1. Introduction
The subject of protein N- and O-glycosylation in the yeast
In the
On the other hand, there is a growing literature describing the involvement of cell wall carbohydrates in fungus-host interactions (for review see [8]), as well as in the maintenance of cell wall integrity [9]. Thus, one can predict a functional link between N-glycosylation and O-mannosylation of cell wall proteins, cell wall integrity and/or fungus–host interactions. Nonetheless, open questions remain concerning the regulatory mechanisms of early events of protein glycosylation and their impact on the synthesis of outer glycan chain in cell surface glycoproteins of
A model of the
2. Contribution of the mevalonate pathway to protein glycosylation and cell wall integrity
Mevalonate pathway in yeasts is important not only for ergosterol biosynthesis but also for the production of nonsterol molecules, deriving from farnesyl diphosphate. Formation of cell wall proteins, i.e. the glycosyl- phosphatidylinositol (GPI) anchored (GPI-CWP) and proteins with internal repeats (pir-CWP,) requires, as an initial intermediate, DolP synthesized together with the other isoprenoid lipids in the mevalonate pathway.
Dolichol biosynthetic pathway has multiple levels of regulation. Thus, its cellular level is amenable to alterations affecting protein glycosylation and, in consequence, the cell wall structure. The pathway is shared with other isoprenoid lipids (Fig.1). The most abundant branch of the pathway, leading to sterol biosynthesis, is one of the main targets for anti-fungal drugs, which exploit the differences in the pathways and the end product - ergosterol in fungal cells and cholesterol in animals. The mevalonate pathway diverges after the synthesis of farnesyl diphosphate by farnesyl diphosphate synthase (FPPS), encoded by the
Products of the subsequent reactions are shown together with the names of the genes, in capital letters, encoding the enzymes catalyzing them. Enzyme affected by lovostatin, an inhibitor of isoprenoid pathway, is indicated (K. Grabinska PhD thesis, IBB 2002)
Using a yeast based two hybrid system, we have identified the Yta7 protein interacting with FPPS, and showed that it was membrane-associated and localised both to the nucleus and the endoplasmic reticulum (ER). In order to assess the importance of the mevalonate pathway for cell wall synthesis and its role in cell-wall integrity (Fig 2), we investigated the effects of
Moreover, farnesol, which is likely to be derived from dephosphorylation of FPP, inhibits growth of
Our results indicate that FPP or its derivatives regulate the transcription of
Biosynthesis of isopentenyldiphosphate (IPP) from mevalonate diphosphate is catalysed by mevalonate pyrophosphate decarboxylase (Fig.1). Subsequently IPP is converted to dimethylallyl diphosphate (DMAPP) and DMAPP (C5) is condensed with farnesyl diphosphate, composed of 3 isoprene units (C15). This reaction is catalysed by Rer2 and Srt1p. Further elongation of the isoprenol chain occurs, by step wise addition of the 5-carbon isoprene units to reach species-specific chain length. Poliprenol diphosphate is dephosphorylated and reduced by alfa saturase (Dfg10p) to form dolichol. To enter glycosylation pathway dolichol is phosphorylated by CTP-dependent dolichol kinase.
Our data indicate that in the rapidly dividing yeast cells “de novo” biosynthesis of dolichyl phosphate described above is a main source of its supply.
However, it is thought that in non dividing cells dolichyl phosphate might be derived mainly from recycling after each cycle of protein glycosylation, either by transfer of the oligosaccharide from DolPPGlcNAc2Man9Glc3 onto acceptor protein or from the transfer across the ER membranes of single glucose (Glc) and mannose (Man) residues from DolPMan or DolPGlc [22].
The length of dolichol molecules is species-specific and in yeast contains 14-18 isoprene units [23]. Although a great deal of progress has been made in the understanding of the enzymatic steps responsible for polyprenyl chain length termination and conversion of dehydrodolichol to dolichol, some open questions still remain.
3. Cell wall alteration resulting from the defect in dolichol and dolichyl phosphate formation
It has been shown that depletion of GDPMan pyrophosphorylase activity in
In this work we have concentrated on the assembly of mono- and oligo-saccharide lipid carrier (DolP), which is another substrate in protein glycosylation, and on it’s effect on cell wall integrity and cell morphology.
In
By growing the strains in repressive conditions we were able to demonstrate that the defect in dolichol backbone synthesis or its phosphorylation, resulted in the aberrant cell wall structure and increased sensitivity to some antifungal drugs. Moreover, the normal morphogenesis of the fungus, e.g. hyphae formation, was prevented (Juchimiuk et al. 2011, in preparation).
