The Genes for the Golgi Apparatus N-Acetylglucosaminyltransferase and the UDP-N-acetylglucosamine Transporter Are Contiguous in Kluyveromyces lactis*

Eduardo Guillen, Claudia Abeijon, and Carlos B. HirschbergDagger

From the Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118-2394

    ABSTRACT
Top
Abstract
Introduction
References

The mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha (1right-arrow2)-linked N-acetylglucosamine. Previously, Smith et al. (Smith, W. L. Nakajima, T., and Ballou, C. E. (1975) J. Biol. Chem. 250, 3426-3435) characterized two mutants, mnn2-1 and mnn2-2, which lacked terminal N-acetylglucosamine in their mannoproteins. The former mutant lacks the Golgi N-acetylglucosaminyltransferase activity, whereas the latter one was recently found to be deficient in the Golgi UDP-GlcNAc transporter activity. Analysis of extensive crossings between the two mutants led Ballou and co-workers (reference cited above) to conclude that these genes were allelic or tightly linked.

We have now cloned the gene encoding the K. lactis Golgi membrane N-acetylglucosaminyltransferase by complementation of the mnn2-1 mutation and named it GNT1. The mnn2-1 mutant was transformed with a 9.5-kilobase (kb) genomic fragment previously shown to contain the gene encoding the UDP-GlcNAc transporter; transformants were isolated, and phenotypic correction was monitored after cell surface labeling with fluorescein isothiocyanate-conjugated Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescence-activated cell sorter. The above 9.5-kb DNA fragment restored the wild-type lectin binding phenotype of the transferase mutant; further subcloning of this fragment yielded a smaller one containing an opening reading frame of 1,383 bases encoding a protein of 460 amino acids with an estimated molecular mass of 53 kDa, which also restored the wild-type phenotype. Transformants had also regained the ability to transfer N-acetylglucosamine to 3-0-alpha -D-mannopyranosyl-D-mannopyranoside. The gene encoding the above transferase was found to be approximately 1 kb upstream from the previously characterized MNN2 gene encoding the UDP-GlcNAc Golgi transporter. Each gene can be transcribed independently by their own promoter. To our knowledge this is the first demonstration of two Golgi apparatus functionally related genes being contiguous in a genome.

    INTRODUCTION
Top
Abstract
Introduction
References

Kluyveromyces lactis mannoproteins have mannan chains that are similar to those of Saccharomyces cerevisiae except that they have terminal alpha (1right-arrow 2)-linked N-acetylglucosamine and lack mannose phosphate (1). Ballou and co-workers (2) previously isolated and characterized two mutants of K. lactis lacking terminal N-acetylglucosamine in their mannoproteins. One mutant, mnn2-1, lacks the terminal N-acetylglucosaminyltransferase activity, whereas the other mutant, mnn2-2, has wild-type levels of this enzyme (3). Extensive crossings between these two mutants showed parental ditype segregation (Table IV in Ref. 2) leading Ballou and co-workers (2) to hypothesize that the mutations were either allelic or tightly linked. More recently, Abeijon et al. (4) discovered that mutant mnn2-2 lacked the Golgi apparatus membrane UDP-N-acetylglucosamine transporter activity and thereafter cloned the gene that corrected the mutant phenotype (5).

We hypothesized based on the above genetic results and our previous observation that a 9.5-kb1 K. lactis genomic clone contained near one end the open reading frame of the UDP-GlcNAc transporter gene (see Fig. 2 in Ref. 5) that this clone might also contain the open reading frame for the N-acetylglucosaminyltransferase. We therefore transformed the N-acetylglucosaminyltransferase mutant (mnn2-1) with a plasmid containing the above 9.5-kb genomic clone and determined whether the wild-type phenotype had been restored. This was measured by the ability of transformants to bind GSII-FITC, a fluorescent-conjugated lectin that detects terminal alpha - or beta -linked N-acetylglucosamine; this approach had been previously used to detect wild-type phenotypic restoration of this sugar in the Golgi transporter-deficient mutant mnn2-2 (5).

