Grażyna Palamarczyk

Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways

Caffeine is a natural purine analogue that elicits pleiotropic effects leading ultimately to cells death by a largely uncharacterized mechanism. Previous works have shown that this drug induces a rapid phosphorylation of the Mpk1p, the final mitogen‐activated protein (MAP) kinase of the Pkc1p‐mediated cell integrity pathway. In this work, we showed that this phosphorylation did not necessitate the main cell wall sensors Wsc1p and Mid2p, but was abolished upon deletion of ROM2 encoding a GDP/GTP exchange factor of Rho1p. We also showed that the caffeine‐induced phosphorylation of Mpk1p was accompanied by a negligible activation of its main downstream target, the Rlm1p transcription factor. This result was consolidated by the finding that the loss of RLM1 had no consequence on the increased resistance of caffeine‐treated cells to zymolyase, indicating that the cell wall modification caused by this drug is largely independent of transcriptional activation of Rlm1p‐regulated genes. Additionally, the transcriptional programme elicited by caffeine resembled that of rapamycin, a potent inhibitor of the TOR1/2 kinases. Consistent with this analysis, we found that the caffeine‐induced phosphorylation of Mpk1p was lost in a tor1Δ mutant. Moreover, a tor1Δ mutant was, like mutants defective in components of the Pkc1p‐Mpk1p cascade, highly sensitive to caffeine. However, the hypersensitivity of a tor1 null mutant to this drug was rescued neither by sorbitol nor by adenine, which was found to outcompete caffeine effects specially on mutants in the PKC pathway. Altogether, these data indicated that Tor1 kinase is a target of caffeine, whose inhibition incidentally activates the Pkc1p‐Mpk1p cascade, and that the caffeine‐dependent phenotypes are largely dependent on inhibition of Tor1p‐regulated cellular functions. Finally, we found that caffeine provoked, in a Rom2p‐dependent manner, a transient drop in intracellular levels of cAMP, that was followed by change in expression of genes implicated in Ras/cAMP pathway. This result may pose Rom2p as a mediator in the interplay between Tor1p and the Ras/cAMP pathway.

Dolichol biosynthesis in the yeast Saccharomyces cerevisiae: an insight into the regulatory role of farnesyl diphosphate synthase

Dolichol, an isoprenoid lipid, known mainly for its function in protein glycosylation, is synthesised in the mevalonate pathway. The pathway is highly regulated, on multiple levels, by sterol and non-sterol derivatives of mevalonic acid. Farnesyl diphosphate (FPP) and/or FPP-derived molecules have been identified as the main non-sterol compounds regulating degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, one of the regulatory enzymes in the mevalonate pathway. In the present review we concentrate on the effect of overexpression of farnesyl diphosphate synthase on dolichol biosynthesis in yeast. In this context the role of the Yta7 protein, belonging to the AAA ATPase family, in the regulation of FPP flux to the dolichol branch of the mevalonate pathway is discussed, and the effect of FPP and/or derived molecules on the transcription of genes encoding the first enzyme committed to dolichol biosynthesis, i.e. cis-prenyl transferase.

1,4-Dideoxy-1,4-imino-d-mannitol inhibits glycoprotein processing and mannosidase

1,4-Dideoxy-1,4-imino-D-mannitol (DIM) was synthesized chemically from benzyl-alpha-D-mannopyranoside [Fleet et al (1984) J. Chem. Soc. Chem. Commun., 1240-1241], and was tested in vitro as an inhibitor of various alpha-mannosidases and in cell culture as an inhibitor of glycoprotein processing. DIM proved to be an effective inhibitor of jack bean alpha-mannosidase, with 50% inhibition requiring 25 to 50 ng/ml inhibitor. It also inhibited lysosomal alpha-mannosidase, but in this case 50% inhibition required about 1 to 2 micrograms/ml. In both cases, the inhibition was of the competitive type when p-nitrophenyl-alpha-D-mannopyranoside was used as the substrate. The inhibition was better at higher pH values, suggesting that DIM was more effective when the nitrogen in the ring was in the unprotonated form. In addition, rat liver processing mannosidase I was also inhibited by DIM as measured by the release of [3H]mannose from [3H]mannose-labeled Man9GlcNAc. Glycoprotein processing was examined in influenza virus-infected MDCK cells. Infected cells were incubated in various concentrations of DIM and labeled with [2-3H]mannose. Viral and cell pellets were digested with Pronase and glycopeptides were isolated by gel filtration on columns of Bio-Gel P-4. The glycopeptides were then treated with endoglucosaminidase H (Endo H) and rechromatographed on the Bio-Gel column in order to distinguish complex from high-mannose structures. As the DIM concentration in the medium was raised, more and more of the [3H]mannose was incorporated into high-mannose oligosaccharides, and less and less radioactivity was in the complex chains. Most of the Endo H-released oligosaccharides induced by DIM were of the Man9GlcNAc structure, as determined by gel filtration, HPLC, and digestion by alpha-mannosidase. Thus, DIM also appears to inhibit mannosidase I in cell culture. However, about 15% of the Endo H-released oligosaccharides appear to be hybrid types of oligosaccharides, suggesting that DIM may also inhibit mannosidase II.

