Molecular Cloning and Characterization of a Novel Isoform of the Human UDP-Galactose Transporter, and of Related Complementary DNAs Belonging to the Nucleotide-Sugar Transporter Gene Family

Ishida, Nobuhiro; Miura, Nobuhiko; Yoshioka, Shigemi; Kawakita, Masao

doi:10.1093/oxfordjournals.jbchem.a021523

Cited by 91 publications

(67 citation statements)

References 11 publications

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“…UGTrel1 is a gene of unknown function that has similarity with human UDP-galactose transporter genes (15). In humans, Muraoka et al (24) identified a gene related to UGTrel1 and reported that the product transports both UDPglucuronic acid and UDP-N-acetylgalactosamine.…”

Section: Fig 2 Subcellular Localization Of Papst1 Proteinmentioning

confidence: 99%

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Molecular Cloning and Identification of 3′-Phosphoadenosine 5′-Phosphosulfate Transporter

Kamiyama

Suda

Ueda

et al. 2003

Journal of Biological Chemistry

127

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Nucleotide sulfate, namely 3 -phosphoadenosine 5 -phosphosulfate (PAPS), is a universal sulfuryl donor for sulfation. Although a specific PAPS transporter is present in Golgi membrane, no study has reported the corresponding gene. We have identified a novel human gene encoding a PAPS transporter, which we have named PAPST1, and the Drosophila melanogaster ortholog, slalom (sll). The amino acid sequence of PAPST1 (432 amino acids) exhibited 48.1% identity with SLL (465 amino acids), and hydropathy analysis predicted the two to be type III transmembrane proteins. The transient expression of PAPST1 in SW480 cells showed a subcellular localization in Golgi membrane. The expression of PAPST1 and SLL in yeast Saccharomyces cerevisiae significantly increased the transport of PAPS into the Golgi membrane fraction. In human tissues, PAPST1 is highly expressed in the placenta and pancreas and present at lower levels in the colon and heart. An RNA interference fly of sll produced with a GAL4-UAS system revealed that the PAPS transporter is essential for viability. It is well known that mutations of some genes related to PAPS synthesis are responsible for human inherited disorders. Our findings provide insights into the significance of PAPS transport and post-translational sulfation.Glycosylation, phosphorylation, and sulfation are essential post-translational alterations of glycoproteins, proteoglycans, and glycolipids for normal growth and development. For sulfation, an activated form of sulfate, 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS), 1 is used as a common sulfate donor (1). Sulfate is transferred from PAPS to a defined position on the sugar residue by sulfotransferases. In higher organisms, PAPS is synthesized in the cytosol (2, 3) by a bifunctional PAPS synthetase having both ATP-sulfurylase and adenosine-phosphosulfate kinase activities (4). Since sulfation occurs in the lumens of the endoplasmic reticulum and Golgi apparatus (5), PAPS must be translocated from the cytosol into the Golgi lumen through a specific transporter localized in the microsomal membrane.Earlier studies had shown a saturable transport activity of PAPS using isolated Golgi vesicles (6) or reconstituted proteoliposome (7). To identify this transporter protein, the proteins responsible for PAPS translocating activity have been purified (8 -10). The characterization of these purified proteins revealed the kinetic behavior of a PAPS-specific transport through an antiport mechanism (8 -10); however, cloning of the transporter has not been reported.Recently, several nucleotide-sugar transporters (NSTs) have been cloned and characterized in mammals, yeast, protozoa, and plants (11-13). Nucleotide-sugars are the donor substrates for glycosylation, which is catalyzed by glycosyltransferases. These NST proteins are highly hydrophobic Type III multitransmembrane proteins localized in the Golgi or endoplasmic reticulum membrane and provide a specific substrate for the glycosylation. The structural conservation among NSTs has contributed to the identifi...

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Section: Fig 2 Subcellular Localization Of Papst1 Proteinmentioning

confidence: 99%

“…NST gene homologous to human UGTrel1 (UDP-galactose transporter-related isozyme 1) (15). Unexpectedly, the heterologous expression of PAPST1 in yeast Saccharomyces cerevisiae did not result in any nucleotide-sugar transport activity but revealed PAPS transport activity.…”

mentioning

confidence: 96%

Molecular Cloning and Identification of 3′-Phosphoadenosine 5′-Phosphosulfate Transporter

