Nucleotide Sugar Transporters: Elucidation of Their Molecular Identity and Its Implication for Future Studies

Kawakita, Masao; Ishida, Nobuhiro; Miura, Nobuhiko; Sun‐Wada, Ge‐Hong; Yoshioka, Shigemi

doi:10.1093/oxfordjournals.jbchem.a022004

Cited by 54 publications

(43 citation statements)

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“…Chimeric transporters such as E2 and G1, which are dually specific for UDP-Gal and CMP-Sia, should be very helpful for achieving an understanding of these mechanisms. Some pertinent questions include: 1) to what extent do the binding sites of UDP-Gal and CMP-Sia overlap in the dually specific chimeric transporters? 2) How many and which helices are actually involved in constructing the whole specific substrate-binding sites and the transport channel?…”

Section: Discussionmentioning

confidence: 99%

Substrate Recognition by UDP-galactose and CMP-sialic Acid Transporters

Aoki

Ishida

Kawakita

2001

Journal of Biological Chemistry

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Human UDP-galactose transporter (hUGT1) and CMPsialic acid transporter (hCST) are related Golgi membrane proteins with 10 transmembrane helices. We have constructed chimeras between these proteins in order to identify submolecular regions responsible for the determination of substrate specificity. To assess the UGT and CST activities, chimeric cDNAs were transiently expressed in either UGT-deficient mutant Lec8 cells or CST-deficient mutant Lec2 cells, and the binding of plant lectins, GS-II or PNA, respectively, to these cells was examined. During the course of analysis of various chimeric transporters, we found that chimeras whose submolecular regions contained helices 1, 8, 9, and 10, and helices 2, 3, and 7 derived from hUGT1 and hCST sequences, respectively, exhibited both UGT and CST activities. The dual substrate specificity for UDP-galactose and CMP-sialic acid of one such representative chimera was directly confirmed by in vitro measurement of the nucleotide sugar transport activity using a heterologous expression system in the yeast Saccharomyces cerevisiae. These findings indicated that the regions which are critical for determining the substrate specificity of UGT and CST resided in different submolecular sites in the two transporters, and that these different determinants could be present within one protein without interfering with each other's function.Nucleotide sugar transporters are a family of related proteins located in the endoplasmic reticulum and the Golgi membranes (for recent reviews, see Refs. 1 and 2). Their substrates, nucleotide sugars, are synthesized in either the cytoplasm or the nucleus. These nucleotide sugars have to be delivered into the lumen of the endoplasmic reticulum or the Golgi apparatus by appropriate transporters before they are utilized by glycosyltransferases as substrates for glycoconjugate biosynthesis. The transporters are thus indispensable for this process. In fact, nucleotide sugar transporter deficiency leads to a pleiotropic aberration in cellular glycoproteins and glycolipids (3, 4), and may be responsible for a congenital disorder, leukocyte adhesion deficiency type II (5). These transporters may also be involved in controlling the spectrum of glycoconjugates synthesized by a cell, by regulating the amounts of nucleotide sugars delivered into the Golgi lumen. We need to learn much more about the molecular basis of nucleotide sugar transporter function and its regulation to better understand the mechanisms and significance of transporter-mediated glycoconjugate control.Several cDNAs encoding nucleotide sugar transporters have been cloned so far, including cDNAs for UDP-galactose (UDPGal) transporter (UGT) 1 (6 -9), CMP-sialic acid (CMP-Sia) transporter (CST) (7, 10, 11), UDP-N-acetylglucosamine (UDPGlcNAc) transporter (12-14), and GDP-mannose (GDP-Man) transporter (15-18), each from a few species. These transporters constitute a family of related membrane proteins that are localized in the Golgi apparatus and have similar hydropathy profiles. Regarding their...

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Section: Discussionmentioning

confidence: 99%

Substrate Recognition by UDP-galactose and CMP-sialic Acid Transporters

Aoki

Ishida

Kawakita

2001

Journal of Biological Chemistry

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“…], and 62% identity with an ortholog in the moss Physcomitrella patens). NSTs constitute a family of transmembrane proteins that transport nucleotide-activated sugars from cytoplasm to ER and Golgi lumens, where activated sugars are used as substrates for various glycosyl transferases that synthesize polysaccharides and modify proteins and lipids (Kawakita et al, 1998;Berninsone and Hirschberg, 2000). Although its own substrate specificity is unknown at present, in a multispecies phylogenetic tree of the NST family, TEX2 clusters together with another Arabidopsis protein, At5g41760 (Bakker et al, 2005), capable of transporting CMPsialic acid into the Golgi apparatus (Bakker et al, 2008).…”

