant knowledge has been gained with regard to regulation and cell biology of the SMVT system, there is little known about its structure-function relationships. Here we examined the role of each of the ten conserved (among species) cysteine residues in the function of the human SMVT (hSMVT) using site-directed mutagenesis. Our results showed a significant impairment in biotin uptake only in cells transfected with hSMVT mutated at Cys(294), but not at the other conserved cysteine residues; the impairment in biotin uptake caused by mutating Cys(294) was not related to the polar status of substituting amino acid. The inhibition in hSMVT function upon mutating Cys(294) was mediated via a significant reduction in the V(max), but not the apparent K(m), of the biotin uptake process, suggesting a decrease in the number (and/or activity) of hSMVT but not affinity. Biotinylation assay confirmed this suggestion by showing a marked reduction in the level of expression of the mutated protein at the cell membrane, without affecting total cellular level of induced hSMVT. These results show an important role for Cys(294) in the function and cell biology of hSMVT.     

Cys294 is essential for the function of the human sodium-dependent multivitamin transporter

The sodium-dependent multivitamin transporter (SMVT) plays an important role in biotin uptake in the intestine and other cell types. While significant knowledge has been gained with regard to regulation and cell biology of the SMVT system, there is little known about its structure-function relationships. Here we examined the role of each of the ten conserved (among species) cysteine residues in the function of the human SMVT (hSMVT) using site-directed mutagenesis. Our results showed a significant impairment in biotin uptake only in cells transfected with hSMVT mutated at Cys²⁹⁴, but not at the other conserved cysteine residues; the impairment in biotin uptake caused by mutating Cys²⁹⁴ was not related to the polar status of substituting amino acid. The inhibition in hSMVT function upon mutating Cys²⁹⁴ was mediated via a significant reduction in the V_max, but not the apparent K_m, of the biotin uptake process, suggesting a decrease in the number (and/or activity) of hSMVT but not affinity. Biotinylation assay confirmed this suggestion by showing a marked reduction in the level of expression of the mutated protein at the cell membrane, without affecting total cellular level of induced hSMVT. These results show an important role for Cys²⁹⁴ in the function and cell biology of hSMVT.     

Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system

It has been well established that human intestinal and liver epithelial cells transport biotin via an Na+-dependent carrier-mediated mechanism. The sodium-dependent multivitamin transport (SMVT), a biotin transporter, is expressed in both cell types. However, the relative contribution of SMVT toward total carrier-mediated uptake of physiological (nanomolar) concentrations of biotin by these cells is not clear. Addressing this issue is important, especially in light of the recent identification of a second human high-affinity biotin uptake mechanism that operates at the nanomolar range. Hence, we employed a physiological approach of characterizing biotin uptake by human-derived intestinal Caco-2 and HepG2 cells at the nanomolar concentration range. We also employed a molecular biology approach of selectively silencing the endogenous SMVT of these cells with specific small interfering RNAs (siRNAs), then examining carrier-mediated biotin uptake. The results showed that in both Caco-2 and HepG2 cells, the initial rate of biotin uptake as a function of concentration over the range of 0.1 to 50 nM to be linear. Furthermore, we found that the addition of 100 nM unlabeled biotin, desthiobiotin, or pantothenic acid to the incubation medium had no effect on the uptake of 2.6 nM [3H]biotin. Pretreatment of Caco-2 and HepG2 cells with SMVT specific siRNAs substantially reduced SMVT mRNA and protein levels. In addition, carrier-mediated [3H]biotin (2.6 nM) uptake by Caco-2 and HepG2 cells was severely (P 0.01) inhibited by the siRNAs pretreatment. These results demonstrate that the recently described human high-affinity biotin uptake system is not functional in intestinal and liver epithelial cells. In addition, the results provide strong evidence that SMVT is the major (if not the only) biotin uptake system that operates in these cells.     

