Plasma membrane

Fig. 4.32 A diagrammatic cross section the cell wall of yeast (redrawn from Kreutzfeldt and Witt. 1991).

Increasingly, much is known about the cell wall and its many roles, functions and responsibilities. In particular, the cell wall provides osmotic protection, a point well made when on its removal the cell lyses! The cell wall is analogous to a 'thick overcoat' providing general protection from the environment as well as limiting (through its permeability ) what can enter or leave the cell. Further, it is instrumental in determining cell shape and morphology. The cell wall also provides a matrix for a variety of enzymes involved in wall maintenance and development together with hydrolytic proteins. Somewhat inevitably it is also involved in attachment to surfaces and, importantly in brewing, molecules from the outside world are attached to the wall. Finally, the role of the cell wall in flocculation has attracted perhaps more than its fair share of attention from brewing scientists (see Section 4.4.4). This, of course, is to be expected and encouraged. Despite this, it is fair to say that the global impact of brewery fermentations and serial repitching on the cell wall and its physiological roles are poorly understood. This special case would benefit from some structured investigation.

4.4.2 Composition

The yeast cell wall is a carbohydrate chemist's dream! As can be seen from the review by Fleet (1991), the fractionation and structural analysis of cell wall carbohydrates is usually a harsh affair involving acid and alkali extraction. Consequently, although gross interactions are understood, 'judgements about the molecular organisation of the wall become difficult if not impossible' (Fleet, 1991). Glucans. The 'glucans' are the major polymers in the cell wall, accounting for between 30-60% of the wall. Although they cover the entire cell in the form of a microfibrillar net they are restricted to the inner layer of the wall (Fig. 4.32). Fleet (1991) has described three classes of glucans: (i) alkali insoluble-acetic acid insoluble p-(l->3) (35% of the cell wall), (ii) alkali soluble p-(l->3) (20%) and (iii) highly branched p-(l->6) (5%). The p-(l->3) glucans are branched via p-(l->6) linkages whereas the p-(l->6) glucans are branched through p-(l->3) linkages. With so much glucan in the wall it is no surprise to find that these polymers play an important role in the cell architecture. Fleet (1991) proposed that the alkali insoluble-acetic acid insoluble (3-(l ->3) glucan maintains wall rigidity and shape, whereas the alkali soluble |3-(l->3) glucan 'may confer flexibility to the wall'. More recently, Stratford (1994) has reported that the two types of glucan are one and the same, differing only in the extent of cross linkage with chitin (see Section The role and location of the (3-(l->6) glucan is less clear. Cabib et al. (1982) have speculated that it forms a layer between mannoprotein and p-(l->3) glucan. More recently ((Collar et al., 1997) have developed this theme and have shown the (3-(l ->6) glucan to play a central role in the organisation of the cell wall. Essentially the p-(l->6) glucan interconnects with the cell wall polysaccharides, p-(l->3) glucan, mannoprotein (Section and chitin (Section

How these polymers might confer rigidity and strength is perhaps explained by considering p-(l->3) glucan as 'rope' rather than 'steel reinforcing rods'. Ballou (1982) has proposed that two chains of p-(l->3) glucan wind around each other to form a rope-like double helix. These, presumably, form the microfibrillar net seen on removal of the mannoprotein outer layer of the cell wall. If Ballou (1982) is right, elaboration of new wall through cell growth could simply be achieved through the chains sliding along each other. Mannoprotein. The outer layer of the cell wall is composed of mannoprotein. This glycoprotein is a major player, accounting for 25-50% of the cell wall. There is evidence that the 'structural' mannoproteins are anchored to the cell wall through linkage with p-(l->6) glucan (Fleet & Manners, 1976; Cid et al., 1995). In addition, extracellular enzymes such as invertase (that hydrolyse sucrose to fructose and glucose) are mannoproteins.

Although, their function is not clear, cell wall mannoproteins are essential for cell survival (Fleet, 1991) but play no role in cell shape or rigidity. Not surprisingly, given their location, mannoproteins appear to have an 'interactive' role acting as antigenic determinants (Section, as receptors for 'killer toxins' (see Section and sexual agglutination. More importantly in the brewing context, mannoproteins are the receptors in the flocculation process (Section

Mannoproteins have also been implicated in cell wall porosity. As noted by Nobel and Barnett (1991), the size and shape of molecules passing through or out of the cell wall has been subject to much debate. Early work in the 1950s and '60s, which suggested that only relatively small molecules could permeate the cell wall, has been overturned by the realisation that proteins are easily secreted out of yeast cells. Indeed, as described elsewhere (Section 4.3.4), yeast is an excellent host for the genetic manipulation and subsequent secretion of 'foreign' heterologous proteins. The current picture of cell wall porosity remains somewhat speculative. Nobel and Barnett (1991) suggest that the confusion over what can and cannot diffuse may be explained by a mix of predominately small pores in the cell wall with a few 'large' holes that allow the diffusion of big heterologous and homologous proteins. The mannoproteins in the cell wall are thought to obstruct diffusion through ionic interactions and the web of mannan side chains.

The structure of mannoprotein is complex. The review of Fleet (1991) notes that mannoprotein consists of about 90% mannose and 10% protein together with small amounts of phosphate located on the outside of the molecule (Cawley et al., 1972). In passing, it is noteworthy that this phosphate is the major source of negative charge on the cell surface (see Sections and The polysaccharide part of the complex consists of a-(l->6)-linked mannose with side chains of a-(l->2) and a-(1 ->3) linked residues (see Fig. 4.33). Although at first glance beyond the scope of this book, it is appropriate to dwell a little on the structure of these side chains because of their involvement as flocculation receptors. The side chains on the much-repeated 'outer chain' are all linked a-(l->2) to the backbone. Characterisation after cleavage and fractionation shows there to be four types of side chain: (i) mannobiose (M2-*M), (ii) mannotriose (M2-^2-^), (iii) mannotriose (M2-^3-^) and (iv) mannote-traose (M2-^2-^3-^). The mix of the side chains varies within the repeating unit of the outer chain. It is likely that immunochemical differentiation of strains and


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