FEBS Letters 580 (2006) 4246–4251

Characterization of AtNST-KT1, a novel UDP-galactosetransporter from Arabidopsis thaliana

Inga Rollwitz, Marcella Santaella, Diana Hille, Ulf-Ingo Flugge, Karsten Fischer*

Botanisches Institut, Universitat zu Koln, Gyrhofstr. 15, 50931 Koln, Germany

Received 22 May 2006; revised 21 June 2006; accepted 23 June 2006

Available online 5 July 2006

Edited by Michael R. Sussman

Abstract Nucleotide sugar transporters (NST) mediate thetransfer of nucleotide sugars from the cytosol into the lumen ofthe endoplasmatic reticulum and the Golgi apparatus. Becausethe NSTs show similarities with the plastidic phosphate translo-cators (pPTs), these proteins were grouped into the TPT/NSTsuperfamily. In this study, a member of the NST-KT family,AtNST-KT1, was functionally characterized by expression ofthe corresponding cDNA in yeast cells and subsequent transportexperiments. The histidine-tagged protein was purified by affinitychromatography and reconstituted into proteoliposomes. Thesubstrate specificity of AtNST-KT1 was determined by measur-ing the import of radiolabelled nucleotide mono phosphates intoliposomes preloaded with various unlabelled nucleotide sugars.This approach has the advantage that only one substrate hasto be used in a radioactively labelled form while all the nucleotidesugars can be provided unlabelled. It turned out that AtNST-KT1 represents a monospecific NST transporting UMP in count-erexchange with UDP-Gal but did not transport other nucleotidesugars. The AtNST-KT1 gene is ubiquitously expressed in all tis-sues. AtNST-KT1 is localized to Golgi membranes. Thus,AtNST-KT1 is most probably involved in the synthesis of galac-tose-containing glyco-conjugates in plants.� 2006 Federation of European Biochemical Societies. Publishedby Elsevier B.V. All rights reserved.

Keywords: Nucleotide sugar transporters; UDP-galactose;Golgi; Phosphate translocator

1. Introduction

One of the main functions of the endoplasmatic reticulum

(ER) and the Golgi apparatus in plant cells is the transfer of

sugars to glycoproteins, glycolipids and to polysaccharides of

the cell wall like xyloglucans, arabinoxylans, and glucomann-

ans [1]. These non-cellulosic polysaccharides and numerous

cell wall associated glycoproteins contain a number of different

sugars such as galactose, glucose, mannose and others. Synthe-

Abbreviations: AtUTr1, Arabidopsis thaliana UDP-galactose trans-porter 1; ER, endoplasmatic reticulum; GDP-Fuc, GDP-fucose; GDP-Man, GDP-mannose; GONST, golgi nucleotide sugar transporter;GUS, b-glucuronidase; NST, nucleotide sugar transporter; pPT,plastidic phosphate translocators; UDP-Gal, UDP-galactose; UDP-GalNAc, UDP-N-acetyl-galactosamine; UDP-Glc, UDP-glucose;UDP-GlcA, UDP-glucuronic acid; UDP-GlcNAc, UDP-N-acetyl-glu-cosamine

*Corresponding author. Fax: +49 221 4705039.E-mail address: Karsten.Fischer@uni-koeln.de (K. Fischer).

0014-5793/$32.00 � 2006 Federation of European Biochemical Societies. Pu

doi:10.1016/j.febslet.2006.06.082

ses of polysaccharides and proteoglucans are performed by

synthases and glycosyltransferases of the ER and Golgi lumen

that use nucleotide sugars as substrates, i.e. sugars that are

activated by the addition of UDP or GDP [2]. Most nucleotide

sugars are synthesized in the cytosol from UDP-Glc, GDP-

Man or myo-inositol [3,4]. Therefore, the nucleotide sugars

must be transported from the cytosol into the lumen of the

ER and Golgi apparatus. While most, but not all of the sugars

are transported across the ER membranes attached to doli-

chol, the transport into the Golgi lumen is mediated by nucle-

otide sugar transporters (NSTs) [5].

In the last decade, several NSTs from different organisms

were characterized at the molecular level [6], e.g. the yeast

UDP-GlcNAc transporter [7], the human UDP-Gal trans-

porter [8] and the Arabidopsis GDP-Man transporter [9].

