Laboratoire de Microbiologie et Génétique Moléculaire, CNRS-Institut National Agronomique Paris-Grignon-INRA, 78850 Thiverval-Grignon, France
Correspondence
Stéphanie Barnay-Verdier
barnay{at}grignon.inra.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the YlANL1 and YlOCH1 sequences reported in this paper are DS55218/138556 and DS55232/138580, respectively.
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INTRODUCTION |
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Indeed, in the Golgi, the extension of the oligosaccharide chain involves many specific glycosyltransferases. This specificity generates the observed diversity of glycan structures between different species and cell types (Drickamer & Taylor, 1998). In the yeast Saccharomyces cerevisiae, the glycosyltransferases are mannosyltransferases which lead to the formation of two main types of mannan outer chain. Many proteins of the cell wall and periplasm receive a large mannan structure that contains a long
-1,6-linked backbone of about 50 mannoses with short
-1,2 and
-1,3 side chains. In contrast, the proteins of the internal organelles display a smaller core-type structure, with only a few mannoses (Munro, 2001
).
The structure of the mannan outer chain has been investigated by the study of mnn (mannan defective) mutants isolated by Ballou and co-workers (Ballou, 1982, 1990
). The analysis of the partial N-glycan structures in these mutants has allowed the ordering of the steps of mannan synthesis. Studies of other glycosylation mutants (van, och, vrg) have shown that mutants severely affected in mannan outer-chain extension have additional phenotypes: enhanced hygromycin B sensitivity and sodium orthovanadate resistance (Ballou et al., 1991
; Kanik-Ennulat et al., 1995
). The degree of sensitivity or resistance is correlated with the severity of the glycosylation defect in these mutants. Underglycosylated cells are not affected in protein secretion.
The recent work of Munro suggests a model for the complete N-glycosylation pathway in S. cerevisiae (Munro, 2001; Stolz & Munro, 2002
). In this model, the formation of the mannan outer chain is initiated by the Och1p protein, a type II membrane
-1,6-mannosyltransferase that defines an early Golgi compartment (Nakanishi-Shindo et al., 1993
; Romero et al., 1994
; Nakayama et al., 1997
). Upon arrival in the Golgi, Och1p adds a single
-1,6-mannose to all N-glycan cores. The formation of the long
-1,6-linked backbone is generated by two enzyme complexes. The M-Pol I complex contains two
-1,6-mannosyltransferases, Mnn9p and Van1p. It is responsible for the first committed step in the generation of the mannan structure by adding about ten mannoses to the nascent
-1,6-linked chain. The M-Pol II complex consists of five proteins, of which two
-1,6-mannosyltransferases, Mnn9p and Anp1p, are involved in the last step of the extension of the long
-1,6-linked backbone by adding a large number of mannoses (about 50). The side chains are then completed by the action of two
-1,2-mannosyltransferases, Mnn2p and Mnn5p, and a single
-1,3-mannosyltransferase, Mnn1p. In contrast, formation of the small core-type N-glycan involves only Och1p, one unknown
-1,2-mannosyltransferase and Mnn1p. MNN9 (Yip et al., 1994
), ANP1 (Chapman & Munro, 1994
) and VAN1 (Kanik-Ennulat et al., 1995
) are three members of a gene family. They encode
-1,6-mannosyltransferases co-localized within the cis Golgi compartment (Jungmann & Munro, 1998
). Null alleles of the three genes are viable, and the strains mutated in mnn9 show the most severe underglycosylation and osmotic fragility. The anp1 mutants are the least severely affected.
We wished to investigate this specific part of the secretory pathway in the yeast Yarrowia lipolytica. The non-conventional yeast Y. lipolytica is a good alternative model organism for fundamental and applied studies (Barth & Gaillardin, 1996, 1997
). It has a secretion process closer to that of mammals than does S. cerevisiae (Beckerich et al., 1998
; Boisramé et al., 1998
) and it is a better host for the expression of heterologous secreted proteins in an active form (Madzak et al., 2000
). At the beginning of this project, only one Y. lipolytica N-glycosylation pathway gene sequence, a homologue of the S. cerevisiae MNN9 gene, was available in the public databases. Additionally, the N-glycan structure of Y. lipolytica was not as yet determined. To perform a functional analysis of the Golgi glycosylation pathway in Y. lipolytica, we characterized gene sequences encoding homologues of proteins known to be involved in this pathway in S. cerevisiae, such as mannosyltransferases. The objective of the work presented in this paper was to confirm the function of the encoded proteins in Y. lipolytica by the construction and analysis of the glycosylation mutants. Our aim is (i) to establish a model for the glycosylation pathway, and (ii) to be able to modify it in order to permit production and secretion of mammalian-type proteins in Y. lipolytica.
