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

Abstract

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.

Defect in dolichol‐dependent glycosylation increases sensitivity of Saccharomyces cerevisiae towards anti‐fungal drugs

“…As we reported previously, the yeast dpm1-6 mutant similarly to sec 59 -1, has an aberrant cell wall composition and ultrastructure (Orłowski et al, 2007). This strain showed double sensitivity towards amphotericin B compared to the isogenic wild-type, but RER2 overexpression decreased the sensitivity to the wild-type level (Table 3).…”

“…We have demonstrated previously that point mutations in the genes encoding two essential proteins involved in early steps of glycosylation in yeast, Sec59 responsible for CTP-dependent dolichol phosphorylation and Dpm1 involved in the assembly of dolichyl phosphate mannose, lead to a cell wall defect (Orłowski et al, 2007). Our results indicated a functional interrelationship between the dolichol biosynthetic (mevalonate) pathway, synthesis of DolP-linked mono-and oligosaccharides, which are intermediates in protein glycosylation, and the cell wall assembly.…”

“…However, in S. cerevisiae the mevalonate pathway contributes to cell wall integrity, not only by producing the isoprenoid groups for dolichol biosynthesis but also for prenylation of proteins involved in stress signal transduction. In our earlier work (Orłowski et al, 2007) we reported alterations of the cell wall composition and integrity in two thermosensitive S. cerevisiae strains, sec 59-1 and dpm1-6, impaired in CTP-dependent dolichol kinase activity and in DolP-Man formation, respectively.…”

“…Protein concentration was determined using the Bradford assay. Proteins (50-100 µg) were separated on Orłowski et al, 2007dpm1-6 MATa dpm1::LEU2 leu2-3, 112 lys-801 trp1-∆ura3-52 Orlean, 1992 DPM1 Orłowski et al, 2007 dpm1-6 pRER2 MATa dpm1::LEU2 leu2-3, 112 lys-801 trp1-∆ ura3-52[pNEV] RER2 Orłowski et al, 2007 10% SDS-polyacrylamide gel and transferred to Immobilon-P membrane (Millipore), washed with methanol and dried. The membrane was first incubated with mouse primary antibodies: anti-diphosphorylated MAP kinase (1 : 2000; Signaling Technology) and then with a secondary anti-mouse alkaline phosphatase-conjugated antibody (1 : 2000; Sigma).…”

“…Our previous studies on dolichol biosynthesis and dolichol-dependent glycosylation in Saccharomyces cerevisiae and filamentous fungi have shed light on the relationship between these processes and cell wall integrity (Orłowski et al, 2007). Formation of cell wall proteins, i.e.…”