Interestingly, the NSTs show sequence similarities to phos-

phate translocators of the inner envelope membrane of plastids

(pPTs) that transport inorganic phosphate in exchange with

phosphorylated compounds [10,11]. Therefore the pPTs and

NSTs belong to the same superfamily named TPT/NST that

in turn is part of the drug/metabolite superfamily [12]. In the

GDP-Man transporters from yeast and Arabidopsis that con-

stitute the golgi nucleotide sugar transporter (GONST) family,

a conserved GALNK motif has been identified that is required

for binding of the nucleotide sugar [9,13]. This consensus motif

is located in a region that is highly conserved between the pPTs

and NSTs. Remarkably, the lysine residue at the end of this

motif is conserved in all proteins of the TPT/NST superfamily

[11]. In the case of the GDP-Fuc transporter from human, a

mutation in that region leads to a defect in GDP-Fuc transport

into the lumen of the Golgi [14] suggesting that this region is

part of the substrate binding site.

Besides sequence similarities, further evidence corroborates

the close relationship between pPTs and NSTs. Both the size

of the NST and pPT proteins and the number of membrane

spanning regions are similar. The pPTs and NSTs consist of

320–340 amino acids and possess 8–10 transmembrane do-

mains [5,11,15]. Both groups of transporters function as

antiporters. NSTs exchange nucleotide sugars with the corre-

sponding nucleotide monophosphates [16] that originate, fol-

lowing transfer of the sugars by glycosyltransferases, from a

Golgi nucleotide diphosphatase.

The Arabidopsis genome contains about 50 genes encoding

members of the TPT/NST superfamily. Several of the NST-like

plant proteins can be divided into different families, that were

named the NST-KT-, NST-KVAG- and the NST-KD families

according to specific dipeptides at the putative substrate bind-

ing site [11]. These three families comprise more than 20

blished by Elsevier B.V. All rights reserved.

I. Rollwitz et al. / FEBS Letters 580 (2006) 4246–4251 4247

proteins of mostly unknown function. Only recently, the first

three proteins of these groups have been functionally charac-

terized. One protein of the NST-KT family and one of the

NST-KVAG family were shown to be UDP-Gal transporters

[17] while a second protein of the NST-KVAG family might

transport GDP-Man but also other nucleotide sugars [18].

These results and further extensive analysis of NST proteins

revealed that the degree of sequence identity is not indicative

for the substrate specificity of the transporters [6], i.e. the pre-

diction of the activities of these transporters by sequence anal-

ysis seems to be infeasible for most NSTs. Therefore, we

determined the function of a transporter of the NST-KT fam-

ily by measuring its transport activity after reconstitution into

proteoliposomes. Here we show that this protein functions as a

UDP-Gal transporter localized to Golgi membranes.

2. Materials and methods

2.1. MaterialsRadioactive nucleotide mono phosphates (a-33P-UMP) were pur-

chased from Hartmann Analyticals (Braunschweig, Germany). Nucle-otide sugars were obtained from Sigma (St. Louis, MO). The Gatewaydestination vectors used in this work (pGWB3, and pGWB5) werekindly provided by Tsuyoshi Nakagawa (Shimane University, Japan).The mouse sialyltransferase-GFP fusion in pGWB5 was kindly pro-vided by Martin Hulskamp (University of Cologne, Germany).

2.2. Cloning proceduresRNA was isolated from whole plants. First strand cDNA was syn-

thesized using SuperScript� II reverse transcriptase (Invitrogen LifeTechnologies). For expression in yeast, the coding regions of thecDNA encoding AtNST-KT1 and AtUDP-GalT2 (lacking the stop-co-dons) were amplified using the primers AtNST-KT1for (5 0-C ACCAAA ATG TTT AAA AAA ATG AGT GCT ACT-3 0), AtNST-KT1rev (5 0-CAC CTT TTC ATC AGA TTC GTT T-3 0), AtUG-alT2for (5 0-C AAC AAA ATG GAG AAA CCG GAG AGC GAGA-3 0) and AtUGalT2rev (5 0-TGG TTT AGT GTC ACC GAGTTC-3 0). The cDNA fragments were cloned into pENTR�/D-TOPO(Invitrogen Life Technologies) resulting in vector pENTR-AtNST-KT1 and pENTR-AtUGalT2. The DNA fragment of pENTR-AtNST-KT1 was subsequently cloned into the yeast expression vectorpYES-Dest52 (Invitrogen Life Technologies) using the LR-clonasereaction resulting in clone pYES-AtNST-KT1. This clone containeda His-6 tag fused to the C-terminus of the protein.

