Genetic Techniques for Biological Research Corinne A. Michels Copyright q 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic) 3 Saccharomyces Cell Structure Saccharomycescerevisiue is aeukaryote and as such containsthe subcellular organelles commonlyfound in eukaryotes.The structure and function of these organelles is fundamentally the same as it is in other eukaryotes with less versatile systems for genetic analysis, and for this reason Saccharomyces is the organism of choice for many cell biologists. For a truly in-depth review of Saccharomyces cell structure and function, the reader is referred to The Molecular and Cellular Biology of theYeast Saccharomyces: Vol. 3: CellCycle and Cell Biology (Broach etal., 1997). The discussion here will provide a very brief overview of the cell structure and will focus on certain unique features of Saccharomyces cell structure in order to facilitate reading of the literature. CELL SHAPE AND GROWTH PATTERNS Underusual culture conditions, Saccharomyces is ellipsoidaUovoid in shapeand approximately 5-10 pm long by 3-7 pm wide. This is referred to as the yeast form. Figure 3.1 shows a scanning electron micrograph (SEM) of a cell in the yeast form. Cell division is by budding; that is, a smaller ovoid daughter cell formsas a projection from the surface of the mother cell. Haploid cells are generally about one-half the volume of diploid cells. The characteristic shape is maintained by a rigid cell wall that completely surrounds the plasma membrane of Saccharomyces. Changes in this shape involve remodeling of the cell wall and occur during budding, mating,and pseudohyphal differentiation. Under nutrient stressed conditions certain strains undergo a shape change, forming filaments consisting of short chains of individual cells called pseudohyphe. In thepseudohyphal form the cells are elongated rather than ovoid in shape, and formchains of cells because the daughters do not detach from the mother even though cytokinesis is complete (Figure 3.2). Pseudohyphaepenetrate into the solid substrategrowth medium makingthem difficult to wash off, so-called invasive growth. The plasma membrane surrounds a cytoplasm that is organized structurally by a cytoskeleton and is divided intomembrane-bound compartments including a nucleus, mitochondria, pexoisomes, a vacuole, a Golgi complex, vesicles of various types, andthe endoplasmic reticulum (ER). In many respects the ER, Golgi, vacuole, secretory vesicles and endocytic vesicles, and plasma membrane should be considered to be part of an interconnected system. Newly synthesized membrane and membrane proteins move from the ER to the Golgi and then to either the plasma membrane or the vacuole via secretory vesicles. Additionally, plasma mem- brane is internalized to form endocytic vesicles that fuse eventually with the vacuole. These combined processes of secretion (or exocytosis) and endocytosis are often referred to as membrane trafficking processes. Various particulate structures are found in the cytosol including ribosomes and proteasomes (essential protease complexes involved in the degradation of soluble proteins). 44 GENETIC TECHNIQUES FOR BIOLOGICAL RESEARCH Figure 3.1 SEM of S. cerevisiae. A dividing cell exhibiting the yeast form is shown using SEM. Arrows point to the birth scar (BirS) and bud scar (BS), indicating that this cell has formed one bud prior to the one that is currently forming. Taken from Walker (1998). Reproduced with permission of John Wiley & Sons Limited Figure 3.2 Pseudohyphal growth pattern. In starvation conditions, certain Saccharomyces strains alter their shape and budding pattern. This altered growth pattern, shown in this figure, is referredto as the pseudohyphal morphology. An elongated cell shape and a unipolar pattern of budding characterize the pseudohyphal growth. In the unipolar budding pattern, new buds form onlyat the end of the cell opposite to the birth end (the distal pole) in both the mother and the daughter cell. Adapted from Taheriet al. (2000) by permission of Oxford University Press CELL WALL, CELL SURFACE MORPHOLOGY, AND MORPHOLOGICAL VARIATION The Saccharomyces cell wall is about 200nm thick and completely surrounds the cell. Itsfunction is to preservethe osmotic integrity of the cell and define morphology but it has several otherroles. Proteins involved in cell-cell recognition andadhesion, such as occursduring mating, are found in the cellwall. Other proteins are immobilized or retainedin the periplasm, the spacebetween the plasma membrane and thecell wall, by the cell wall. These are secreted proteinsand localize specifically to this region. Finally, the cell wall may also be a partial permeability barrier. The cell wall is a very dynamic structure that is synthesized during bud CELL STRUCTURE 45 SACCHAROMYCESSTRUCTURE CELL growthbut must also undergo remodeling during cell division,mating, and sporulation. For a thorough reviewof the cellwall and cellwall biogenesis the reader is referred to Orlean (1997). CELL WALL COMPOSITION