The yeast cell wall contains an unusually high number of different mannoproteins. The physiological role of most of them is unknown and gene disruptions leading to depletion of different proteins do not affect major functions of the wall. In this work the phenotype of different single and multiple cell wall protein mutants was observed at the level of individual cells. It was found that the lack of the non-covalently bound wall proteins Scw4p, Scw10p and Bgl2p increases the mortality of Saccharomyces cerevisiae cells grown exponentially under standard laboratory conditions, as assayed by methylene blue staining. Mutation of SCW11, however, suppressed the phenotype of scw4scw10, or scw4scw10bgl2, indicating that Scw4p, Scw10p and Bgl2p act synergistically while Scw11p has an activity antagonistic to that of the other three proteins. Mutants lacking major covalently bound proteins, either all four described Pir-proteins or the five most abundant glycosylphosphatidylinositol (GPI)-anchored proteins (Ccw12p, Ccw13p/Dan1p, Ccw14p/Icwp1p, Tip1p and Cwp1p), also had increased mortalities, the first somewhat more and the latter less than that of scw4scw10bgl2. In all cases the observed phenotype was suppressed by the addition of an osmotic stabilizer to the growth medium, indicating that cells died due to decreased osmotic stability. If cells were grown to stationary phase, Scw-mutants showed only slightly increased mortality, but mutants lacking Pir-or GPI-anchored proteins had significantly increased sensitivity, suggesting that their physiological function is primarily expressed in stationary-phase cells. In many cases structures consisting of a living ccw5ccw6ccw7ccw8 (multiple Pir-protein mutant) mother with two methylene blue-stained daughters could be seen.
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.
Yeast cell walls have two major roles, to preserve physical integrity of the cell, and to ensure communication with surrounding molecules and cells. While the first function requires evolutionary conserved polysaccharide network synthesis, the second needs to be flexible and provide adaptability to different habitats and lifestyles. In this study, the comparative in silico analysis of proteins required for cell wall biosynthesis and functions containing 187 proteins of 92 different yeasts was performed in order to assess which proteins were broadly conserved among yeasts and which were more species specific. Proteins were divided into several groups according to their role and localization. As expected, many Saccharomyces cerevisiae proteins involved in protein glycosylation, glycosylphosphatidylinositol (GPI) synthesis and the synthesis of wall polysaccharides had orthologues in most other yeasts. Similarly, a group of GPI anchored proteins involved in cell wall biosynthesis (Gas proteins and Dfg5p/Dcw1p) and other non-GPI anchored cell wall proteins involved in the wall synthesis and remodeling were highly conserved. However, GPI anchored proteins involved in flocculation, aggregation, cell separation, and those of still unknown functions were not highly conserved. The proteins localized in the cell walls of various yeast species were also analyzed by protein biotinylation and blotting. Pronounced differences were found both in the patterns, as well as in the overall amounts of different groups of proteins. The amount of GPI-anchored proteins correlated with the mannan to glucan ratio of the wall. Changes of the wall proteome upon temperature shift to 42 °C were detected.
The state of anhydrobiosis is linked with the reversible delay of metabolism as a result of strong dehydration of cells, and is widely distributed in nature. A number of factors responsible for the maintenance of organisms' viability in these conditions have been revealed. This study was directed to understanding how changes in cell wall structure may influence the resistance of yeasts to dehydration-rehydration. Mutants lacking various cell wall mannoproteins were tested to address this issue. It was revealed that mutants lacking proteins belonging to two structurally and functionally unrelated groups (proteins non-covalently attached to the cell wall, and Pir proteins) possessed significantly lower cell resistance to dehydration-rehydration than the mother wildtype strain. At the same time, the absence of the GPI-anchored cell wall protein Ccw12 unexpectedly resulted in an increase of cell resistance to this treatment; this phenomenon is explained by the compensatory synthesis of chitin. The results clearly indicate that the cell wall structure/composition relates to parameters strongly influencing yeast viability during the processes of dehydration-rehydration, and that damage to cell wall proteins during yeast desiccation can be an important factor leading to cell death.
Enzyme immobilization to solid matrices often presents a challenge due to protein conformation sensitivity, desired enzyme purity, and requirements for the particular carrier properties and immobilization technique. Surface display of enzymes at the cell walls of microorganisms presents an alternative that has been the focus of many research groups worldwide in different fields, such as biotechnology, energetics, pharmacology, medicine, and food technology. The range of systems by which a heterologous protein can be displayed at the cell surface allows the appropriate one to be found for almost every case. However, the efficiency of display systems is still quite low. The most frequently used yeast for the surface display of proteins is Saccharomyces cerevisiae. However, apart from its many advantages, Saccharomyces cerevisiae has some disadvantages, such as low robustness in industrial applications, hyperglycosylation of some heterologous proteins, and relatively low efficiency of surface display. Thus, in the recent years the display systems for alternative yeast hosts with better performances including Pichia pastoris, Hansenula polymorpha, Blastobotrys adeninivorans, Yarrowia lipolytica, Kluyveromyces marxianus, and others have been developed. Different strategies of surface display aimed to increase the amount of displayed protein, including new anchoring systems and new yeast hosts are reviewed in this paper.
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