The AtNST-KT1-GFP and AtUGalT2-GFP constructs were gener-ated as translational fusions by cloning the DNA fragments from vec-tors pENTR-AtNST-KT1 and pENTR-AtUGalT2 into vector pGWB5resulting in clones pGWB5-AtNST-KT1 and pGWB5-AtUGalT2.

For the generation of the AtNST-KT1 promoter-GUS construct, thepromoter of the AtNST-KT1 gene including the first exon of the cod-ing region was amplified using the primers AtNST-KT1promfor(5 0-CAC CTC TTT AAC CAA ACG GAA CCT-3 0) and AtNST-KT1promrev (5 0-GAT GAT TCC AAC GGA AGT AA-3 0). Theresulting DNA fragment was cloned into pENTR�/D-TOPO (Invitro-gen Life Technologies) resulting in pENTR-AtNST-KT1prom, se-quenced and then cloned into the vector pGWB3 resulting in clonepGWB3-AtNST-KT1prom that contained a translational fusion ofAtNST-KT1 with the uidA gene from E. coli.

2.3. Plant transformation and gene expression analysisTransgenic plants were generated by vacuum infiltration of Arabid-

opsis thaliana plants using Agrobacterium cultures containing pGWB3-AtNST-KT1. Transformants were selected with kanamycin andverified by PCR analysis. Histochemical localization of GUS activitywas performed as described previously [19].

For analysis of subcellular protein localization, tobacco BY2 cellswere transformed with vectors pGWB5-AtNST-KT1, pGWB5-AtUG-alT2 and pGWB5-sialyltransferase essentially as described by Negrutiuet al. [20]. Protoplasts were incubated at 22 �C in the dark. Aliquots for

analysis of GFP fluorescence were taken 18–24 h after transformationof the protoplasts. Fluorescence microscopy was performed using a Ni-kon eclipse E800 microscope coupled to a Digital camera (ky- F1030,JVC, Japan). Detection of GFP was achieved using a GFP filter (exci-tation 460–500 nm and emission 510–560 nm).

2.4. Yeast transformation, reconstitution of transport activities, and

in vitro transport assayThe uracil-auxotrophic yeast strain InvSc1 was transformed with

pYES-AtNST-KT1 and cells containing the plasmid were selected onplates lacking uracil. For membrane preparation, cells were grown insynthetic complete dropout liquid media lacking uracil and the expres-sion of AtNST-KT1 was induced by the addition of galactose (2% (w/v)). Cells were harvested 6 h after induction, disrupted in a buffer con-taining 10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 300 lg mL�1 phenyl-methylsulfonyl-fluoride and the 100000 · g membrane fractioncontaining the expressed AtNST-KT1-His6 protein was prepared byultracentrifugation.

Reconstitution of the transport activity was performed essentially asdescribed [21], with slight modifications. Liposomes were preparedfrom acetone-washed phosphatidylcholine (120–130 mg mL�1) by son-ication for 4 min on ice in 100 mM potassium phosphate, pH 7.8,50 mM potassium gluconate, and 0.2 mM of substrate as indicated.Yeast membranes were solubilized using 1.5–3% (w/v) Triton X-100as detergent and directly reconstituted or subjected to purificationvia metal-affinity chromatography on Ni2+-nitrilotriacetic acid agarose(Qiagen, Hilden, Germany) before being added to the liposomes. Affin-ity purification was performed essentially as described [22]. Incorpora-tion of the purified protein into the liposomes was achieved by afreeze–thaw step. After sonication, the external medium was removedby passing the liposomes over Sephadex G-25 columns. Eluted prote-oliposomes were used for transport, which was initiated by addition of[33P] UMP (20 lM) as the external counter-exchange substrate and ter-minated at different time points (15–120 s) by passing the liposomesover a Dowex AG1-X8 anion exchange column. The radioactivity ofthe eluate was determined by liquid scintillation counting.