AND SYNTHESIS The major component (80%-90%) of the cell wall is polysaccharide. This includesp- glucans,mannoproteins, and chitin. P-Glucan is a glucose homopolymer. In Saccharomyces, one finds /3-1,3 straight chains up to 1500 residues long with some p-1,6 branches. These long polymers are intertwined to form microfibrils that are interwoven into the meshwork that makes the basic support structure of the cell wall, much like the steel rods in reinforced concrete. Imbedded into this meshwork are the mannoproteins or mannans. These are secreted proteins with large, highly branched, covalently bound carbohydrateside groups consisting mostlyof mannose residues but also includingglucose and N-acetylglucosamine residues. Some of these glycoproteins are also attached to lipids of the plasma membrane via a GP1 anchor (glycosyl phosphatidylinositol) at the C-terminus of the protein. Cell wall proteins include the agglutinins and flocculins that play important roles incell adhesion. Enzymes such as invertase, a heavily glycosylated protein, are found in the peri- plasmicspace. Chitin is ahomopolymer of P- l,4-linked N-acetylglucosamine residues. It is a minor component of the cell wall (1%-2%0) and is largely found at the site of bud formation and in bud scars. Many of the cell wall components are cross-linked to one another to form this very complex and interconnecting rigid structure. Figure 3.3 shows the organization of the Saccharomyces cell wall. Synthesis of the major cell wall components takes place in the ER and Golgi, and transit to the cell surface is achieved via secretory vesicles. Glycosylationand mannosylation of cellwall proteinsinitiates in theER and is completed in the Golgi. The same is true for the formation of the GP1 anchor. Polymerization of P- glucan initiates in the ER but continues in the Golgi and completes at the plasma membrane and involves some membrane-bound protein components of these com- partments. Chitin synthesis is different. Chitin synthase is a membrane enzyme. It uses an intracellular precursor, UDP-N-acetylglucosamine, to synthesize extracellu- lar chitin by some type of transmembrane process that is as yet not well understood. Since chitin is found in selected regions of the cell wall, chitin synthase must be active only in certain sites and is apparently regulated by the processes of bud site selection. BUD SCARS, BIRTH SCARS, AND BUDDING PATTERNS Bud scars are chitin-rich turtleneck-like raised rings that form at the site of bud formation and surround the budneck. The bud scar remainson the cell wall surface on the mother cell even after the bud has detached.Since an individual cell can divide 25 or more times, the surfaceof a maturecell will be studded with multiple bud scars. The site on the daughter cell that had been the attachment to the mother is also visible on the surface and is called the birth scar. Both can be seen in Figure 3.1. Budding patterns differ in haploid versus diploid cells. In haploid cells, both a- and a-mating type, the new bud forms near the site of the previous bud scar on the ns an m lic Jic )ic Figure 3.3 Composition and structure of the cell wall. The various components of the Saccharomyces cell wall and their complex intermolecular interactions to form a mesh-like organization are depicted. Taken from Schreuder et al. (1996) with permission from Elsevier Science CELL STRUCTURE 47 SACCHAROMYCESSTRUCTURE CELL mother and the birth scar on the daughter, asis shown in Figure 3.1 and depicted in Figure 3.5. This is referred to as an axial budding pattern. In ala diploid cells, the bud forms at or near either pole in the mother but only at the opposite pole in the daughter giving what is referred to as a bipolar budding pattern (Figure 3.4 and depicted in Figure 3.5). A third type of budding pattern, unipolar budding, is seen in cells undergoing pseudohyphal growth. Here the bud always forms in the mother at a site near the attachment of mother and daughter and always at the opposite pole in the daughter cell (Figure 3.4 and depicted in Figure 3.5). In budding yeast, the daughter cell (bud) is smaller than the mother cell at the time of cell separation. Therefore, before it can produce its own bud it must growto a certain minimal size and does this by lengthening the time spent in GI of the cell cycle. This G1 delay is evidentfrom the finding thatmother cells budbefore daughter cells. This is indicated in Figure 3.5. SCHMOO FORMATION AND MATING Inresponse to matingpheromone in themedium, haploid cellswill arrest cell division in G1 and begin to form a protrusion on the side of highest pheromone concentration. This gives the cell a pearlike shape, jokingly called a schmoo after a defunct syndicated newspaper cartoon character, and the process is called schmoo- ing.The shmoo isseen in Figure 3.6 andone should note that schmoosare unbudded.Schmoos of oppositemating type will attachto oneanother at the schmoo tip, a region of the cell wall containing a high concentration of agglutinin proteins.