We found that the above 9.5-kb genomic clone did, indeed, restore the wild-type phenotype of the N-acetylglucosaminyltransferase mutant and that this phenotype was the result of the mutants having regained their ability to transfer N-acetylglucosamine to mannose acceptors. After subcloning of the above 9.5-kb genomic fragment, a smaller one containing an ORF of 1,383 kb encoding a protein of 460 amino acids with an estimated molecular mass of 53 kDa also restored the wild-type phenotype. Together, these studies demonstrate that this latter protein is the K. lactis alpha (1right-arrow2) N-acetylglucosaminyltransferase and that the genes for this enzyme as well as the UDP-GlcNAc transporter, each with their own promoter, are contiguous in the K. lactis genome.

    MATERIALS AND METHODS

[alpha -32P]dCTP (3000 Ci/mmol) and UDP[3H]GlcNAc (20 Ci/mmol) were purchased from NEN Life Science Products. Griffonia simplicifolia II lectin labeled with fluorescein isothiocyanate (GSII-FITC) was purchased from EY Laboratories. Restriction enzymes were purchased from New England Biolabs. 3-O-alpha -D-mannopyranosyl-D-mannopyranose was purchased from Sigma.

DNA ligations were performed with T4 DNA ligase (Life Technologies, Inc.). Escherichia coli plasmid DNA was isolated with either Wizard Plus Miniprep kit (Promega) or Nucleobond AX (the Nest Group, Southport, MA). All transformations were performed by electroporation as described before (5). Polymerase chain reactions were made using Pfu DNA polymerase (Stratagene) in a PTC-100 thermal controller (MJ Research, Watertown, MA).

Strains, Media, and Growth Conditions-- K. lactis strain MW103-1C (Mat alpha , uraA, lysA, K+, pKD+) (6) was a gift from Dr. H. Fukuhara (Institute Curie, France). Y-43alpha (Mat a, his3) and Y-43alpha (3-55) (Mat a, mnn2-1, his3) (2) were obtained from Dr. C. Ballou (UCLA). KL11 (Mat a, mnn2-1, uraA, lysA, K+, pKD+) was obtained by crossing MW103-1C with Y-43alpha (3-55) as described in Ref. 7 followed by sporulation and tetrad dissection.

K. lactis cells were grown at 26 °C either in yeast extract/peptone/dextrose media or SD-U synthetic uracyl-free media (0.67% yeast nitrogen base without amino acids, 2% glucose, and 2% SCM-URA (synthetic mix of amino acids and inositol) (8) (Bufferad, Inc.)

Plasmids-- Two additional plasmids were derived from the initial genomic clone in pCA66 to narrow the position of the N-acetylglucosaminyltransferase. After digestion of pCA66 with NheI and SalI, the resulting 4.3-kb Nhe-Nhe fragment was subcloned into the Nhe site of the K. lactis expression vector Kep 6 (9) generating pCE1. The 2.3-kb Nhe-Sal fragment was subcloned into Kep 6 linearized with Nhe and Sal, generating pCE2. Plasmid pCA73 was described previously (5).

Labeling of K. lactis with GSII-FITC and Fluorescence-activated Cell Sorting-- K. lactis cells, grown either in yeast extract/peptone/dextrose media or uracyl-free media, were labeled with GSII-FITC and run through a FACScan (Becton Dickinson) as described previously (10).

Subcellular Fractionation and Assay for alpha (1 right-arrow 2) N-acetylglucosaminyltransferase-- Golgi-enriched membranes from K. lactis were obtained as described (4). Enzyme assays were performed as described previously (2) using 50 µg of membrane protein and 2 µM UDP-[3H]GlcNAc (240 cpm/pmol) and 0.5 mM 3-0-alpha -D-mannopyranosyl-D-mannopyranoside in 20 mM imidazole-HCl, 20 mM MgCl2, 0.2% Triton X-100, pH 6.5. Incubations were at 30 °C for 20 min.