O-glycosylation of proteins by membrane fractions of Trichoderma reesei QM 9414

In order to investigate O-glycosylation of proteins in the fungus Trichoderma reesei QM 9414, a membrane preparation was isolated and used to study the glycosylation of endogenous proteins. Exogenously added GDP-[U-14C]mannose was used to mannosylate both endogenous lipid and protein. The kinetics of mannosylation together with pulse-chase experiments with cold GDP-mannose revealed that lipid was labelled before protein. The lipid was identified as mannosyl phosphoryl dolichol (Dol-P-Man) by TLC together with an authentic standard from yeast. Addition of tsushimycin, a specific inhibitor of Dol-P-Man synthesis, completely blocked transfer of mannose from GDP-[U-14C]mannose to endogenous lipid. The mannosyl units transferred to endogenous protein could be released by β-elimination, and were shown to consist mainly of tetra-, di- and monomannosyl chains. Mannosylation of endogenous proteins occurred at a lower rate with membranes isolated from glycerol-grown cells. This could be overcome by addition of cold GDP-mannose, suggesting a limitation of endogenous GDP-mannose and/or dolichol phosphate in glycerol-grown (i.e. catabolite-repressed) cells.

Stimulation of exoprotein secretion by choline and Tween 80 in Trichoderma reesei QM 9414 correlates with increased activities of dolichol phosphate mannose synthase

Summary: Addition of choline (20 mM) or Tween 80 (0.06%) to the culture medium of Trichoderma reesei QM 9414 increased (a) the secretion of protein under both carbon-catabolite-repressed and -derepressed conditions, and (b) cellulase secretion under carbon-catabolite-derepressed conditions. In contrast, no stimulation by choline or Tween 80 was observed with the hypersecretory strain T. reesei RUT C-30. In view of the obligatory role of O-glycosylation in protein secretion by this fungus, an investigation was made into the effects on this process of choline and Tween 80. A membrane preparation was isolated from both strains of T. reesei and used to assay enzymes involved in O-glycosylation. Significant differences were observed with respect to the activity of dolichol phosphate mannose (Dol-P-Man) synthase only. Strain QM 9414, grown on media supplemented with choline or Tween 80 exhibited a two- to threefold higher activity of Dol-P-Man synthase compared to a control lacking these supplements. This stimulatory effect was observed during growth under both carbon-catabolite-repressed and -derepressed conditions. In contrast, strain RUT C-30 exhibited decreased activities of Dol-P-Man synthase when grown in media supplemented with choline. Choline had no effect on Dol-P-Man synthase in vitro, whereas Tween 80 decreased the activity. Thus the effect of Tween 80 or choline on protein secretion by T. reesei may be due to a stimulation of formation and/or activity of Dol-P-Man synthase, thereby elevating the level of O-glycosylation and protein secretion.

Dissecting the role of dolichol in cell wall assembly in the yeast mutants impaired in early glycosylation reactions.