3. Results and discussion

3.1. Molecular characterization of AtNST-KT1

Plants possess a remarkable high number of NST proteins as

revealed by database searches (http://aramemnon.botanik.uni-

koeln.de/) that can be grouped into five different families

[11,17]. In Arabidopsis, 43 distinct genes encoding NSTs and

seven genes (including one pseudogene) encoding pPTs could

be identified while 43 NST genes and ten pPT genes were de-

tected in rice. Only nine of the 43 NST proteins in Arabidopsis

have been analyzed in detail so far, namely members of the

GONST family, GONST1 to GONST4, that transport GDP-

Man [9,18] and two members of a family that was named

UTR according to the AtUTr proteins which have been de-

scribed by Norambuena and colleagues [23,24]. AtUTr1 was

the first NST from plants shown to transport UDP-activated

sugars, namely UDP-Gal and UDP-Glc [23] while a second

transporter, AtUTr2, was shown to solely transport UDP-

Gal [24]. Three NST families have been named NST-KD,

NST-KT and NST-KVAG according to a specific dipeptide

that is part of the putative substrate binding site [11]. Two pro-

teins belonging to the NST-KVAG family have been shown to

transport UDP-Gal (AtUDP-GalT1) [17] and GDP-Man

(GONST5) [18]. In contrast, the monospecific UDP-Gal trans-

porter AtUDP-GalT2 [17] is a member of the NST-KT family.

To characterize the substrate specificities and the physiolog-

ical function of NSTs, three different experimental strategies

have been employed. First, mutants of yeast or other

organisms that are defective in a particular nucleotide sugar

transport activity were functionally complemented by

4248 I. Rollwitz et al. / FEBS Letters 580 (2006) 4246–4251

(heterologous) NSTs thereby allowing the characterization of

the corresponding substrate specificities [9,17,18,23,25]. Sec-

ond, most of the NSTs have been characterized by measuring

the transport of radiolabelled nucleotide sugars into micro-

somes or Golgi enriched vesicles isolated from rat liver or yeast

that overexpress particular NST proteins [9,10,17,23,24]. How-

ever, characterization of NSTs in transport assays using Golgi

vesicles could be complicated by a high background of endog-

enous transport activities and by competing glycosyltransfe-

rases that also use nucleotide sugars as substrates [26]. Third,

Fig. 1. Expression of AtNST-KT1 in yeast cells and purification of theHis-tagged protein. (A) Western blot analysis of yeast membraneproteins. Membrane proteins were isolated from cells of clone pYES-AtNST-KT1 and pYES-Dest52. Proteins were separated by SDS–PAGEand transferred onto nylon membranes. His-tagged AtNST-KT1 proteinwas detected by an anti-His antibody. M, Molecular weight marker; 1,pYES-AtNST-KT1; 2, pYES-Dest52. (B) Purification of the AtNST-KT1 protein by Ni2+-NTA chromatography. Yeast membrane pro-teins were solubilized by detergent and the AtNST-KT1 protein waspurified by affinity chromatography (see Section 2). Total yeastmembrane proteins and aliquots of different fractions were analyzed bySDS/PAGE and silver staining and by Western blotting using an anti-his antibody. M, Molecular weight marker; lanes 1 and 5, totalmembrane proteins; lanes 2 and 6, flow-through of the Ni2+-NTAcolumn; lanes 3 and 7, proteins eluted by washing buffer; lanes 4 and 8,proteins eluted with 150 mM imidazole.

transporters were solubilized by detergents and reconstituted

into artificial liposomes. Hirschberg and colleagues were the

first who used this approach to characterize NSTs from rat li-

ver Golgi membranes and to purify the UDP-GalNAc and

GDP-Fuc transporters to almost homogeneity by different

chromatographic techniques [27–29]. Here, we used the recon-

stitution assay for the molecular characterization of AtNST-

KT1, a transporter that belongs to the NST-KT family and

that is encoded by the gene At4g39390. The corresponding

cDNA was extended by a DNA-fragment encoding a C-termi-

nal His6-tag by cloning into the yeast expression vector pYES-

Dest52. Yeast cells were transformed with this construct as

well as with the empty vector as a control. Heterologous

expression of AtNST-KT1 was verified by Western blot analy-

sis using the anti-His antibody detecting a protein of 34 kD in

isolated membranes of pYES-AtNST-KT1 clones exclusively

(Fig. 1A).