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METHODS |
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All disrupted strains of Y. lipolytica presented in this paper were constructed in the strain INAG136463 (MatB, scr1 : : ADE1, SCR2, his-1, leu-2, ura3). Yeast strains were grown on rich YPD medium, with 0·5 M sorbitol when necessary, or on minimal YNB medium supplemented with the appropriate amino acids plus proline as nitrogen source, at 28 °C. When required, 1·25 mg ml1 5'-fluoroorotic acid was added to solid media for selection of ura strains.
The pGAA plasmid containing the Arxula adeninivorans glucoamylase (GAA) coding sequence under the control of the hp4d hybrid promoter (Swennen et al., 2002) was used to transform wild-type and mutant haploid strains. To express GAA, the Y. lipolytica cells were cultivated on rich YPD in 50 mM phosphate buffer, pH 6·8, at 28 °C.
Transformation of strains, DNA isolation and sequencing.
Basic DNA manipulation and transformation in E. coli were performed according to standard methods. Yeast transformation was carried out by the lithium acetate method (Xuan et al., 1988). Plasmid DNA from E. coli was prepared using the Qiaprep Kit (Qiagen) and DNA fragments were purified from agarose gels using the Gel Extraction Kit (also from Qiagen). Sequencing was performed by Eurogentec (Belgium). The oligonucleotides used for sequencing are shown in Table 1
.
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Two-hybrid experiment.
The S. cerevisiae strain PJ694 MAT
, trp1-901, leu2-3,112, ura3-52, his3-200, gal4
, gal80
, LYS2 : : GAL1-HIS3, GAL2-ADE2, met2 : : GAL7-lacZ (James et al., 1996
) was used for the two-hybrid experiment.
The YlANL1 and YlMNN9 ORFs were amplified with two primer pairs: ANLhyb5'-Nco and ANLhyb3'-Bgl or MNNhyb5'-Nco and MNNhyb3'-Bam (Table 1) in order to clone them in-frame with either the DNA-binding domain of Gal4p in pAS2
or the activating domain in pACT2. The first primer of each couple contains a NcoI restriction site, and a BglII or a BamHI restriction site is present in the second one. After restriction of the two amplified fragments, the digested products, of 1250 and 920 bp, respectively, were ligated with the pAS2
or pACT2 vector cut with NcoI and BamHI.
Immunofluorescence.
Cells grown in rich YPD medium to an OD600 of 12 were fixed by addition of 5 % formaldehyde to the culture. After centrifugation, cells were incubated for 2 h at room temperature without shaking in 50 mM potassium phosphate buffer, pH 6·5, 0·5 mM MgCl2, 5 % formaldehyde. Cells were then resuspended in 10 ml 0·1 M potassium phosphate buffer, pH 7·5, containing 25 mM -mercaptoethanol and 1·2 M sorbitol, and permeabilized using 10 mg Zymolyase 20T (Seikagaku) and 20 mg Cytohelicase (Sigma) for 40 min at 37 °C. Cells were washed in PBS, and 10 µl of the cell suspension in PBS was transferred to wells of immunofluorescence slides pretreated with polylysine. Cells were treated with 10 µl PBS, 0·5 % BSA and 0·05 % Nonidet P-40 for 15 min and washed before the addition of 10 µl 1 : 300-diluted anti-Hap (Santa Cruz Biotechnology) or anti-cmycp antibodies (Upstate Biotechnology). After 1 h of incubation and washes in PBS, bound primary antibodies were reacted with 1 : 300-diluted goat anti-rabbit Texas red-conjugated IgG (Jackson ImmunoResearch). Slides were treated with 300 µg ml1 4,6-diamidino-2-phenylindole (DAPI) and mounted in one drop of mounting medium (1 : 10 PBS, 9 : 10 glycerol, 1 mg p-phenylenediamine ml1).
Calcofluor White staining.