Kamiyama

Suda

Ueda

et al. 2003

Journal of Biological Chemistry

127

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“…cerevisiae HUT1 (scHUT1) encodes a putative hydrophobic protein with approximately 30% identity to human UDP-galactose transporter-related gene (UGTrel1) product, which is somewhat similar to human UDP-galactose transporter gene (UGT1) product, but the function is unknown (Ishida et al, 1996). We have found that the overexpression of scHUT1 in S. cerevisiae cells expressing S. pombe galactosyltransferase (Gma12p), stimulates in vivo galactosylation and in vitro UDP-galactose transport activity (Kainuma et al, this Issue).…”

Section: Introductionmentioning

confidence: 99%

Hut1 proteins identified in Saccharomyces cerevisiae and Schizosaccharomyces pombe are functional homologues involved in the protein‐folding process at the endoplasmic reticulum

Nakanishi

Nakayama

Yokota

et al. 2001

Yeast

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The Saccharomyces cerevisiae HUT1 gene (scHUT1) and the Schizosaccharomyces pombe hut1+ gene (sphut1 + ) encode hydrophobic proteins with approximately 30% identity to a human UDP-galactose transporter-related gene (UGTrel1) product. These proteins show a significant similarity to the nucleotide sugar transporter and are conserved in many eukaryotic species, but their physiological functions are not known. Both scHUT1 and sphut1+ genes are non-essential for cell growth under normal conditions, and their disruptants show no defects in the modification of O-and N-linked oligosaccharides, but are sensitive to a membrane-permeable reducing agent, dithiothreitol (DTT). Consistent with this phenotype, scHUT1 has genetic interaction with ERO1, which plays an essential role in the oxidation of secretory proteins at the endoplasmic reticulum (ER). Overexpression of the MPD1 or MPD2 genes, which were isolated as multicopy suppressors of protein disulphide isomerase (PDI) depletion, could not replace the essential function of PDI in Dhut1 S. cerevisiae cells. Our results indicate that scHut1p and spHut1p are functional homologues, and their physiological function is to maintain the optimal environment for the folding of secretory pathway proteins in the ER.

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“…Using PCR-based approaches, the murine UGT (11), a second isoform of the human UGT (12), and a full-length S. pombe UGT cDNA (13) were isolated. Sequence alignments reveal strong conservation of UGTs, since species as distant as human and S. pombe are 40% identical (see Fig.…”

mentioning

confidence: 99%

Point Mutations Identified in Lec8 Chinese Hamster Ovary Glycosylation Mutants That Inactivate Both the UDP-galactose and CMP-sialic Acid Transporters

Oelmann

Stanley

Gerardy‐Schahn

2001

Journal of Biological Chemistry

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Nucleotide-sugar transporters (NSTs) are critical components of glycosylation pathways in eukaryotes. The identification of structural elements that are involved in NST functions provides an important task. Chinese hamster ovary glycosylation mutants defective in nucleotide-sugar transport provide access to inactive transporters that can define such structure/function relationships. In this study, we have cloned the hamster UDP-galactose transporter (UGT) and identified defects in UGT gene transcripts from nine independent Chinese hamster ovary mutants that belong to the Lec8 complementation group. Reverse transcription polymerase chain reaction with primers that span the UGT open reading frame showed that three Lec8 mutants express a full-length open reading frame, while six Lec8 mutants predominantly express truncated UGT gene transcripts. Sequencing identified different single or triplet nucleotide changes in full-length UGT transcripts from three of the mutants. These mutations translate into three different amino acid changes at positions that are highly conserved in all the known mammalian NSTs. Transfection of a cDNA encoding either of the mutations ⌬serine 213 or G281D failed to correct the UDP-galactose transport defect in Lec8 transfectants. Most importantly, introducing these same mutations into the homologous region of the murine CMP-sialic acid transporter caused inactivation of this transporter. Thus, identifying point mutations that inactivate UGT in Lec8 mutants resulted in the discovery of amino acids that are critical to the activity of both UGT and CST, the two most divergent mammalian NSTs.

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Molecular Cloning and Characterization of a Novel Isoform of the Human UDP-Galactose Transporter, and of Related Complementary DNAs Belonging to the Nucleotide-Sugar Transporter Gene Family

Cited by 91 publications

References 11 publications

Molecular Cloning and Identification of 3′-Phosphoadenosine 5′-Phosphosulfate Transporter

Molecular Cloning and Identification of 3′-Phosphoadenosine 5′-Phosphosulfate Transporter

Hut1 proteins identified in Saccharomyces cerevisiae and Schizosaccharomyces pombe are functional homologues involved in the protein‐folding process at the endoplasmic reticulum

Point Mutations Identified in Lec8 Chinese Hamster Ovary Glycosylation Mutants That Inactivate Both the UDP-galactose and CMP-sialic Acid Transporters

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