Sequencing and Data Analysis-- The 4.3-kb NheI fragment derived from pCA66 and containing the complete ORF for the N-acetylglucosaminyltransferase was subcloned into pBluescript II SK (+) (Stratagene) linearized with XbaI. Sequencing was performed in an Applied Biosystems DNA sequencer by using the dideoxy chain termination method (Molecular Genetics Facility, University of Georgia, Athens, GA). Sequences were assembled and analyzed using DNAstar programs (DNAstar, Madison, WI), and data base searches were made through the NCBI Blast server (National Center for Biotechnology Information, Bethesda, MD)

Northern Blot Analysis-- Total RNA was obtained from Y-43 alpha  (3-55) and Y-43 alpha  by the acidic phenol method (11), and 15 µg/lane were separated on a 1% formaldehyde-agarose gel and capillary-transferred to a nylon Hybond-N+ membrane (Amersham Pharmacia Biotech) (12). Prehybridization was performed at high stringency conditions by including the membrane in 50% formamide, 10× Denhardt's, 6× saline/sodium phosphate/EDTA, 0.5% SDS, and 0.2 mg/ml salmon sperm single-stranded DNA at 42 °C for 4 h. Hybridization was done overnight at 42 °C with double-stranded DNA probes labeled with an oligolabeling kit (Amersham Pharmacia Biotech) and [alpha -32P]dCTP as indicated by the manufactures. Membranes were washed at 42 °C in 2× SSC (1× SSC= 0.15 M NaCl and 0.015 M sodium citrate), 0.1% SDS two times, and then two times in 1× SSC, 0.1% SDS. The final wash was done twice in 0.5× SSC, 0.1% SDS.

Probes were obtained by polymerase chain reaction using pCA66 as template and the primers 7N0740 (5'-CAGAGTTCATCCCAGATTTGAC-3') and 7H2017 (5'-TATGGTACTACCTTTTACGCACGG-3') for the-N-acetylglucosaminyltransferase and 3P1234 (5'-AGCTCATTTCTGGTACTATTTCG-3') and 7H0286 (5'-ATTTACGCACTAGAAGCACGACAG-3') for the UDP N-acetylglucosamine transporter. In both cases the product included most of the ORF.

Polymerase chain reaction reaction products were separated on a 1.2% agarose gel, and the band of interest was cut and eluted by freeze-thaw and centrifugation in Spin-X centrifuge tube filters (Costar Inc., Corning, NY). DNA was purified by phenol/chloroform extraction and ethanol-precipitated before being used in the labeling reaction.

    RESULTS

The 9.5-kb Genomic Clone Correcting the K. lactis Golgi UDP-GlcNAc Transporter Defective Mutant (mnn2-2) Also Corrects the N-Acetylglucosaminyltransferase-defective Mutant (mnn2-1)-- Based on the previous observation by Ballou and co-workers (2) that crossings between what is now known to be the UDP-GlcNAc transporter mutant and the N-acetylglucosaminyltransferase mutant indicated that both genes were in close proximity, and the existence of sufficient DNA at either side of the transporter gene in the genomic clone to contain the transferase ORF led us to hypothesize that the 9.5-kb genomic clone might also contain the transferase gene. Our strategy to isolate the genomic DNA encoding the N-acetylglucosaminyltransferase relied on the isolation of those K. lactis mnn2-1 transformants that had acquired wild-type binding to GSII-FITC lectin after transformation with the genomic insert-containing plasmid encoding also the K. lactis Golgi UDP-GlcNAc transporter (5).

K. lactis KL11, an N-acetylglucosaminyltransferase-deficient strain, was transformed with the above pCA66 plasmid containing the 9.5-kb genomic DNA fragment. Cells were plated in uracyl-free media, individual colonies were isolated and replated, and cells were labeled with GSII-FITC lectin. Fig. 1 shows that the transformed mutant K. lactis cells, growing in uracyl-free media, had acquired fluorescence in the wild-type range. This suggests a restoration of N-acetylglucosamine to cell wall mannans and that the corresponding transferase activity was now functional (see below).