Cells grown in rich YPD medium to 5x106 cells ml1 were fixed by addition of 4 % formaldehyde to the culture at room temperature for 10 min. After centrifugation, cells were incubated for 1 h at room temperature without shaking in PBS containing 4 % formaldehyde. Cells were then pelleted, washed and resuspended in 500 µl PBS. A 100 µl sample of cells was treated with 10 µl of 1 mg Calcofluor White ml1 in the dark for 1 h, and mounted in one drop of mounting solution (1 part PBS, 9 parts glycerol, 1 mg p-phenylenediamine ml1, 50 ng DAPI ml1, pH 8, adjusted with 0·5 M sodium carbonate).
Western blot analysis.
Aliquots of supernatant from yeast cultures were digested by 1 µl endo H (Biolabs) overnight at 37 °C. Digested and undigested aliquots of supernatant were mixed with sample buffer (100 mM Tris/HCl, pH 6·8, 2 % -mercaptoethanol, 20 % glycerol, 4 % SDS, 0·02 % Bromophenol Blue), heated for 10 minutes at 65 °C and loaded on a 10 % polyacrylamide denaturing gel. After migration, separated proteins were transferred onto a nitrocellulose membrane. Rabbit anti-Gaap antibodies, kindly provided by Dr D. Swennen (INRA UMR 1238, France), were used as primary antibodies, peroxidase-conjugated anti-IgG antibodies as secondary ones, and detection was done by the ECL method (Amersham).
Nucleotide sequence accession numbers.
We used the YlMNN9 sequence, isolated and characterized by Zueco and co-workers (Jaafar et al., 2003), available from EMBL/GenBank/DDBJ under accession no. AF44127. The accession numbers for the YlANL1 and YlOCH1 sequences reported in this work are given in the footnote.
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RESULTS |
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Effect of YlMNN9, YlANL1 and YlOCH1 deletion on sensitivity to hygromycin B and SDS
Previous work with yeasts has shown that mutants affected specifically in Golgi N-glycosylation processing display characteristic phenotypes, such as enhanced hygromycin B sensitivity, SDS sensitivity and sodium orthovanadate resistance (Ballou et al., 1991; Kanik-Ennulat et al., 1995
; Dean, 1995
). The degree of sensitivity or resistance is correlated with the severity of the glycosylation defect in these mutants. In order to show that the Y. lipolytica proteins Mnn9p, Anl1p and Och1p operate in the glycosylation pathway, we performed phenotypic analysis. To determine the sensitivity of the parental 136463 and mutant strains, 5 µl aliquots of serial dilutions of overnight cultures of the strains were plated on YPD plates containing various amount of SDS. All the mutant strains exhibited a higher sensitivity to SDS than the parental 136463 strain (Fig. 6a
).
Ylmnn9 was the most affected mutant: its growth was severely inhibited from 0·015 % SDS upwards. The
Ylanl1 strain was the least severely affected mutant, with SDS sensitivity at a concentration of 0·05 %. It is interesting to note that the double-mutant strains,
Ylmnn9
Ylanl1 and
Ylmnn9
Yloch1, did not display a higher sensitivity to SDS than the
Ylmnn9 mutant. These results suggest that the three Y. lipolytica
-1,6-mannosyltransferases YlMnn9p, YlAnl1p and YlOch1p are actually involved in the N-glycosylation pathway. Moreover, the deletion of the YlMNN9 gene induces similar glycosylation defects in the S. cerevisiae (Ballou, 1990
; Yip et al., 1994
) and C. albicans
mnn9 strains (Southard et al., 1999
). The fact that the double-mutant strains are no more severely affected than the single-mutant
ylmnn9 strain proves that Mnn9p has a major function in N-glycosylation in Y. lipolytica, as postulated by Munro for S. cerevisiae (Munro, 2001
; Stolz & Munro, 2002
).
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To confirm that the cell-wall structure is affected in these mutant strains, we performed Calcofluor-White staining, which allows a qualitative estimation of the accumulation of chitin in the cell wall. In a wild-type context, this accumulation occurs only in damaged or fragile regions of the cell wall, such as in the bud scar, but in an underglycosylation context a high and continuous chitin accumulation is visible all around the cell wall. This allows the cell to maintain cell-wall integrity. As the cell wall is mainly composed of mannan proteins which are hypermannosylated, underglycosylation induces a general cell-wall fragility that can be highlighted by this specific chitin accumulation. Wild-type and defective cells of Ylmnn9,
Ylanl1 and
Yloch1 strains were treated with 10 µl 1 mg Calcofluor White ml1 and mounted in a drop of mounting solution. As expected, in the parental 136463 strain, chitin accumulation was observed only in the bud scar; conversely, the mutant strains displayed chitin accumulation specific to the underglycosylation context, as shown in Fig. 7
. Taken together, these results indicate that the cell-wall structure of the mutant strains is characteristic of an underglycosylated cell wall.
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DISCUSSION |
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Following a sequence-dependent approach, we found one ORF (YlANL1) encoding a 451 amino acid protein that has a high degree of similarity with Sch. pombe and S. cerevisiae Anp1p proteins. Similarly to these Anp1ps, a predicted transmembrane domain is present (amino acids 42 to 63). In order to characterize the Y. lipolytica YlAnl1p, we performed a two-hybrid system analysis of the YlAnl1p and YlMnn9p interaction. Using this system, the Y. lipolytica Anl1p and Mnn9p were shown to interact directly, as has been demonstrated (by co-immunoprecipitation) for S. cerevisiae (Jungmann & Munro, 1998). A recent analysis of the phylogenetic tree of the identified members of the ANP family in Y. lipolytica, S. cerevisiae, C. albicans and Sch. pombe revealed an interesting point. In contrast to S. cerevisiae, Y. lipolytica possesses two Anp1p-like proteins, Anl1p and Anl2p, but no Van1p-like protein (Fig. 9
). These data demonstrate the particularity of the Y. lipolytica ANP family and raise the possibility of a specific role for its protein members. In order to confirm this hypothesis, a two-hybrid screening study, using YlMnn9p and YlAnl1p as baits, will elucidate the multi-protein complexes involved in the Golgi N-glycosylation pathway in this yeast.
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The Ylmnn9,
Ylanl1,
Yloch1,
Ylanl1
Ylmnn9 and
Ylmnn9
Yloch1 mutant strains displayed sensitivity to SDS. This is a characteristic phenotype expected for mutants defective in some aspect of protein glycosylation. Moreover,
Ylanl1 or
Yloch1 deletions induce some alteration in the cell-wall structure, as shown by Calcofluor-White sensitivity and chitin accumulation for the
Ylmnn9 deletion. These results provide indirect evidence that YlAnl1p and YlOch1p are involved in the specific Golgi N-glycosylation process.
To explore more directly the role of these -1,6-mannosyltransferases in this process, we examined the secreted N-glycosylated protein Gaap from A. adeninivorans in wild-type and mutant strains. This experiment demonstrated that YlMnn9p and YlAnl1p have a direct effect on N-glycosylation of the Gaap secreted protein. Moreover, the most severe glycosylation defect was observed in the
Ylmnn9 mutant. Taken together, all presented results suggest a major role for YlMnn9p and a probable secondary role for YlAnl1p in the Golgi N-glycosylation pathway, as postulated in S. cerevisiae.
However, no effect on Gaap glycosylation has been shown in the Yloch1 mutant. Thus, our current data suggest a minor role for YlOch1p in Golgi N-glycosylation. We cannot exclude, however, the possibility that YlOCH1 has a severe effect on the glycosylation of a specific protein that we have not assayed. In S. cerevisiae, two tightly related
-1,6-mannosyltransferase homologues, Och1p and Hoc1p, have been identified and characterized. Och1p has been described as a critical protein in the extension of N-linked oligosaccharide chains, and catalyses the addition of the first
-1,6-linked mannose to the core oligosaccharide (Nakanishi-Shindo et al., 1993
; Romero et al., 1994
; Nakayama et al., 1997
). In contrast, Hoc1p has been defined as a regulator protein belonging to the M-Pol II complex, and is involved in the last step of the long
-1,6-linked backbone extension (Neiman et al., 1997
; Munro, 2001
; Stolz & Munro, 2002
). However, in the complete Y. lipolytica genome sequence we found only one sequence homologue to these two genes. This specific characteristic of Y. lipolytica allows us to propose that YlOCH1 might have a different role in the N-glycosylation pathway from those described for the S. cerevisiae proteins. To define the functional role of YlOch1p we will determine its membership of the M-Pol II type complex by the two-hybrid system.
In order to establish a model for the Y. lipolytica Golgi N-glycosylation pathway, future work will consist of further investigating the modification of the native Y. lipolytica glycosylated chain in all mutant strains by analysis of the oligosaccharidic chains of secreted proteins using mass spectrometry.
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ACKNOWLEDGEMENTS |
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Received 4 November 2003;
revised 3 March 2004;
accepted 7 April 2004.
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