Recently, we have cloned an ortholog of
Double deletion of the
However, based on our results, only 30% of dehydrodolihol is reduced to dolichol in
We have also studied alterations in cell wall composition and integrity in
For the
We have shown that the
As already mentioned, a defect in protein O-mannosylation in fungi results in impaired cell wall integrity [9]. This process is initiated at the luminal side of the ER. The key enzyme of O-mannosylation is protein -O-mannosyltransferase (Pmtp) catalysing direct transfer of Man from DolPMan into the serine/threonine OH group in acceptor protein. This is followed by the addition of a short linear glycan, composed of mannosyl residues, directly from GDPMan. Whereas O-mannosylation is initiated in the ER, further modifications of the glycan chain occur in the Golgi apparatus. In
Studies of the effect of tunicamycin revealed the effect of the dolichol dependent protein N-glycosylation on
4. Cell morphology in Golgi glycosylation mutants
A number of data indicates that a defect in glycosylation process occurring in the Golgi stack might affect cell morphology and virulence.
Initial steps of protein N- and O-glycosylation described so far occur in the ER. “Dolichol-dependent” glycosylation ends with the formation of DolPP-oligosaccharide (DolPPGlcNAc2Man9Glc3) and subsequent transfer of the oligosaccharide to the beta amido group of asparagine within the N-glycosylation site (Asn/X/Ser). Such a glycosylated peptide undergoes partial trimming, which is species-specific and in yeast involves removal of the three Glc and one Man residues. Partially processed glycopeptide is transported to the Golgi stack where the saccharide part (GlcNAc2Man8) undergoes further processing and maturation. Whereas glycosylation reactions occurring in the ER are well conserved in the eukaryotic cells, N-glycan processing in the Golgi is greatly diverse. In yeast, the core structure of glycoproteins is hypermannosylated (up to 200 Man residues), forming a backbone made up of alpha 1,6 linked residues, branched by alpha 1,2- and alpha 1,6 mannosyl residues. In addition, another mature core structure occurs in the Golgi, i.e. GlcNAc2Man8-13 [51]. A number of Golgi mannosyltransferases is involved in the synthesis of the sugar backbone and branching. It offers a vast majority of modifications of the sugar structure. The
Mannan branching involves Mnn1p, one of five
5. Evidence for the role of glycan in cell defence mechanisms
The fungal cell wall is a highly dynamic structure, essential for the shape and stability of the cell. Thus in yeast and fungi cell wall integrity is tightly controlled by the activation of the protein kinase C –dependent MAP-kinase pathway [57] (Fig. 3).
The sensors of the cell wall damage located in the plasma membrane (Mid2p, Wsc1-3p) pass the signal thorough the Rom1p to Rho1p, which in turn activates the Pkc1p- dependent cascade of MAP kinases (Bck1p, Mkk1/2p) Mpk1 kinase activates its main targets Swi4/6p and Rlm1p, transcription factors that activate expression of the cell wall genes (adopted from [57]).
Efficiency of this process depends, among others, on the glycosylation status of the receptor proteins located in the plasma membrane [58]. It has been demonstrated that the plasma membrane protein Mid2, a putative mechanosensor, responds to cell wall stresses and changes in the cell morphology induced by pheromone treatment. The response is related to the glycosylation status of the Mid2 protein, which is highly O-glycosylated and contains two potential glycosylation sites, one of them at Asn-35, carrying N-linked sugars. It was demonstrated that O-glycosylation is responsible for the stability of the protein, whereas the presence of the N-linked sugar chain is a prerequisite for its function as a sensor of external stimuli.
Our data [35] indicate that impairement of dolichol kinase (Sec59p) activity is concomitant with a defect in plasma membrane/cell wall Gas1p glycosylation and the activation of the CWIP. These results were further confirmed by the analysis of the cell wall composition of the
An activation of the cell defence mechanisms was also observed in
Together, based on the knowledge acquired so far, it can be assumed that the glycosylation pathway in yeast and fungi offers many levels of regulation which might influence the final quality and quantity of cell wall glycoproteins and consequently cell surface immunogeneity and the fungal-host interaction. This includes also the processes occurring in the ER i.e. dolichol biosynthesis and glycosylation steps involving dolichol.
Acknowledgement
The experimental work was supported by the grant N N303 577238 from Ministry of Science and Higher Education, Poland, to G.P.
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