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Fig. 1.   The plasmid pCA66 containing the ORF for the UDP N-acetylglucosamine transporter also includes the ORF for the N-acetylglucosamine transferase. K. lactis strains were surface-labeled with GSII-FITC lectin and separated by fluorescence-activated cell sorter. Cells of wild-type phenotype (MW103-1C) show higher fluorescence than the mutant mnn2-1 lacking N-acetylglucosamine transferase activity (KL-11). When the latter is transformed with the plasmid pCA66, also containing the ORF for the UDP N-acetylglucosamine transporter, the fluorescence is restored to values similar to those of the wild type.

The N-Acetylglucosaminyltransferase and the UDP-GlcNAc Transporter Genes Are in Different Regions of the pCA66 Plasmid-- To further determine the region of the genomic 9.5-kb fragment encoding the N-acetylglucosaminyltransferase, the DNA was digested with restriction enzymes so that an Nhe-Nhe fragment of 4.3-kb (pCE1) and one Nhe-SalI of 2.3 Kb (pCE2) were obtained (Fig. 2A). The 4.3-kb insert-containing plasmid was still able to restore fully the GSII-FITC binding phenotype as wild-type cells, whereas the one containing the 2.3 kb was not, as did pCA73 containing the entire ORF for the transporter (Fig. 2B).


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Fig. 2.   Panel A, positions of pCE1, pCE2, and pCA73 on the K. lactis genomic clone pCA66. Panel B, K. lactis mnn2-1 mutant for N-acetylglucosamine transferase activity (KL11), was transformed with pCA66-derived plasmids pCE1, pCE2, and pCA73. Cells were labeled with GSII-FITC lectin and separated by fluorescence-activated cell sorter. Only the plasmid pCE1 comprising a 4.3-kb fragment of pCA66, including the complete ORF for K1-GNT1, restores the wild-type phenotype in the mutant.

To determine directly whether this wild-type phenotype recovery was indeed the result of restoration of N-acetylglucosaminyltransferase activity, Golgi-enriched membranes were prepared from KL11 transformed with pCE1, and enzymatic activity was measured in vitro using 3-0-alpha -D-mannopyranosyl-D-mannopyranoside as exogenous acceptor. As can be seen in Fig. 3, pCE1 was able to restore the transferase activity to levels comparable or even slightly higher than wild-type cells.


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Fig. 3.   In vitro N-acetylglucosaminyl transferase activity. Golgi-enriched membranes were obtained as described under "Materials and Methods" from wild-type (wt) K. lactis MW103-1C, K. lactis N-acetylglucosamine transferase mutant KL11, or the former transformed with the plasmid pCE66, which restores the in vivo phenotype. When indicated, 3-0-alpha -D-mannopyranosyl-D-mannopyranose was used as acceptor. Valves are the mean of three to six determinations of two independent membrane preparations.

Sequencing the genomic DNA of pCE1 revealed an ORF of 1383 bp encoding a protein of 460 amino acids (Fig. 4). The predicted molecular mass of the polypeptide is 53 kDa with a pI of 4.98, in excellent agreement with the pI of 4.9 previously determined during the enzyme purification (3). The reported molecular mass of the partially purified native protein, obtained by size-exclusion chromatography of detergent-solubilized enzyme in detergent-free buffer, is 300 kDa (3), raising the possibly that the enzyme is multimeric or tightly associated with other proteins. The ORF is located upstream from the gene encoding the UDP-N-acetylglucosamine transporter and separated by an intergenic region of 948 bp. Both genes are transcribed in the same direction. Analysis of the primary sequence reveals a putative type II protein; toward the amino terminus there is a likely noncleavable signal peptide determining a transmembrane-anchoring segment separating 10 amino acids, probably a cytoplasmic tail, from a 431-luminal domain presumptively containing the substrate recognition and catalytic site. There are four consensus N-glycosylation sites through the protein (Fig. 5, panel A); most likely some of them are utilized, as Douglas and Ballou (3) reported binding of enzymatic activity to concanavalin A (3).