Section: Disruption Of the Exine Gene Network Can Have Many Morphologmentioning

confidence: 99%

A Large-Scale Genetic Screen in Arabidopsis to Identify Genes Involved in Pollen Exine Production

et al. 2011

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Exine, the outer plant pollen wall, has elaborate species-specific patterns, provides a protective barrier for male gametophytes, and serves as a mediator of strong and species-specific pollen-stigma adhesion. Exine is made of sporopollenin, a material remarkable for its strength, elasticity, and chemical durability. The chemical nature of sporopollenin, as well as the developmental mechanisms that govern its assembly into diverse patterns in different species, are poorly understood. Here, we describe a simple yet effective genetic screen in Arabidopsis (Arabidopsis thaliana) that was undertaken to advance our understanding of sporopollenin synthesis and exine assembly. This screen led to the recovery of mutants with a variety of defects in exine structure, including multiple mutants with novel phenotypes. Fifty-six mutants were selected for further characterization and are reported here. In 14 cases, we have mapped defects to specific genes, including four with previously demonstrated or suggested roles in exine development (MALE STERILITY2, CYP703A2, ANTHER-SPECIFIC PROTEIN6, TETRAKETIDE α-PYRONE REDUCTASE/DIHYDROFLAVONOL-4-REDUCTASE-LIKE1), and a number of genes that have not been implicated in exine production prior to this screen (among them, fatty acid ω-hydroxylase CYP704B1, putative glycosyl transferases At1g27600 and At1g33430, 4-coumarate-coenzyme A ligase 4CL3, polygalacturonase QUARTET3, novel gene At5g58100, and nucleotide-sugar transporter At5g65000). Our study illustrates that morphological screens of pollen can be extremely fruitful in identifying previously unknown exine genes and lays the foundation for biochemical, developmental, and evolutionary studies of exine production.

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“…This mutant is deficient in UDP-Gal transport, leading to decreased glycoprotein galactosylation, deficiency in sialylation, and a failure to react with the sialic acid (␣2,3)-galactose-specific M. amurensis agglutinin (41,51). When a functional UDP-Gal transporter is expressed in these cells, M. amurensis agglutinin binding is restored (52), a widely used assay to functionally identify and characterize UDP-Gal transporters from a variety of species (19,21,22).…”

Section: Lpg5a and Lpg5b Rescue The Glycosylation Defect Of The Udp-gmentioning

confidence: 99%

Two Functionally Divergent UDP-Gal Nucleotide Sugar Transporters Participate in Phosphoglycan Synthesis in Leishmania major

Capul

Barron

Dobson

et al. 2007

Journal of Biological Chemistry

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In the protozoan parasite Leishmania, abundant surface and secreted molecules, such as lipophosphoglycan (LPG) and proteophosphoglycans (PPGs), contain extensive galactose in the form of phosphoglycans (PGs) based on (Gal-Man-PO 4 ) repeating units. PGs are synthesized in the parasite Golgi apparatus and require transport of cytoplasmic nucleotide sugar precursors to the Golgi lumen by nucleotide sugar transporters (NSTs). GDP-Man transport is mediated by the LPG2 gene product, and here we focused on transporters for UDP-Gal. Data base mining revealed 12 candidate NST genes in the L. major genome, including LPG2 as well as a candidate endoplasmic reticulum UDP-glucose transporter (HUT1L) and several pseudogenes. Gene knock-out studies established that two genes (LPG5A and LPG5B) encoded UDP-Gal NSTs. Although the single lpg5A ؊ and lpg5B ؊ mutants produced PGs, an lpg5A ؊ /5B ؊ double mutant was completely deficient. PG synthesis was restored in the lpg5A ؊ /5B ؊ mutant by heterologous expression of the human UDP-Gal transporter, and heterologous expression of LPG5A and LPG5B rescued the glycosylation defects of the mammalian Lec8 mutant, which is deficient in UDP-Gal uptake. Interestingly, the LPG5A and LPG5B functions overlap but are not equivalent, since the lpg5A ؊ mutant showed a partial defect in LPG but not PPG phosphoglycosylation, whereas the lpg5B ؊ mutant showed a partial defect in PPG but not LPG phosphoglycosylation. Identification of these key NSTs in Leishmania will facilitate the dissection of glycoconjugate synthesis and its role(s) in the parasite life cycle and further our understanding of NSTs generally.

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Nucleotide Sugar Transporters: Elucidation of Their Molecular Identity and Its Implication for Future Studies

Cited by 54 publications

References 0 publications

Substrate Recognition by UDP-galactose and CMP-sialic Acid Transporters

Substrate Recognition by UDP-galactose and CMP-sialic Acid Transporters

A Large-Scale Genetic Screen in Arabidopsis to Identify Genes Involved in Pollen Exine Production

Two Functionally Divergent UDP-Gal Nucleotide Sugar Transporters Participate in Phosphoglycan Synthesis in Leishmania major

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