Proteins of total yeast membranes were solubilized by deter-

gent and reconstituted into liposomes which had been pre-

loaded with different substrates. We used a new approach for

the measurement of the transport activities that is based on

particular properties of the NSTs. These transporters are

shown to act through an antiport mechanism involving an

one-for-one exchange between the external nucleotide sugar

and the corresponding lumenal nucleotide monophosphate

[5]. Therefore, the substrate specificities of NSTs could be ana-

lyzed by measuring the transport of radiolabelled nucleotide

monophosphates into liposomes instead of radiolabelled nucle-

otide sugars as it has been the case in previous studies. This ap-

proach has the advantage that only one substrate needs to be

used in a radioactively labelled form while all the nucleotide

sugars (which are either not commercially available or rather

expensive as radiolabelled compounds) can be provided unla-

Fig. 2. Determination of substrate specificities of AtNST-KT1. Totalmembrane proteins from yeast cells transformed with pYES-AtNST-KT1 or pYES-Dest52 (vector control) or the purified AtNST-KT1-His6 protein (P-) were reconstituted into liposomes which had beenpreloaded with substrates as indicated. Transport activities weremeasured as described in Section 2. The results shown are means of 3independent measurements except for liposomes containing the puri-fied protein but without preloading.

I. Rollwitz et al. / FEBS Letters 580 (2006) 4246–4251 4249

belled. Thus, the transport of all nucleotide sugars that are

potentially involved in the synthesis of glycoconjugates can

be easily tested.

As shown in Fig. 2, the recombinant expressed AtNST-KT1

protein transports UMP only when the liposomes were pre-

loaded with UDP-Gal but not with other nucleotide sugars like

UDP-Glc or UDP-GlcNAc. Most importantly, no transport

into empty liposomes without any counter-exchange substrate

or into liposomes containing proteins from the vector control

could be detected indicating that AtNST-KT1 transports only

Fig. 3. Intracellular localization of AtNST-KT1 and AtUDP-GalT2. TobaccKT1, pGWB5-AtUGalT2, pGWB5-sialyltransferase and, as control, with pGthe dark and aliquots for analysis of GFP fluorescence were taken 18–24 h apGWB5-AtNST-KT1; row B, cells transformed with pGWB5-AtUGalT2; rtransformed with pGWB5; row E, non-transformed cells. Column 1, Fluorescof cells treated with brefeldin A; column 4, fluorescent images of cells treate

UDP-Gal and UMP in a strict counter-exchange mode but

none of other UDP activated sugars tested. To support this

finding, the solubilized AtNST-KT1 protein was purified from

the membrane fraction by Ni2+-NTA chromatography

(Fig. 1B) and reconstituted into liposomes. The data shown

in Fig. 2 clearly confirm that the purified protein indeed trans-

ports UMP in a strict exchange with UDP-Gal. To our knowl-

edge, the reconstitution of an affinity purified recombinant

protein, a technique that has been successfully used for

the molecular characterization of the plastidic phosphate

o BY2 cells were transiently transformed with vectors pGWB5-AtNST-WB5 containing the GFP gene. Protoplasts were incubated at 22 �C infter transformation of the protoplasts. Row A, Cells transformed withow C, cells transformed with pGWB5-sialyltransferase; row D, cellsent images; column 2, bright field images; column 3, bright field imagesd with brefeldin A.

Fig. 4. Expression of AtNST-KT1 in Arabidopsis. Transgenic plants containing the AtNST-KT1-promoter-GUS construct were analyzed asdescribed in Section 2. (a) 7-day-old seedling, (b) leaf of 7-day-old seedling, (c) 12-day-old seedling, (d) leaf of 12-day-old seedling, (e) mature leaf,(f) hypocotyl and shoot apex of 7-day-old seedling, (g) root tip, (h) flower, (i,j) siliques.

4250 I. Rollwitz et al. / FEBS Letters 580 (2006) 4246–4251

translocators [22], has been applied to the characterization of

only one NST so far, a protein from Leishmania donovani that

transports GDP-Man and other GDP activated sugars (26).

