Renata Teparić

Proteins involved in building, maintaining and remodeling of yeast cell walls

The cell wall defines the shape and provides osmotic stability to the yeast cell. It also serves to anchor proteins required for communication of the yeast cell with surrounding molecules and other cells. It is synthesized as a complex structure with β-1,3-glucan chains forming the basic network to which β-1,6-glucan, chitin and a number of mannoproteins are attached. Synthesis, maintaining and remodeling of this complex structure require a set of different synthases, hydrolases and transglycosidases whose concerted activities provide necessary firmness but at the same time flexibility of the wall moiety. The present state of comprehension of the interplay of these proteins in the yeast cell wall is the subject of this article.

Binding assay for incorporation of alkali-extractable proteins in the Saccharomyces cerevisiae cell wall

Yeasts have developed three different ways of attaching proteins to cell wall glucan. Some proteins are bound to β‐1,3‐glucan non‐covalently, while others are attached covalently, through GPI‐anchor and β‐1,6‐glucan, or directly to β‐1,3‐glucan by alkali‐labile ester linkage between the γ‐carboxyl groups of glutamic acid and the hydroxyl groups of glucoses (Pir proteins). In order to obtain further insight into the binding mechanism, a novel, simple binding assay for Pir‐family proteins was developed. It has been shown that PIR, as well as SCW4 mutants, can bind externally added Ccw5p to their cell walls. A study of appropriate binding conditions revealed the requirement of the native conformation of Ccw5p. The presence of EDTA blocked the binding of Ccw5p, indicating the cation dependence of the reaction. Both wild‐type and mutant cells showed enhanced binding of the Ccw5p in 0.6 M KCl. After disruption of all Pir genes (CCW5, CCW6, CCW7 and CCW8), 67 kDa protein still remained in NaOH extract. SCW4 disruption in the ccw5ccw6ccw7ccw8 mutant resulted in disappearance of the 67 kDa band from the extract, indicating that Scw4p could also be covalently linked to the cell wall by a so‐far unidentified alkali‐labile linkage. Copyright

Carbendazim impends hepatic necrosis when combined with imazalil or cypermethrin.

Imazalil, cypermethrin and carbendazim are detected in plants for human nutrition. To explore whether their combinations, applied orally in low doses, would induce changes in metabolic patterns and hepatotoxicity, a subchronic in vivo experiment was conducted. Doses of 10mg/kg of imazalil (im) and cypermethrin (cy) and 20 mg/kg of carbendazim (car) and their combinations (im, 10 mg/kg+cy, 10mg/kg; im, 10mg/kg+car, 20mg/kg; car, 20 mg/kg + im, 10 mg/kg) were given to Swiss mice daily over 28 days. After 24 hr from the last dose, the relationships of cytotoxicity biomarkers were analysed: serum lactate dehydrogenase, aspartate transaminase, alanine transferase, amylase, alkaline phosphatase, creatine kinase, creatinine and total proteins. Individual pesticides showed different toxic potential (cy>im car) generally characterized by increase in enzyme activities. Histological analysis showed that cypermethrin, but not imazalil or carbendazim, alone can cause mild necrosis. Combinations generally caused decrease in the activity of enzymes, indicating liver damage. Low doses of carbendazim in combination with low doses of imazalil or cypermethrin caused very pronounced hepatic necrosis, more than any of the three individually applied pesticides or combination of imazalil and cypermethrin. In fruits and vegetables for human consumption, residues of these three pesticides and prolonged combined intake of low doses, which by themselves acutely would not cause any effect, may have similar hepatotoxic effects.

Proteolytic processing of the Saccharomyces cerevisiae cell wall protein Scw4 regulates its activity and influences its covalent binding to glucan.

Yeast cell wall contains a number of proteins that are either non-covalently (Scw-proteins), or covalently (Ccw-proteins) bound to β-1,3-glucan, the latter either through GPI-anchors and β-1,6-glucan, or by alkali labile ester linkages between γ-carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir-proteins). It was shown that a part of Scw4, previously identified among the non-covalently bound cell wall proteins, was covalently attached to wall polysaccharides by a so far unknown alkali sensitive linkage. Thus Scw4 could be released from cell walls by treatments with hot SDS, mild alkali, or β-1,3-glucanases, respectively. It was further shown that non-covalently bound Scw4 (SDS released) underwent the Kex2 proteolytic processing. In this paper it was demonstrated that Scw4 was also processed by yapsins at a position 9 amino acids downstream of the Kex2 cleavage site. Scw4 cleaved at the yapsin site had a markedly lower potential for covalent attachment to glucan. The overproduction of the fully processed form of Scw4 lead to high mortality, particularly in the stationary phase of growth, and to markedly increased cell size. On the other hand, the overproduction of Scw4 processed only by Kex2 or not processed at all had no apparent change in mortality indicating that only the smallest, completely mature form of Scw4 had the activity leading to observed phenotype changes.

Purification and Characterization of a Novel Cold-Active Lipase from the Yeast Candida zeylanoides.

Cold-active lipases have attracted attention in recent years due to their potential applications in reactions requiring lower temperatures. Both bacterial and fungal lipases have been investigated, each having distinct advantages for particular applications. Among yeasts, cold-active lipases from the genera Candida, Yarrowia, Rhodotorula, and Pichia have been reported. In this paper, biosynthesis and properties of a novel cold-active lipase from Candida zeylanoides isolated from refrigerated poultry meat are described. Heat-sterilized olive oil was found to be the best lipase biosynthesis inducer, while nonionic detergents were not effective. The enzyme was purified to homogeneity using hydrophobic chromatography and its enzymatic properties were tested. Pure enzyme activity at 7°C was about 60% of the maximal activity at 27°C. The enzyme had rather good activity at higher temperatures, as well. Optimal pH of pure lipase was between 7.3 and 8.2, while the enzyme from the crude extract had an optimum pH of about 9.0. The enzyme was sensitive to high ionic strength and lost most of its activity at high salt concentrations. Due to the described properties, cold-active C. zeylanoides lipase has comparative advantages to most similar enzymes with technological applications and may have potential to become an industrially important enzyme.