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Fig. 4.   Genomic K. lactis sequence containing the UDP N-acetylglucosamine transferase gene. The sequence shown is located directly upstream of GenBankTM U48413 containing the coding region for the UDP N-acetylglucosamine transporter (5). The one-letter amino acid code is used on the translation of the coding region. The putative transmembrane domain is underlined. The combined sequences can be accessed from GenBankTM under accession number AF106080.


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Fig. 5.   A, hydrophilicity plot of the K. lactis N-acetylglucosaminyl transferase as determined with the Kyte-Doolittle algorithm (24) and a window size of 17 amino acids. Dots are asparagines that are part of N-glycosylation consensus sequences. The horizontal bar underlines the sequence shown in panel B. B, alignment of a domain highly conserved between Kl-GNT1p and glycogenins using the Jotum-Hein algorithm (25). The consensus sequence hhhhDXDXh (18) is boxed.

UDP-N-Acetylglucosaminyltransferase and UDP N-Acetylglucosamine Transporter Are Translated into Different Messengers-- Northern blots of total RNA from wild-type strain Y-43alpha show two transcripts of 2.4 and 1.5 kb using as probe the UDP-GlcNAc transferase gene (Fig. 6). The same two transcripts are in the mnn2-1 transferase mutant but with lesser intensity (Fig. 6). Preliminary results from our laboratory2 indicate that there is a nonsense mutation in the ORF of the transferase gene. This type of mutation is known to induce message instability, and the results of Fig. 5 appear to suggest that this indeed has occurred.


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Fig. 6.   Northern blot of total RNA for K. lactis wild type (wt; Y-43alpha ) or mutant (mut.) for the a N-acetylglucosaminyl transferase Y-43alpha (3-55) using as probe the transferase and UDP-GluNAc transporter genes.

Although the 2.4-kb transcript has the size to contain the ORFs of the transporter and the transferase, when the same membrane is probed for the transporter, only a band of 1.5 kb could be seen (Fig. 6). Furthermore, the intensity of the transcript for the transporter was similar both in the wild type and in the mutant for the transferase; therefore, it can be concluded that the UDP-N-acetylglucosamine transporter and the N-acetylglucosamine transferase are transcribed into independent messenger RNAs.

    DISCUSSION

Besides the N-acetylglucosaminyltransferase (Kl-GNT1) reported in this paper, the only other known eukaryotic UDP-N-acetylglucosamine:oligosaccharide alpha -N-acetylglucosaminyltransferases are alpha -GlcNAc TI and TII, which participate in the synthesis of heparin/heparan sulfate, transferring alpha -N-acetylglucosamine to glucuronic acid in the core or in the elongating polymer, respectively (13). Recently Lind et al. (14) reported that the alpha -N-acetylglucosaminyltransferase and glucuronyl transferase activities necessary to elongate glycans in heparan sulfate-type proteoglycans were contained in a single polypeptide of 70 kDa. This polypeptide shows no significant overall homology with Kl-GNT1p (15% identity and 23% similarity). Sequence analysis of the glycosyltransferases cloned so far detects little if any sequence homology between members of catalytically distinct families of enzymes (15, 16). This is true even in most of the cases where they have in common either the sugar donor or the acceptor. We found extensive homology only with YOR320p of S. cerevisiae. (36% identical, 55% similar at amino acid level throughout the entire ORF). This is a 491-amino acid protein with a putative noncleavable endoplasmic reticulum translocation signal determining a 10 amino acid cytoplasmic tail and a large lumenal domain whose function is still undetermined. It doesn't seem likely that YOR320p has a similar enzymatic activity than its K. lactis homolog, and no oligosaccharide or glycolipid modified with N-acetylglucosamine has been described so far in S. cerevisiae, although the possibility that such modification is transiently present at some point during the cell cycle or under certain growth conditions cannot be ruled out.

An interesting possibility is that both YOR320p and YEL004p (the S. cerevisiae sequence homolog of the K. lactis UDP N-acetylglucosamine transporter) operate on the same, yet unknown, pathway in a similar manner as their K. lactis homologs do. In this case, probably solving the problem of the biochemical role of one of the above proteins will answer the function of the other as well.