To analyze the substrate affinities in more detail, the apparent

kinetic constants of AtNST-KT1 for the transport of UMP

and the apparent inhibition constants for UDP-Gal and

UDP-Glc were determined. The KM for UMP is

4.2 ± 1.7 lM, while the apparent Ki values for the competitive

inhibition of UMP transport are 5.1 ± 1.7 lM for UDP-Gal

and 12.2 ± 0.4 lM for UDP-Glc which is in the same range

shown for other NSTs [5]. The low Ki for UDP-Glc indicates

that it could bind to the protein without being transported.

3.2. Expression pattern of AtNST-KT1 gene and subcellular

localization of the corresponding protein

Most of the NSTs are located in Golgi membranes while

some are in addition or exclusively located in ER membranes

[5]. To analyze the intracellular localization of AtNST-KT1,

the coding region of the cDNA was translationally fused to

the GFP cDNA and the construct was introduced into tobacco

BY2 cells. In addition, the closely related AtUDP-GalT2 was

also fused to GFP (AtUGalT2-GFP). As marker for the Golgi

apparatus, a sialyltransferase-GFP fusion protein (ST-GFP)

was used while GFP (pGWB5) served as negative control.

The pattern of fluorescence was analyzed by confocal micros-

copy and compared to the pattern of ST-GFP and AtUGalT2-

GFP (Fig. 3). All three fusion proteins produced a dot-like

pattern typical for the Golgi apparatus while GFP localized

to the cytosol and the nucleus. Treatment with brefeldin A

caused a significant change in the fluorescence pattern of the

transformed cells. Because brefeldin A induces a fusion of Gol-

gi and ER membranes and the formation of hybrid ER-Golgi

stacks [30], it can be concluded that both AtNST-KT1 and

AtUDP-GalT2 are located within the Golgi membrane system.

To determine the tissue-specific expression of the AtNST-

KT1 gene, the AtNST-KT1 promoter region (1884 bp) and

102 bp of the coding region were used to generate a transla-

tional fusion with the E. coli uidA (GUS) reporter gene. This

construct was stably transferred to Arabidopsis plants and

three of the resulting lines were analyzed for GUS activity.

All lines showed similar GUS histochemical staining. High

GUS expression was observed in young leaves while in older

leaves GUS activity was restricted to the vasculature (Fig. 4).

Strongest staining was detected in young, growing parts of

roots and stems, especially in the elongation zone of the roots

and in lateral root primordia. In the floral tissue, GUS activity

is restricted to the style and to the filaments, while no activity

was observed in sepals, petals, anthers and the stigma. In sili-

ques, staining was detectable mainly in the abscission zone

while seeds do not show any GUS activity.

3.3. Concluding remarks

AtNST-KT1 represents the fifth NST from Arabidopsis that

transports UDP-Gal. However, it is reasonable to assume that

Arabidopsis possesses more than five UDP-Gal transporters.

These could be other members of the TPT/NST family but

there may be further nucleotide sugar transporters that belong

to families other than TPT/NST. The redundancy of nucleo-

tide sugar transporters in plants offers an explanation for the

absence of any mutant phenotype in Arabidopsis mutant lines

lacking AtNST-KT1, AtUDP-GalT2 or any other transporter

of the NST-KT family (data not shown). Even mutant lines

with more than one disrupted NST-KT gene do not show a

phenotype different from the wild-type. Indeed, in plants lack-

ing any particular NST no mutant phenotypes have been de-

scribed so far. In contrast, in animals and fungi, the loss of

only one NST can lead to a deficiency in the synthesis of gly-

coconjugates, to developmental abnormalities and congenital

disorders [14,31,32]. However, the fact that Arabidopsis and

rice contain much more NSTs than animals points to their par-

ticular importance for plants in general. In this study, a novel

Arabidopsis UDP-Gal transporter localized in the Golgi mem-

brane system has been functionally characterized by use of a

newly employed experimental approach. Using this approach,

the identification of other nucleotide sugar transport activities

in A. thaliana and other organisms appears feasible.

Acknowledgements: This work was supported by the Deutsche Fors-chungsgemeinschaft. We thank Kirsten Bell for technical support.

References

[1] Dupree, P. and Sherrier, D.J. (1998) The plant Golgi. Biochim.Biophys. Acta 1404, 259–270.

[2] Seitz, B., Klos, C., Wurm, M. and Tenhaken, R. (2000) Matrixpolysaccharide precursors in Arabidopsis cell walls are synthe-

I. Rollwitz et al. / FEBS Letters 580 (2006) 4246–4251 4251

sized by alternate pathways with organ-specific expressionpatterns. Plant J. 21, 537–546.

[3] Seifert, G.J. (2004) Nucleotide sugar interconversions and cellwall biosynthesis: how to bring the inside to the outside. Cur.Opin. Plant Biol. 7, 277–284.

[4] Kanter, U., Usadel, B., Guerineau, F., Li, Y., Pauly, M. andTenhaken, R. (2005) The inositol oxygenase gene family ofArabidopsis is involved in the biosynthesis of nucleotide sugarprecursors for cell wall polysaccharides. Planta 221, 243–254.

[5] Hirschberg, C.B., Robbins, P.W. and Abeijon, C. (1998) Trans-porters of nucleotide sugars, ATP, and nucleotide sulfate in theendoplasmatic reticulum and golgi apparatus. Annu. Rev.Biochem. 67, 49–69.

[6] Gerardy-Schahn, R., Oelmann, S. and Bakker, H. (2001) Nucle-otide sugar transporters: biological and functional aspects.Biochimie 83, 775–782.

[7] Abeijon, C., Robbins, P.W. and Hirschberg, C.B. (1996) Molec-ular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis. Proc.Natl. Acad. Sci. USA 93, 5963–5968.

[8] Miura, N., Ishida, N., Hoshino, M., Yamauchi, M., Hara, T.,Ayusawa, D. and Kawakita, M. (1996) Human UDP-galactosetranslocator: molecular cloning of a complementary DNA thatcomplements the genetic defect of a mutant cell line deficient inUDP-galactose translocator. J. Biochem. 120, 236–241.

[9] Baldwin, T.C., Handford, M.G., Yuseff, M.I., Orellana, A. andDupree, P. (2001) Identification and characterization ofGONST1, a Golgi-localized GDP-mannose transporter in Ara-bidopsis. Plant Cell 13, 2283–2295.

[10] Ma, D., Russell, D.G., Beverley, S.M. and Turco, S.J. (1997)Golgi GDP-mannose uptake requires Leishmania LPG2. J. Biol.Chem. 272, 3799–3805.

[11] Knappe, S., Flugge, U.I. and Fischer, K. (2003) Analysis of theplastidic phosphate translocator gene family in Arabidopsis andidentification of new phosphate translocator homologous trans-porters, classified by their putative substrate binding site. PlantPhysiol. 131, 1178–1190.

[12] Jack, D.L., Yang, N.M. and Saier, M.H. (2001) The drug/metabolite transporter superfamily. Eur. J. Biochem. 268, 3620–3639.

[13] Gao, X.D., Nishikawa, A. and Dean, N. (2001) Identification of aconserved motif in the yeast golgi GDP-mannose transporterrequired for binding to nucleotide sugar. J. Biol. Chem. 276,4424–4432.

[14] Lubke, T., Marquardt, T., Etzioni, A., Hartmann, E., von Figura,K. and Korner, C. (2001) Complementation cloning identifiesCDG-IIc, a new type of congenital disorders of glycolysation, as aGDP-fucose transporter deficiency. Nat. Genet. 28, 73–76.

[15] Eckhardt, M., Gotza, B. and Gerardy-Schahn, R. (1999) Mem-brane topology of the mammalian CMP-sialic acid transporter. J.Biol. Chem. 274, 8779–8787.

[16] Capasso, J.M. and Hirschberg, C.B. (1984) Mechanisms ofglycosylation and sulfation in the Golgi apparatus: evidence fornucleotide sugar/nucleoside monophosphate and nucleotide sul-fate/nucleoside monophosphate antiporters in the Golgi appara-tus membrane. Proc. Natl. Acad. Sci. USA 81, 7051–7055.

[17] Bakker, H., Routier, F., Oelmann, S., Jordi, W., Lommen, A.,Gerardy-Schahn, R. and Bosch, D. (2005) Molecular cloning oftwo Arabidopsis UDP-galactose transporters by complementa-tion of a deficient Chinese hamster ovary cell line. Glycobiology15, 193–201.

[18] Handford, M.G., Silicia, F., Brandizzi, F., Chung, J.H. andDupree, P. (2004) Arabidopsis thaliana expresses multiple Golgi

localised nucleotide-sugar transporters related to GONST1. Mol.Gen. Genet. 272, 397–410.