Role of the putative N-glycosylation and PKC-phosphorylation sites of the human sodium-dependent multivitamin transporter (hSMVT) in function and regulation

The sodium-dependent multivitamin transporter (SMVT) is a major biotin transporter in a variety of tissues including the small intestine. The human SMVT (hSMVT) polypeptide is predicted to have four N-glycosylation sites and two putative PKC phosphorylation sites but their role in the function and regulation of the protein is not known and was examined in this investigation. Our results showed that the hSMVT protein is glycosylated and that this glycosylation is important for its function. Studies utilizing site-directed mutagenesis revealed that the N-glycosylation sites at positions Asn(138) and Asn(489) are important for the function of hSMVT and that mutating these sites significantly reduces the V(max) of the biotin uptake process. Mutating the putative PKC phosphorylation site Thr(286) of hSMVT led to a significant decrease in the PMA-induced inhibition in biotin uptake. The latter effect was not mediated via changes in the level of expression of the hSMVT protein and mRNA or in its level of expression at the cell membrane. These findings demonstrate that the hSMVT protein is glycosylated, and that glycosylation is important for its function. Furthermore, the study shows a role for the putative PKC-phosphorylation site Thr(286) of hSMVT in the PKC-mediated regulation of biotin uptake.     

Inhibition of intestinal biotin absorption by chronic alcohol feeding: cellular and molecular mechanisms

The water-soluble vitamin biotin is essential for normal cellular functions and its deficiency leads to a variety of clinical abnormalities. Mammals obtain biotin from exogenous sources via intestinal absorption, a process mediated by the sodium-dependent multivitamin transporter (SMVT). Chronic alcohol use in humans is associated with a significant reduction in plasma biotin levels, and animal studies have shown inhibition in intestinal biotin absorption by chronic alcohol feeding. Little, however, is known about the cellular and molecular mechanisms involved in the inhibition in intestinal biotin transport by chronic alcohol use. These mechanisms were investigated in this study by using rats and transgenic mice carrying the human full-length SLC5A6 5'-regulatory region chronically fed alcohol liquid diets; human intestinal epithelial Caco-2 cells chronically exposed to alcohol were also used as models. The results showed chronic alcohol feeding of rats to lead to a significant inhibition in carrier-mediated biotin transport events across jejunal brush border and basolateral membrane domains. This inhibition was associated with a significant reduction in level of expression of the SMVT protein, mRNA, and heterogenous nuclear RNA. Chronic alcohol feeding also inhibited carrier-mediated biotin uptake in rat colon. Studies with transgenic mice confirmed the above findings and further showed chronic alcohol feeding significantly inhibited the activity of SLC5A6 5'-regulatory region. Finally, chronic exposure of Caco-2 cells to alcohol led to a significant decrease in the activity of both promoters P1 and P2 of the human SLC5A6 gene. These studies identify for the first time the cellular and molecular parameters of the intestinal biotin absorptive processes that are affected by chronic alcohol feeding.     

Human placental Na+-dependent multivitamin transporter. Cloning, functional expression, gene structure, and chromosomal localization.

We have cloned the human Na+-dependent multivitamin transporter (SMVT), which transports the water-soluble vitamins pantothenate, biotin, and lipoate, from a placental choriocarcinoma cell line (JAR). The cDNA codes for a protein of 635 amino acids with 12 transmembrane domains and 4 putative sites for N-linked glycosylation. The human SMVT exhibits a high degree of homology (84% identity and 89% similarity) to the rat counterpart. When expressed in HRPE cells, the cDNA-induced transport process is obligatorily dependent on Na+ and accepts pantothenate, biotin, and lipoate as substrates. The relationship between the cDNA-specific uptake rate of pantothenate or biotin and Na+ concentration is sigmoidal with a Na+:vitamin stoichiometry of 2:1. The human SMVT, when expressed in Xenopus laevis oocytes, induces inward currents in the presence of pantothenate, biotin, and lipoate in a Na+-, concentration-, and potential-dependent manner. We also report here on the structural organization and chromosomal localization of the human SMVT gene. The SMVT gene is approximately 14 kilobase pairs in length and consists of 17 exons. The SMVT gene is located on chromosome 2p23 as evidenced by somatic cell hybrid analysis and fluorescence in situ hybridization.     

PLEKHA5, PLEKHA6, and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis

Copper homeostasis is crucial for cellular physiology and development, and its dysregulation leads to disease. The Menkes ATPase ATP7A plays a key role in copper efflux, by trafficking from the Golgi to the plasma membrane upon cell exposure to elevated copper, but the mechanisms that target ATP7A to the cell periphery are poorly understood. PDZD11 interacts with the C-terminus of ATP7A, which contains sequences involved in ATP7A trafficking, but the role of PDZD11 in ATP7A localization is unknown. Here we identify PLEKHA5 and PLEKHA6 as new interactors of PDZD11 that bind to the PDZD11 N-terminus through their WW domains similarly to the junctional protein PLEKHA7. Using CRISPR-KO kidney epithelial cells, we show by immunofluorescence microscopy that WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) recruit PDZD11 to distinct plasma membrane localizations and that they are required for the efficient anterograde targeting of ATP7A to the cell periphery in elevated copper conditions. Pull-down experiments show that WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. However, WW-PLEKHAs and PDZD11 are not necessary for ATP7A Golgi localization in basal copper, ATP7A copper-induced exit from the Golgi, and ATP7A retrograde trafficking to the Golgi. Finally, measuring bioavailable and total cellular copper, metallothionein-1 expression, and cell viability shows that WW-PLEKHAs and PDZD11 are required for maintaining low intracellular copper levels when cells are exposed to elevated copper. These data indicate that WW-PLEKHAs-PDZD11 complexes regulate the localization and function of ATP7A to promote copper extrusion in elevated copper.     

Holocarboxylase synthetase regulates expression of biotin transporters by chromatin remodeling events at the SMVT locus

The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, biotin is covalently linked to histones in a reaction catalyzed by holocarboxylase synthetase (HCS); biotinylation of lysine 12-biotinylated histone H4 (K12Bio H4) causes gene silencing. Here, we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesized that nuclear translocation of HCS increases in response to biotin supplementation; HCS then biotinylates histone H4 at SMVT promoters, silencing biotin transporter genes. Jurkat lymphoma cells were cultured in media containing 0.025, 0.25, or 10 nmol/l biotin. The nuclear translocation of HCS correlated with biotin concentrations in media; the relative enrichment of both HCS and K12Bio H4 at SMVT promoter 1 (but not promoter 2) increased by 91% in cells cultured in medium containing 10 nmol/l biotin compared with 0.25 nmol/l biotin. This increase of K12Bio H4 at the SMVT promoter decreased SMVT expression by up to 86%. Biotin homeostasis by HCS-dependent chromatin remodeling at the SMVT promoter 1 locus was disrupted in HCS knockdown cells, as evidenced by abnormal chromatin structure (K12Bio H4 abundance) and increased SMVT expression. The findings from this study are consistent with the theory that HCS senses biotin, and that biotin regulates its own cellular uptake by participating in HCS-dependent chromatin remodeling events at the SMVT promoter 1 locus in Jurkat cells.     

Electrogenic nature of rat sodium-dependent multivitamin transport

We report on the electrogenic nature of the transport process mediated by the rat sodium-dependent multivitamin transporter. In Cos-7 cells, the relationship of Na(+) concentration versus biotin and pantothenate uptake rate was sigmoidal with a Na(+):substrate stoichiometry of 2:1. In Cos-7 cells expressing rat SMVT biotin transport was significantly higher when the membrane was hyperpolarized and considerably reduced when the membrane was depolarized. Similarly, biotin uptake in X. laevis oocytes expressing rat SMVT was inhibited with depolarized oocyte membrane by altering the K(+) permeability across the membrane. It is concluded that the transport of biotin and pantothenate mediated by rat SMVT is electrogenic with a Na(+):substrate coupling ratio of 2:1 and that the transport process is associated with the transfer of one net positive charge across the membrane per transport cycle.     

The PLEKHA7–PDZD11 complex regulates the localization of the calcium pump PMCA and calcium handling in cultured cells

The plasma membrane calcium ATPase (PMCA) extrudes calcium from the cytosol to the extracellular space to terminate calcium-dependent signaling. Although the distribution of PMCA is crucial for its function, the molecular mechanisms that regulate the localization of PMCA isoforms are not well understood. PLEKHA7 is implicated by genetic studies in hypertension and the regulation of calcium handling. PLEKHA7 recruits the small adapter protein PDZD11 to adherens junctions, and together they control the trafficking and localization of plasma membrane associated proteins, including the Menkes copper ATPase. Since PDZD11 binds to the C-terminal domain of b-isoforms of PMCA, PDZD11 and its interactor PLEKHA7 could control the localization and activity of PMCA. Here, we test this hypothesis using cultured cell model systems. We show using immunofluorescence microscopy and a surface biotinylation assay that KO of either PLEKHA7 or PDZD11 in mouse kidney collecting duct epithelial cells results in increased accumulation of endogenous PMCA at lateral cell–cell contacts and PDZ-dependent ectopic apical localization of exogenous PMCA4x/b isoform. In HeLa cells, coexpression of PDZD11 reduces membrane accumulation of overexpressed PMCA4x/b, and analysis of cytosolic calcium transients shows that PDZD11 counteracts calcium extrusion activity of overexpressed PMCA4x/b, but not PMCA4x/a, which lacks the PDZ-binding motif. Moreover, KO of PDZD11 in either endothelial (bEnd.3) or epithelial (mouse kidney collecting duct) cells increases the rate of calcium extrusion. Collectively, these results suggest that the PLEKHA7–PDZD11 complex modulates calcium homeostasis by regulating the localization of PMCA.     

Receptor for activated C-kinase 1, a novel binding partner of adiponectin receptor 1

Adiponectin is an adipose tissue derived hormone with anti-diabetic and insulin-sensitizing properties. Two adiponectin receptors, AdipoR1 and AdipoR2, have recently been identified, yet the signaling pathways triggered through adiponectin receptors remain to be elucidated. Using a yeast two-hybrid screen, we identified an adaptor protein, receptor for activated protein kinase C1 (RACK1), as an interacting partner of human AdipoR1. RACK1 was confirmed to interact with AdipoR1 by co-immunoprecipitation and co-localization analysis in mammalian cells. The interaction was enhanced by adiponectin stimulation. In addition, the knockdown of RACK1 by RNA interference inhibited adiponectin-stimulated glucose uptake in HepG2 cells. These results suggest that RACK1 may act as a key bridging factor in adiponectin signaling transduction through interacting with AdipoR1.     

Human placental brush-border membrane Na(+)-biotin cotransport.

Membrane transport pathways for transplacental transfer of the water-soluble vitamin biotin were investigated by assessing the possible presence of a Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells. The presence of Na(+)-biotin cotransport was determined from radiolabeled tracer flux measurements of biotin uptake using preparations of purified brush-border membrane vesicles. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of biotin to levels approximately 25-fold greater than those observed at equilibrium. The voltage sensitivity of Na+ gradient-driven biotin uptake suggested Na(+)-biotin cotransport is electrogenic occurring with net transfer of positive charge. A kinetic analysis of the activation of biotin uptake by increasing Na+ was most consistent with an interaction of Na+ at 2 sites in the transport protein. Static head determinations used to identify the magnitude of opposing driving forces bringing flux through the cotransport mechanism to equilibrium indicated a coupling ratio of 2 Na+ per biotin. Substrate specificity studies using chemical analogues of biotin suggested both the terminal carboxylic acid of the valeric acid side chain and a second nucleus of anionic charge were important determinants for substrate interaction with the cotransport protein. Initial rate determinations of biotin uptake indicate biotin interacts with a single saturable site (Km = 21 microM) with a maximal transport rate of 4.5 nmol/mg/min. The results of this study provide evidence for an electrogenic Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells.     

Identification and Characterization of MT-1X as a Novel FHL3-Binding Partner

Four and a half LIM domain protein 3 (FHL3) is a member of the FHL protein family that plays roles in the regulation of cell survival, cell adhesion and signal transduction. However, the mechanism of action for FHL3 is not yet clear. The aim of present study was to identify novel binding partner of FHL3 and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, FHL3 was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Methionine-1X was identified as a novel FHL3 binding partner. The interaction between FHL3 and the full length MT-1X was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore,the result demonstrated that MT-1X knockdown promoted the FHL3-induced inhibitory effect on HepG2 cells by regulating FHL3-mediated Smad signaling and involving in the modulation the expression of G2/M phase-related proteins through interaction with FHL3. These findings suggest that functional interactions between FHL3 and MT-1X may provide some clues to the mechanisms of FHL3-regulated cell proliferation.