Surprisingly, a domain with 23% identity and 48% similarity spanning amino acids 97 through 210 in the putative Golgi luminal portion of K1-GNT1p was found to be conserved with glycogenins of diverse origin (Fig. 5B). This domain is also highly conserved among a group of glycogenin-like proteins from nematodes, plants, fungi, and bacteria (17). Interestingly, this domain also contains the sequence IVYFDSDSI, comprising amino acids 193 to 201 (Fig. 5B). This is a highly conserved motif found among a very broad group of glycosyltransferases in the form of a consensus sequence hhhhDXDXh (where h is a hydrophobic amino acid) (18). Aspartates in the motif DXD were found to be essential for the activity of the S. cerevisiae Mnn1p alpha -1,3-mannosyltransferase, with folding or oligomerization not being affected (18). Furthermore, mutations of the aspartic acids of the same motif in the lethal toxin of Clostridium sordelli abolishes its glucosyltransferase activity and toxicity (19). Affinity-labeling with azido UDP-glucose is also suppressed. It has been hypothesized (18) that this domain may play a role in the coordination of the interactions between the divalent cation required for these enzymes to be active and the pyrophosphate in the sugar nucleotides they use as donors of the glycosidic moiety. Thus, it could be the case that this homologous domain in K1-GNT1p may play a similar role, interacting with the UDP-GlcNAc, although the significance of the more extended homology with glycogenins is unclear.

It is very interesting to find two genes of related functions being contiguous in the genome of a eukaryote such K. lactis. Although such occurrence is common in prokaryotes, with their widespread occurrence of operons, its incidence in eukaryotes is much rarer. The norm in eukaryotes is that genes of related functions are transcribed individually and are scattered throughout the genome. However, there are several examples of clustered genes of related functions. Some even are cotranscribed from a single promoter into a polycistronic messenger as in bacterial operons. Examples are found in trypanosomatides, nematodes, mammals, and plants (reviewed in Ref. 20.)

Of particular appeal is the case of the MAL loci in S. cerevisiae. Each locus comprises at least three genes: a trans-acting activator, a maltose permease, and maltase (21). They determine a unit comprising a regulatory gene adjacent to the genes of related functions it controls, translated as independent transcripts (22). It is very tempting to speculate that this may very well be the case for the genes for the UDP-GlcNAc transporter and transferase in K. lactis, and studies are currently under way in our laboratory to address this question.

It has been proposed (23) that S. cerevisiae ancestors arose from the fusion of two diploid cells to form a tetraploid, thus leading to genome duplication, followed by extensive gene deletions and reciprocal translocation between chromosomes leading to gene rearrangement. This event seemingly happened after the common ancestor of K. lactis and S. cerevisiae had diverged. This hypothesis may explain why the homologue genes of the transferase and the transporter of UDP-N-acetylglucosamine, although contiguous in the genome of K. lactis, are in a single copy on different chromosomes in S. cerevisiae.

It remains to be seen if the contiguity in the genome of the UDP-N-acetylglucosamine transporter gene and K1-GNT1 has any physiological significance. Nothing is known about the possible regulation of the expression of either protein depending upon particular physiological conditions of the organism. If such a variation exists, it will be interesting to see if the levels of mRNA and/or activity for these two proteins are related.

    ACKNOWLEDGEMENT

We thank Glenn Paradis, Michael Jennings, and Michael Connelly (MIT flow/cytometry Core Facility) for help with cell sorting and Dr. Hiroshi Fukuhara, Institute Curie, Orsai, France, for K. lactis strains and plasmids.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM 30365 (NIGMS).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, 700 Albany St., W200, Boston, MA 02118-2594. Tel.: 617-414-1040; Fax: 617-414-1041; E-mail: chirschb{at}bu.edu.

2 L. Puglielli, personal communication.

    ABBREVIATIONS

The abbreviations used are: kb, kilobase(s); ORF, open reading frame; FITC, fluorescein isothiocyanate; GSII-FITC, G. simplicifolia II lectin conjugated to FITC.

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