[19] Knappe, S., Lottgert, T., Schneider, A., Voll, L., Flugge, U.I. andFischer, K. (2003) Characterization of two functional phospho-enolpyruvate/phosphate translocator (PPT) genes in Arabidopsis:AtPPT1 may be involved in the provision of signals for correctmesophyll development. Plant J. 36, 411–420.

[20] Negrutiu, I., Shillito, R., Potrykus, I., Biasini, G. and Sala, F.(1987) Hybrid genes in the analysis of transformation conditions.Plant Mol. Biol. 8, 363–373.

[21] Fischer, K., Arbinger, B., Kammerer, B., Busch, C., Brink, S.,Wallmeier, H., Sauer, N., Eckerskorn, C. and Flugge, U.I. (1994)Cloning and in vivo expression of functional triose phosphate/phosphate translocators from C3- and C4-plants: evidence for theputative participation of specific amino acid residues in therecognition of phosphoenolpyruvate. Plant J. 5, 215–226.

[22] Loddenkotter, B., Kammerer, B., Fischer, K. and Flugge, U.I.(1993) Expression of the functional mature chloroplast triosephosphate translocator in yeast internal membranes and purifi-cation of the histidine tagged protein by a single metal affinitychromatography step. Proc. Natl. Acad. Sci. USA 90, 2155–2159.

[23] Norambuena, L., Marchant, L., Berninsone, P., Hirschberg, C.B.,Silva, H. and Orellana, A. (2002) Transport of UDP-galactose inplants: Identification and functional characterization of AtUTr1,an Arabidopsis thaliana UDP-galactose/UDP-glucose transporter.J. Biol. Chem. 277, 32923–32929.

[24] Norambuena, L., Nilo, R., Handford, M., Reyes, F., Marchant,L., Meisel, L. and Orellana, A. (2005) AtUTr2 is an Arabidopsisthaliana nucleotide sugar transporter located in the Golgi appa-ratus capable of transporting UDP-galactose. Planta 222, 521–529.

[25] Oelmann, S., Stanley, P. and Gerardy-Schahn, R. (2001) Pointmutations identified in Lec8 chinese hamster ovary glycosylationmutants that inactivate both the UDP-galactose and CMP-sialicacid transporters. J. Biol. Chem. 276, 26291–26300.

[26] Segawa, H., Soares, R.P., Kawakita, M., Beverley, S.M. andTurco, S.J. (2005) Reconstitution of GDP-mannose transportactivity with purified Leishmania LPG2 protein in liposomes. J.Biol. Chem. 280, 2028–2035.

[27] Milla, M.E. and Hirschberg, C.B. (1989) Reconstitution of Golgivesicle CMP-sialic and adenosine 30-phosphate 5 0 phosphosulfatetransport into proteoliposomes. Proc. Natl. Acad. Sci. USA 86,1786–1790.

[28] Milla, M.E., Clairmont, C.A. and Hirschberg, C.B. (1992)Reconstitution into proteoliposomes and partial purification ofthe Golgi apparatus membrane UDP-galactose, UDP-xylose, andUDP-glucuronic acid transport activities. J. Biol. Chem. 267, 103–107.

[29] Puglielli, L. and Hirschberg, C.B. (1999) Reconstitution, identi-fication and purification of the rat liver Golgi membrane GDP-fucose transporter. J. Biol. Chem. 274, 35596–35600.

[30] Nebenfuhr, A., Ritzenthaler, C. and Robinson, D.G. (2002)Brefeldin A: deciphering an enigmatic inhibitor of secretion. PlantPhysiol. 130, 1102–1108.

[31] Berninsone, P., Hwang, H., Zemtseva, I., Horvitz, H.R. andHirschberg, C.B. (2001) SQV-7, a protein involved in Caenorhab-ditis elegans epithelial invagination and early embryogenesis,transports UDP-glucuronic acid, UDP-N-acetylgalactosamin andUDP-galactose. Proc. Natl. Acad. Sci. USA 98, 3738–3743.

[32] Luhn, K., Wild, M.K., Eckhard, M., Gerardy-Schahn, R. andVestweber, D. (2001) The gene defective in leukocyte adhesiondeficiency II encodes a putative GDP-fucose transporter. Nat.Genet. 28, 69–72.