Evidence is presented that temperature‐sensitive Saccharomyces cerevisiae mutants, impaired in dolichol kinase (Sec59p) or dolichyl phosphate mannose synthase (Dpm1p) activity have an aberrant cell wall composition and ultrastructure. The mutants were oversensitive to Calcofluor white, an agent interacting with the cell wall chitin. In accordance with this, chemical analysis of the cell wall alkali‐insoluble fraction indicated an increased amount of chitin and changes in the quantity of β1,6‐ and β1,3‐glucan in sec59‐1 and dpm1‐6 mutants. In order to unravel the link between the formation of dolichyl phosphate and dolichyl phosphate mannose and the cell wall assembly, we screened a yeast genomic library for a multicopy suppressors of the thermosensitive phenotype. The RER2 and SRT1 genes, encoding cis‐prenyltransferases, were isolated. In addition, the ROT1 gene, encoding protein involved in β1,6‐glucan synthesis (Machi et al., 2004 ) and protein folding (Takeuchi et al., 2006 ) acted as a multicopy suppressor of the temperature‐sensitive phenotype of the sec59‐1 mutant. The cell wall of the mutants and of mutants bearing the multicopy suppressors was analysed for carbohydrate and mannoprotein content. We also examined the glycosylation status of the plasma membrane protein Gas1p, a β1,3‐glucan elongase, and the degree of phosphorylation of the Mpk1/Slt2 protein, involved in the cell wall integrity pathway. Copyright

Comparison of polyprenyl derivatives in yeast glycosyl transfer reactions.

Abstract Seven homogeneous, semi-synthetic polyprenyl phosphates ranging in chain length from 20 to 95 carbon atoms and differing in the presence of saturated isoprene residues, were tested in several glycosyl transfer reactions catalyzed by yeast membranes. They were compared not only as acceptors of sugar phosphate from nucleoside diphosphate sugars but also as donors of the carbohydrate units to endogenous protein. All polyprenyl phosphates tested were effective substrates for the synthesis of mannosyl-lipid and differed by no more than a factor of two in activity. In contrast, a 5–8-fold preference for derivatives containing a saturated rather than an unsaturated α-isoprene residue was observed for utilization of the mannosyl-lipids as substrates for mannosylation of protein. The enzyme catalyzing the synthesis of N-acetylglucosaminylpyrophosphoryl-polyprenol showed the highest specificity and only polyprenyl phosphates resembling natural yeast dolichyl phosphate in nearly all respects were effective substrates. Mono-N-acetylglucosaminyl-lipids were glycosyl donors to protein only after conversion to more polar lipids containing additional residues of N-acetylglucosamine and mannose. In all reactions tested, changes in the chain length of the polyprenyl moiety had little or no effect on activity.

Overexpression of the Gene Encoding GTP:Mannose-1-Phosphate Guanyltransferase, mpg1, Increases Cellular GDP-Mannose Levels and Protein Mannosylation in Trichoderma reesei

ABSTRACT To elucidate the regulation and limiting factors in the glycosylation of secreted proteins, the mpg1 and dpm1 genes from Trichoderma reesei (Hypocrea jecorina) encoding GTP:α-d-mannose-1-phosphate guanyltransferase and dolichyl phosphate mannose synthase (DPMS), respectively, were overexpressed in T. reesei. No significant increases were observed in DPMS activity or protein secretion in dpm1-overexpressing transformants, whereas overexpression of mpg1 led to a twofold increase in GDP-mannose (GDPMan) levels. GDPMan was effectively utilized by mannnosyltransferases and resulted in hypermannosylation of secreted proteins in both N and O glycosylation. Overexpression of the mpg1 gene also increased the transcription of the dpm1 gene and DPMS activity. Our data indicate that the level of cellular GDPMan can play a major regulatory role in protein glycosylation in T. reesei.

cDNA encoding protein O-mannosyltransferase from the filamentous fungus Trichoderma reesei; functional equivalence to Saccharomyces cerevisiae PMT2

Abstract O-Mannosylation is suggested to be essential for protein secretion in Trichoderma reesei. In protein O-glycosylation, the first mannosyl residue is transferred to a serine or threonine hydroxyl group of the protein from dolichyl phosphate mannose by protein O-mannosyltransferase. In Saccharomyces cerevisiae, seven PMT genes have been cloned coding for these enzymes. In the present work, the characterisation of the pmt1 cDNA from T. reesei is reported. Sequence analysis of the predicted protein revealed the highest similarity to Schizosaccharomyces pombe Pmt and to Pmt4p of Saccharomyces cerevisiae. In contrast, expression of the T. reesei cDNA in various S. cerevisiae pmt mutants showed functional similarity to the yeast Pmt2 protein.

Glycoprotein Hypersecretion Alters the Cell Wall in Trichoderma reesei Strains Expressing the Saccharomyces cerevisiae Dolichylphosphate Mannose Synthase Gene

ABSTRACT Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble β-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene.