3

YEAST CYTOLOGY

2.1 INTRODUCTION 2.4.1.3 The Cell Wall

2.2 GENERAL CELLULAR CHARACTERISTICS 2.4.1.4 The Yeast Spore Wall
OF YEASTS 2.4. 1.5 Fimbriae
2.3 CYTOLOGICAL METHODS FOR YEASTS 2.4. 1.6 Capsules
2.3.1 Light and Fluorescence Microscopy 2.4.2 The Cytoplasm and Cytoskeleton
2.3.2 Image Analysis 2.4.3 The Nucleus and
2.3.3 Flow Cytometric Analysis Extrachromosomal Elements
2.3.4 Electron Microscopy 2.4.3.1 The Nucleus
2.3.5 Yeast Cell Fractionation and 2.4.3.2 Extrachromosomal
Organelle Isolation Elements
2.4 YEAST CELL ARCHITECTURE AND FUNCTION 2.4.4 The Secretory System and Vacuoles
2.4.1 The Cell Envelope 2.4.5 Mitochondria
2.4.1.1 The Plasma Membrane 2.5 SUMMARY
2.4. 1.2 The Periplasm 2.6 REFERENCES

2.1 INTRODUCTION will focus on the relationship between subcellular

organization and physiological function.
Yeast cytology refers to the cellular anatomy of General cellular characteristics of yeasts,
yeasts, or ‘a morphological inventory of their including macromolecular constituents and mor-
structural components’ (Robinow and Johnson, phological diversity, will be considered first.
1991). Yeast cells have fascinated cytologists for Cytological methods, including visualization and
many years. This is mainly due to the fact that isolation of cell organelles, will then be reviewed
they are easily grown unicellular eukaryotes before coverage of yeast cell architecture. Ultra-
which portray the ultrastructural features of structural features of yeasts will be described
higher eukaryotic cells. Yeasts are also amenable and their functions discussed in relation to
to a variety of biochemical, genetic and mole- growth and metabolic behaviour. Several yeast
cular biological investigations of their cytology. organelles and macromolecular structures play
Yeast cell biology has been comprehensively key roles in yeast biotechnology and particular
reviewed in Volume 4 of The Yeasts (Rose and attention will be paid to the role of the yeast
Harrison, 1991) and by Kockova-Kratochvilova cell wall in cell-cell interactions and the role of
(1990). Rather than provide an exhaustive cover- organelles in carbon metabolism and protein
age of yeast cell anatomy, the present chapter secretion. Such activities are directly pertinent to
12 YEAST PHYSIOLOGY AND BIOTECHNOLOGY

the production of industrial commodities, display morphological and colorimetric hetero-

including recombinant proteins, by yeast fermen- geneity. Although a certain degree of morpho-
tation. logical variation can be expected within a yeast
culture, profound effects in individual cell mor-
phology are induced by alterations in physical
2.2 GENERAL CELLULAR and chemical conditions.
CHARACTERISTICS OF YEASTS Yeast cell size can vary widely. Phaff et al.
(1978) have stated that some yeasts may be only
Yeast biomass primarily comprises macro- 2-3 pm in length, whereas other species may
molecules which are assembled into the structural attain lengths of 20-50 pm. Cell width appears
components of the cell. The macromolecules in less variable, between 1-10 pm. S. cerevisiae cells
question mainly comprise proteins, poly- are generally ellipsoidal in shape with a large dia-
saccharides, lipids and nucleic acids (Table 2.1). meter ranging from 5-10 pm, and a small dia-
The structural chemistry of S. cerevisiae has been meter 1-3 to 1-7 pm. The mean cell volumes for
described in detail by Kreutzfeldt and Witt a haploid and diploid cell are 29 and 55 pm3,
(1991). The relative concentrations of various respectively (Tschopp et ul., 1987). Brewing
macromolecular constituents will vary from strains of S. cerevisiae are generally bigger than
species to species and will also be greatly influ- laboratory strains; for example, an ale yeast
enced by conditions of growth. (NCYC 1006) possesses a mean diameter of
Yeast cells exhibit great diversity with respect 13.4 pm (Hough et al., 1982). Note that mean
to cell size, shape and colour. Even individual cell size of S. cerevisiae also increases with cell
cells from a pure strain of a single species can age (see Chapter 4).

Table 2.1. Macromolecular constituents of yeast cells.

Class of Comments
macromolecule

Proteins Structural proteins are mainly actin and tubulin of the cytoskeleton, histones and
membrane proteins. Ribosomal proteins are found in the 60s and 40s
ribonucleoprotein subunits. Hormonal proteins are mating pheromones. Functional
proteins include hundreds of enzymes.
Glycoproteins For example, structural mannoproteins in cell walls and functional glycoprotein enzymes
(e.g. invertase).
Polysaccharides Structural polysaccharides are mainly cell wall giucan, mannan and chitin and capsular
heteropolysaccharides. Storage saccharides are glycogen and trehalose.
Polyphosphates Mainly as storage polyphosphate in the vacuole.
Lipids Structural phospholipids are free sterols in membranes; storage lipid particles are sterol
esters and triglycerides; functional lipids include phosphoglyceride derivatives (in signal
transduction) and free fatty acids (in growth and metabolic processes).
Nucleic acids Deoxyribonucleic acid: approx. 80%of total is nuclear genomic DNA, 10-20% is
mitochondria1 genomic DNA and 1-5% is extrachromosomal (e.g. 2pm circles in S.
cerevisiae, killer plasmids in Kluyveromycesspp.). Ribonucleic acid: approx. 80%of total
is in ribonucleoprotein (rRNA); approx. 5% is mRNA in cytoplasm, rough ER and
mitochondria. Some dsRNA is found in killer plasmids (S. cerevisiae) and some small
nuclear RNA (snRNA) is found in the nucleus.
 YEAST CYTOLOGY 13

Some yeasts have distinguishable cell types, the pigments of this yeast have applications as fish
best known of which are the a, a, and a/a cells of food colorants for farmed salmonids, which have
S. cerevisiae (see Chapter 4). The a and a haploid no means of synthesizing these red compounds
cells are able to undergo mating, a process which (Johnston and Gil-Hwan, 1991). High pigment-
culminates in nuclear fusion and creation of the yielding strains of P. rhodozyma can be propa-
a/u diploid. gated on inexpensive feedstocks like molasses to
With regard to cell shape, yeasts can be: produce economic alternatives to chemically
ellipsoidal/ovoid (e.g. Saccharomyces spp.), synthetic astaxanthin.
cylindrical with hemi-spherical ends (e.g. Schizo-
saccharomyces), apiculate/lemon-shaped (e.g.
Hanseniaspora and Saccharomycodes spp.), ogival 2.3 CYTOLOGICAL METHODS FOR
(elongated cell is rounded at one end and pointed YEASTS
at other, e.g. Dekkera and Brettanomyces spp.),
flask-shaped (cells dividing by bud-fission, e.g. 2.3.1 Light and Fluorescence Microscopy
Pityrosporum spp.), triangular (e.g. Trigonopsis
spp.), curved (e.g. Cryptococcus cereanus), fila- Very limited structural information can be
mentous (with pseudohyphae and septate hyphae, gleaned on unstained yeast cells with the light
e.g. Candida albicans, Yarrowia lipolytica), microscope. At 1000-fold magnification, it may
stalked (e.g. Sterigmatomyces spp.), spherical be possible to visualize the yeast vacuole and
(e.g. Debaryomyces spp.) or elongated (many several cytoplasmic ‘inclusion bodies’. The use of
yeasts, depending on growth conditions). Cellular phase-contrast microscopy, together with several
staining techniques, enables a number of cellular
differentiation of vegetative yeast cell forms into
filamentous forms (e.g. germ-tubes, pseudohy- structures of yeasts to be visualized (Table 2.2).
phae and true hyphae) occurs in several yeast Several fluorochromic dyes can also be used with
species. Polymorphic behaviour is often triggered a fluorescence microscope to highlight features
by changes in the nutritional environment and both within yeast cells and on the cell surface
even occurs in the normally ellipsoidal S. cerevi-(Table 2.2). Pringle et al. (1989) have reviewed
siae (see Chapter 4). fluorescence microscopy methods for yeast cells.
Several yeasts are pigmented and the following A relatively recent development (Stearns, 1995)
colours may be visualized in surface-grown colo- involves the use of the green fluorescent protein
nies: (GFP) from the jellyfish (Aequorea victoria) as a
reporter molecule for intracellular localization
Colour Examples and in vivo gene expression studies in S. cerevi-
Cream Many yeasts, including S. cerevisiae siae (e.g. Niedenthal et al., 1996; Waddle et al.,
White Geotrichum spp., albino mutants of 1996); Candida albicans (Cormack et al., 1997)
Phaecoccomyces spp. and Sch. pombe (Sawin and Nurse, 1996). Genes
Black Phaecoccomyces spp. Aureobasidium of interest may be fused with the GFP gene and
pullulans the subcellular destiny of the expressed fusion
Pink Phaffia spp., Oosporidium spp. proteins followed by fluorescence microscopy
Red Rhodotorula spp., adenine mutants of (blue light at 395 nm). Such techniques employ-
S. cerevisiae ing GFP are likely to assist in the functional
Orange Rhodosporidium spp. analysis of yeast genomes.
Yellow Cryptococcus laurentii, Bullera spp. When fluorescent dyes such as fluorescein iso-
thiocyanate and Rhodamine B are conjugated
Some pigmented yeasts, such as Phafla rhodo- with monospecific antibodies raised against yeast
zyma, have uses in biotechnology. The astax- structural proteins, then the range of cellular
anthin (3,3’-dihydroxy-P,P-carotene-4,4‘-dione) features visualized is greatly increased. The
14 YEAST PHYSIOLOGY AND BIOTECHNOLOGY

Table 2.2.Some cytochemical and cytofluorescent dyes for yeast microscopy.

Dye Structures visualized Comments/References

Methylene Blue Whole cells Non-viable cells stain blue (see Chapter 4)
FUN-I~~ Vacuoles/cell walls Non-viable cell vacuoles fluoresce red-orange; cell walls blue
(Haugland, 1996)
ABT' Cell walls Fischer (1 977)
9-Aminoacridine Cell walls Sensitive indicator of surface electrostatic potential (Jones et
a/., 1995)
F C ConA2 Cell walls Binds specifically to mannan (Tkacz and Lampen, 1972)
Alcian blue Cell walls Binds to mannoprotein of yeast cell surface (Friis and
Ottolenghi, 1970)
Calcofluor white Bud scars Chitin in scar tissue fluoresces
Aniline blue Cell walls/septa p1.3 glucan is stained, but not chitin which appears as black
holes in bud scars (Brown et a/., 1994;Kippert and Lloyd.
1995)
India ink Capsules Capsules appear as a clear light zone between the cell wall
and the dark ink (Golubev, 1991)
HCI-Giemsa Nuclei Yeast chromosomes may also stain (Robinow, 1981)
Lomofungin Nuclei Chromosomes stained red (Kopecka. 1977)
DAPI3 Nuclei Renders DNA fluorescent (Williamson and Fennell, 1975)
Toluidine blue Nucleoli Colour intensity diminishes following ribonuclease treatment
(Kockova-Kratochvilova, 1990)
CDCFDC4 Vacuoles Pringle et a/. ( 199 1 )
Neutral red Vacuoles Vacuoles stain red-purple (Streiblova, 1988)
CMAC5 Vacuoles Vacuolar lumen stained blue (Haugland, 1996)
Lucifer yellow Endocytic vesicles Pringle et a/. (1 989)
DASPMI~ Mitochondria Visser et a/. (1995)
Rhodamine 123 Mitochondria Alfa et a/. (1 993);Skowronek et a/. ( 1990)
DAPI Mitochondria Mitochondria fluoresce pinkwhite (Williamson and Fennell,
1975)
Janus reen Mitochondria Mitochondria stained green-blue (Streiblova, 1988)
DiOCs B ER and nuclear envelopes Koning et a/. (1993)
CFDA ~ Cytoplasmic pH Cimprich et a/. (1 995)
Iodine Glycogen deposits Glycogen stained red brown; proteins yellow (Streiblova,
1988)
Sudan black Lipid granules Lipid stained bluegrey; cytoplasm pale pink (Streiblova,
1988)
DAB' Microbodies Stain reacts with catalase (Carson and Cooney, 1990)
RC P'O F-Actin Alfa et a/. (1 993)
GFP' Subcellular localization of e.g. Microtubules (Kahana et a/., 1995);spindle pole bodies
specific proteins (Stearns, 1995)

' *
ABT, aldehyde bisulphite -tohidine blue; F-C ConA, fluoresceinconjugated ConcanavalinA DAPI, 4.6diamidino-2-phenylindole; CDCFDC,
- '
6,[6] carboxy-2.7dichlorodihydrofluorescain diacetata; 'CMAC. 7-amino-4choromethyl coumarin; DASMPI. dimethybmino styrvl-methylpyr-
'
idinium iodine; DiOC6 3.3-dihexyloxacar~anideiodide; CFDk carboxyfluoresceine diacetate; 'DAB, 3.3diaminobenzidina; loR-C P. Rhoda-
mineconjugated phalloidin; " GFP. Green Fluorescent Protein (Jelly fish).

cellular sites of immunochemical reactions can be (Kilmartin and Adams, 1984). Confocal scanning-
differentiated by brilliant fluorescence using a laser immunofluorescence microscopy can also be
dark-ground microscope. For example, immuno- used to detect intracellular distribution of proteins
fluorescence has enabled tubulin and actin to be within yeast cells. For example, Wu et ul. (1996)
localized during the S. cerevisiue cell division cycle have employed such techniques to study the
 YEAST CYTOLOGY 15

spatial organization of mitosis-regulating protein ogy for on-line estimates of morphological

kinases and phosphatases in fission yeast. Con- heterogeneity. Information on vacuolar forma-
focal microscopy has also proved very useful in tion in S. cerevisiae during growth can also be
vizualizing organellar morphology in yeasts and obtained by image processing. Such analyses may
Visser et al. (1995) have used this method to be especially useful for process observation and
produce high-resolution three-dimensional images control of industrial yeast fermentations. This is
of fluorescently labelled yeast mitochondria (see because structural heterogeneity of individual
Fig 2.15). Although, in general, immuno- cells may reflect the physiological condition (or
fluorescence microscopy can provide simple ‘metabolic fitness’) of the culture and this, in
mapping information (e.g. localization of proteins turn, may impact on fermentation productivity.
in the cytoplasm, nucleus or vacuole), higher reso-
lution ultrastructural information on yeast cells
can only be obtained with immunoelectron micro- 2.3.3 Flow Cytometric Analysis
scopy techniques (Clark, 1994).
Note that endogenous, metabolic fluorescence Flow cytometry has several applications in yeast
in yeasts (mainly due to NADH and NADPH) cytological and physiological studies. Davey and
can be continuously determined using commer- Kell (I 996) have reviewed these applications for
cially available fluorosensors. Such devices enable assessment of yeast cell physiological states and
process control of yeast bioreactions by feedback have additionally discussed the value of cell
regulation of biomass levels via oxygen and nutri- sorting technology for isolating high-yielding
ent supply. This is particularly advantageous in strains for biotechnological uses. Fluorescence-
automated control of yeast fed-batch propaga- activated cell sorting (FACS) has been employed
tions (Beyeler et al., 1981; Beyeler and Meyer, by Gift et al. (1996) for isolating slowly growing
1984). Chemiluminescence methods can also be variants of S. cerevisiae. Such procedures are
employed to analyse redox states and vitality of deemed very useful in biotechnology for enrich-
yeast cultures (see Nishimoto and Yamashoji, ing cultures with hyperproducing cells.
1994). In studies of the yeast cell cycle and organelle
biogenesis, flow cytometers have proved particu-
larly useful. For example, Sazer and Sherwood
2.3.2 Image Analysis (1990) have analysed DAPI-stained Sch. pombe
cells to monitor changes in the development of
Information on gross cell morphology (e.g. mean mitochondria. Discrimination between respira-
cell volumes) of yeasts may be obtained using tory-competent and respiratory-deficient cells of
electronic particle counters and size analysers S. cerevisiae stained with Rhodamine 123 has
(e.g. Coulter Multisizer). Digital image proces- also been achieved by flow cytometry (Skow-
sing can also be employed to provide computer- ronek et al., 1990). Cell cycle progression in S.
automated characterization of yeast cell cerevisiae has been tracked by Porro et al.
morphology. This has been accomplished during (1995) using a flow cytometer. In this method,
growth and fermentation with several yeast developed by Porro and Srienc (1995), yeast cell
species (Huls et af., 1992; Pons et al., 1993; walls were labelled with Concanavalin A con-
Hashida et al., 1995; O’Shea and Walsh, 1996). jugated to fluorescein isothiocyanate (FITC) and
For example, O’Shea and Walsh (1996) have cell protein with tetramethylrhodamine iso-
quantitatively monitored the geometry of cells thiocyanate (TRITC). This double-label cyto-
and filaments of Kluyveromyces marxianus during metric tag enables quantitative information on
fermentation of cheese whey using image analy- the growth properties of individual yeast cells as
sis. In S. cerevisiae, Zalewski and Buckholz they progress through their cell cycle. Figure 2.1
(1 996) have used digital image process technol- shows the type of information which can be
16 YEAST PHYSIOLOGY A N D BIOTECHNOLOGY

obtained with the aid of an electron microscope.

Scanning electron microscopy (SEM) is useful in
studies of cell topology, while transmission elec-
tron microscopy (TEM) of ultrathin yeast sections
is essential for the visualization of intracellular
fine structure. SEM and TEM methods for yeasts
have been described by Streiblova (1988).
High-contrast, nanometre resolution of yeast
cell surfaces by non-destructive means may be
achieved with the use of an atomic force
microscope. De Souza Pereira et al. (1996) have
used this technique on uncoated, unfixed pre-
parations of S. cerevisiae and have demonstrated
the possibility of imaging cell surface poly-
saccharide molecules at the distance of atoms.
Figure 2.2 reveals interesting topographical dif-
ferences of the cell walls of several baker's strains
of S. cerevisiae as imaged by atomic force micro-
scopy.

0 1 2 3 4 2.3.5 Yeast Cell Fractionation and

10 10 10 10 10 Organelle Isolation
CELL WALL TAG
Yeast cell wall disruption approaches have been
(CONA-FITC FLUORESCENCE; CHFINNEL NUMBER) reviewed by Kurtzman (1987) and Middleberg
(1993) has discussed various physical, chemical,
Figure 2.1. Flow cytometric analysis of fluorescently
labelled S. cerevisiae cells. The figure shows the
enzymatic and mechanical methods for process-
dynamics of a cell wall-fluorescent tag (ConAFITC) for scale disruption of S. cerevisiae cells. Evaluation
S. cerevisiae cells. The evolution of partially stained of such procedures is particularly important
and unstained cells over time is clearly visible. The spe- when considering the purification of unsecreted
cific growth rate of the overall population and the heterologous proteins from yeasts. The prepara-
length of budded phase were 0.125 h-I and 1.98 h l ,
respectively. T = 0 h, (A); T = 1 h, (B); T = 2 h, (C); T = tion of cell wall-less yeast protoplasts has been
3 h (D); T = 4 h, (E): T = 5 h, (F); T = 6 h, (G); T = 7 h, reviewed by Freeman and Peberdy (1983) and
(H). The inset on the top shows the cell cycle phases of Zimmerman and Sipiczki (1 996) have discussed
a growing yeast cell. Reproduced with permission from methods employed for protoplast fusion in
Porro eta1 (1 995) C s J. Wiley and Sons Ltd. yeasts, including non-Saccharomyces species. The
isolation of yeast cell organelles for biochemical
analyses of subcellular functions has been
obtained from flow cytometric analysis of fluor- reviewed by Lloyd and Cartledge (1991) and
escently labelled S. cerevisiae cells. Zinser and Daum (1995). Table 2.3 provides
examples of published methods for yeast orga-
nelle isolation and purification.
2.3.4 Electron Microscopy The purity of different yeast cell fractions can
be determined by electron microscopy, by mono-
Detailed information on organelle ultrastructure specific antibodies or biochemically using marker
and macromolecular architecture can only be enzymes (Table 2.4).
 YEAST CYTOLOGY 17

’ 10.000

- 5.000

nm
30

. 5.000

nm
5.600 10.000 c, 5.600 1OooO

Figure 2.2. Atomic force microscopy of yeast cell surfaces. The micrographs show different morphological
aspects between commercial baking strains of S. cerevisiae. A, Fermipan yeast (Holland); B, ltaiquara yeast
(Brazil); C, Nishin Seifun Co. Yeast (Japan); D, Fleischman yeast (Brazil). The micrographs were kindly provided by
Dr Ricardo de Souza Pereira, Universidade Estadual Paulista, Sao Paulo, Brazil.

2.4 YEAST CELL ARCHITECTURE AND tures present: nucleus, mitochondria, Golgi appa-
FUNCTlON ratus, secretory vesicles, endoplasmic reticulum,
vacuoles and microbodies. Note that several
Subcellular compartmentalization in yeast typifies organelles derive from an extended intramem-
that of eukaryotic cells with the following struc- branous system and are not completely indepen-
18 YEAST PHYSIOLOGY AND BIOTECHNOLOGY

Table 2.3. Examples of methods for yeast organelle isolation.

Organelle References
~ ~~

Cell walls Catley (1988); Fleet (199 1)

Plasma membranes Rose and Veazey (1988); Panaretou and Piper (1996)
Plasma membrane vesicles Menendez et a/. (1995)
Mitochondria Rickwood et a/. (1988); Herrmann et a/. (1994); Glick and Pon (1995)
Nuclei Mann and Mecke (1982); Lohr (1988)
Nuclear envelopes Stambiode-Castilla et a/. (1995)
Spindle pole bodies Rout and Kilmartin (1994)
Rough ER membranes Sanderson and Meyer ( 1994)
ERderived vesicles Lupashin et a/. (1996)
Vacuoles Wiemken et a/. (1979)
Microsomes KiSppeli et a/. (1982)
Peroxisomes Distel et a/. (1996)
Golgi membranes Lupashin et a/. (1996)

Table 2.4. Marker enzymes for isolated yeast organelles.

Organelle/compartment Marker enzyme

Cell wall: periplasm lnvertase

secretory Acid phosphatase
Plasma membrane Vanadate-sensitive MgZ+-ATPase
Cytoplasm Glucose 6-phosphate dehydrogenase
Nucleus: nucleoplasm DNAdependent RNA polymerase
nuclear envelope TEM or 38 kDa protein antiserum
Endoplasmic light microsomal NADPH: cytochrome c oxidoreductase
reticulum: fraction
Vacuole: membrane u-Mannosidase
sap Proteases A and B carboxypeptidase Y
Golgi apparatus w l u c a n synthetase; mannosyltransferase
Mitochondrion: matrix Aconitase; fumarase
intermembrane space Cytochrome c peroxidase
inner membrane Cytochrome c oxidase
outer membrane Kynurenine hydroxylase
Peroxisome Catalase; isocitrate lyase; flavin oxidases

Adapted from Kreutzfeldt and Witt (1991) and Griffin (1994). who provide references for assaying the different marker enzymes.

dent from each other. The cytoplasm contains further capsular and fibrillar layer may also be
ribosomes and occasionally plasmids and the present in some yeasts.
structural organization of the intracellular milieu Figure 2.3(a) provides a diagrammatic view of
is maintained by a cytoskeleton. The cellular the ultrastructural features of an idealized yeast
contents are encased by an envelope comprising cell. Note that some yeasts (e.g. Candida albicans,
plasma membrane, periplasm and cell wall. A see Figure 2.3(b)) may not possess some of the
 YEAST CYTOLOGY 19

a cell wall is the principal feature which distin-

guishes a yeast cell from an animal cell.

2.4.1 The Cell Envelope

The yeast cell envelope is considered as the struc-

ture which surrounds and encases the yeast cyto-
plasm. That is, from the inside looking out: the
plasma membrane, the periplasmic space, the cell
wall and, in certain yeasts, the capsule and other
extracellular features. In S. cerevisiae, the cell
envelope occupies about 15% of the total cell
volume and plays a major role, as it does in all
yeasts, in controlling the osmotic and perme-
ability properties of the cell. Structural and func-
tional aspects of these cell envelope components
will now be described in turn, with reference
being made to their practical significance.

2.4.1. 1 The Plasma Membrane

Yeasts would not exist as unicellular entities
without plasma membranes. This is because these
structures represent the primary barriers for
passage of hydrophilic molecules and they there-
fore prevent yeast cytoplasmic contents mixing
freely with the aqueous environment. The follow-
ing briefly describes yeast plasma membrane
Figure 2.3. Yeast cell ultrastructural features. (a) Idea-
ultrastructure and discusses membrane functional
lized yeast cell. (b) Candida albicans cell. CW, cell aspects relating to yeast physiology and biotech-
wall; P, periplasm; CM, plasma membrane; CMI, inva- nology. More comprehensive coverage of the
gination; BS, birth scar; C, cytoplasm; N, nucleus; M, chemical composition, molecular anatomy and
mitochondrion; ER, endoplasmic reticulum; G, Golgi functional characteristics of yeast plasma
apparatus, S, secretory vesicles; V, vacuole; PER, per-
oxisome. The Candida a/bicans transmission electron
membranes, especially those of S. cerevisiae, is to
micrograph ( x 21 000) was kindly provided by Pro- be found in Henschke and Rose (1991), van der
fessor Masako Osumi, Japan Women's University, Rest et al. (1995) and Hofer (1997).
Tokyo. The S. cerevisiae plasma membrane is about
7.5 nm thick with occasional invaginations pro-
truding into the cytoplasm. Like other biological
structures indicated and also that some structures membranes, it can be described as a lipid bilayer
may only appear under certain specialized condi- interspersed with globular proteins which form a
tions of growth (e.g. peroxisomes). fluid mosaic. The lipid components comprise
Because yeast cells share many of the struc- mainly phospholipids (principally phosphati-
tural (and functional) features of higher eukary- dylcholine and phosphatidylethanolamine with
otes, yeasts have been considered models for minor proportions of phosphatidylinositol, phos-
eukaryotic cell science. Of course, the presence of phatidylserine and phosphatidyl-glycerol) and
20 YEAST PHYSIOLOGY AND BIOTECHNOLOGY

Figure 2.4. Classes of yeast membrane proteins. Adapted from van der Rest et a/. ( 1 995).

sterols (principally ergosterol and zymosterol the uptake of sugars, nitrogenous sources, ions,
with minor proportions of fecosterol and lanos- etc. is discussed in Chapter 3. Of prime impor-
terol). It is likely that the phospholipids confer tance in active transport of solutes into yeast
fluidity and the sterols rigidity to the membranes. cells is the activity of the plasma membrane
The protein components include those involved proton-pumping ATPase. This enzyme hydro-
in: solute transport (ATPase, permeases, chan- lyses ATP and generates an electrochemical
nels); cell wall biosynthesis (glucan and chitin proton gradient, which together with the mem-
synthases); transmembrane signal transduction brane potential, provides the driving force for
(adenylate cyclase, G-proteins) and cytoskeletal uptake of essential solutes.
anchoring. Figure 2.4 schematically depicts the Another very important physiological function
main classes of yeast plasma membrane proteins. of the yeast plasma membrane is in signal trans-
In addition, S. cerevisiue membranes contain duction of external stimuli to mediate (via second
ATP binding cassette (ABC) transporter proteins messengers) a number of internal biochemical
which are implicated in multidrug transport reactions. Henschke and Rose (1991) have dis-
(Kolaczkowski et ul., 1996). cussed the plasma membrane phosphoinositide
Yeast species differ with regard to their plasma second messenger system which operates in S.
membrane structural make-up and even different cerevisiue. Further aspects of signal transduction
strains of a single species can exhibit variations in in control of yeast growth and metabolic pro-
the membrane lipid composition. For example, cesses are discussed in Chapters 4 and 5, respec-
strains of brewing yeast (S. cerevisiue) possess a tively, and its role specifically in S. cerevisiue has
much higher phosphatidylcholine content been extensively reviewed (Uno, 1992; Thevelein,
(approx. 10-fold) compared with baking strains 1994; van Dam, 1996).
(Vendramin-Pintar et ul., 1995). The yeast plasma Other transport-related functions of the yeast
membrane should also not be regarded as a fixed, plasma membrane relate to exocytosis and endo-
static feature of a pure yeast strain. This is cytosis. In the former, secretory vesicles derived
because it changes both structurally and func- from the endoplasmic reticulum and Golgi appa-
tionally depending on the conditions of growth. ratus fuse with the plasma membrane to deliver
For example, lipid composition, particularly the proteins (e.g. mannoproteins) through the cell
unsaturated fatty acid constitution, can alter envelope (see section 2.44.). The vesicles in ques-
quite dramatically with changing growth rates, tion are also believed to contain constituents
temperature and oxygen availability. Such required for the biosynthesis and assembly of the
changes in lipid composition of the membrane cell envelope (Tschopp et ul., 1987). With regard
change, in turn, its functional properties - espe- to endocytosis, this is a system of internalizing
cially with regard to amino acid and sugar trans- and localizing certain molecules with the aid of
port (see Henschke and Rose, 1991). specialized membranous structures known as
The primary functions of yeast plasma mem- endosomes (Figure 2.5).
branes are to dictate what enters and what leaves In the first stages of endocytosis, plasma mem-
the cytoplasm. These selective permeability prop- brane invaginations pinch off to form vesicles
erties are mediated by specialized membrane which deliver their extracellular cargo to the
proteins. Their role in yeast nutrition, that is, in endosomes. Endocytosis in S. cerevisiae is known
 YEAST CYTOLOGY 21

fatty acids and sterols in their plasma mem-

branes. Thus, cell membranes enriched in linoleyl
(C18:2)fatty acid residues and with ergosterol (as
opposed to other sterols) exhibit a greatly
endosome enhanced tolerance to ethanol (Rose, 1993).
Oxygen availability in fermentations plays
1 a key role in dictating the fatty acid/sterol
make-up of brewing yeast membranes. This is
because oxygen is absolutely required for the
bte synthesis of unsaturated fatty acids and sterols.
In practical terms, this means that brewer’s yeast
requires initial oxygenation before fermentation
to ensure correct plasma membrane biosynthesis.
1 Failure to provide sufficient molecular oxygen
will render cell membranes more susceptible to
ethanol toxicity and this restricts yeast growth
and fermentative capability.

2.4.1.2 The Periplasm

Continuing in the inside-to-outside direction, the

next ‘structure’ encountered as a component
feature of the yeast cell enylope is the peri-
Figure 2.5. Schematic representation of endocytosis plasm. This is. a thin (35-45A), cell wall-asso-
in yeast cells. Adapted from Riezman (1993) who has
discussed the molecular events of the endocytic ciated region external to the plasma membrane
pathway in S. cerevisiae and internal to the wall. It was described by
Arnold (1991) as the periplasmic space, and by
Robinow and Johnson (199 1) as the never-never
land of the yeast cell.
to play an important role in the internalization of The periplasm mainly comprises secreted pro-
mating pheromones and their subsequent trans- teins (e.g. mannoproteins) which are unable to
port from the plasma membrane to the vacuole. permeate the cell wall. These include the glyco-
Endocytic pathways may also be involved in protein enzymes invertase and acid phosphatase
other aspects of yeast physiology, including stress which catalyse the hydrolysis of substrates that
responses and sporulation (Riezman, 1993). In do not cross the plasma membrane. Other
addition, Govindan and Novick (1995) have dis- enzymes which may be located in the periplasm
cussed the possible involvement of endocytosis in of certain yeasts are melibiase and trehalase
regulating budding patterns in S. cerevisiae. Hor- (Arnold, 1991).
azdovsky et al. (1995) have reviewed molecular The biotechnological significance of the yeast
biological aspects of endocytosis and intracellular periplasm is that invertase is an enzyme commer-
protein sorting in yeast cells. cially prepared from baker’s yeast following cell
The plasma membrane is especially significant wall autolysis or hydrolysis. Yeast invertase has
when considering the physiology of industrial applications in the confectionery industry where
yeasts. For example, the ability of brewing yeast it is used to hydrolyse crystalline sucrose to fruc-
strains to produce and tolerate ethanol is tose and glucose in the manufacture of soft-
intimately linked to the nature of unsaturated centred chocolates and candies.
22 YEAST PHYSIOLOGY AND BIOTECHNOLOGY

2.4. 1.3 The Cell Wall 1,2 and a-1,3 side chains. Chitin, a polymer of N-
acetylglucosamine, is present in small quantities in
The wall of yeast cells represents quite a thick S. cerevisiae (approx. 2-4%), mainly in bud scars.
(generally, 100-200 nm) structure comprising Some filamentous yeasts (e.g. C. albicuns) possess
between 15-25% of the total dry mass of the cell higher contents of chitin whereas other yeasts
and is a prominent distinguishing feature of all appear to lack chitin completely. Other compo-
yeasts. The composition, structure and function nents of yeast cell walls include variable propor-
of wall components in C. albicans and S. cerevi- tions of protein, lipids and inorganic phosphate.
siae have been reviewed, respectively, by Shep- The precise proportion of the chemical con-
herd (1987) and Fleet (1991). stituents of yeast cell walls will vary depending on
The main structural constituents of yeast cell yeast strain, cell age and growth conditions.
walls are polysaccharides, which account for 80- The precise molecular arrangements of the S.
90% of the wall. These are principally glucans cerevisiae wall are unresolved and several struc-
and mannans, with a minor proportion of chitin. tural models exist (e.g. Zlotnick et al., 1984;
Yeasts derive their strength from the glucan com- Stratford, 1994; Vukovic and Mrsa, 1995).
ponents of the cell wall which are partly arranged Nevertheless, the general consensus is that the
in a microfibrillar network. Both p-1,6- and /3-1,3- cell wall is a layered structure, the outermost
linked glucans are present, distinguished by their section of which comprises cross-linked manno-
solubility properties in acid and alkali. Mannans proteins (Figure 2.6). These are linked to each
are present as an u-1,6-linked inner core with a- other by hydrophobic interaction or by dis-

Figure 2.6. Composition and structure of the cell wall of Saccharomyces cerevisiae. The cell wall, which is
located outside the plasma membrane, consists of two layers. The inner layer provides cell wall strength, and is
made of &1,3- and B1.6glucan that is complexed with chitin. The outer layer consists of mannoproteins, and
determines most of the surface properties of the cell. The majority of mannoproteins are covalently linked to the
inner glucan layer. Periplasmic enzymes are trapped between the plasma membrane and the inner skeletal layer.
Reproduced with permission from Schreuder et a/. (1996a) and Elsevier Science Ltd.
 YEAST CYTOLOGY 23

ulphide bonds. They are also linked to the inner

fibrillar glucan network by covalent bonds. The
mannoproteins may be important in determining
the porosity of the yeast cell wall which is known
to exclude molecules greater than about 600 Da.
Some P-glucan of the inner layer is cross-linked
to chitin (Brown et ul., 1993). Chitin itself is a
major constituent of bud scars in budding yeasts
and different chitin synthase isozymes play key
roles in septum formation and morphogenesis
(Silverman, 1989). Chitin is also located in
smaller amounts throughout the cell wall of S.
cerevisiue where it serves various functions as: a
killer toxin receptor (Takita and Castilho-Valavi-
cius, 1993) and in the maintenance of osmotic
and morphological integrity (see Stratford, 1994).
Secreted proteins may interact with cell wall
polysaccharides (glucan) and Stratford (1994) has
discussed the structural role of a new class of cell
wall mannoproteins which anchor in the plasma
membrane, span the wall and protrude into the
medium. In S. cerevisiue, these proteins are
involved in flocculation and sexual aggregation.
Stratford (1994) has made the following inter-
esting analogy between yeast cell wall structure
and reinforced concrete: ‘Steel reinforcing rods
are represented by enmeshed alkali-insoluble
(1,3)-P-glucan fibrils, comprising some 35% of
the wall. The reinforcing is surrounded by con- Figure 2.7. Scanning electron micrographs showing
yeast bud and birth scars. (a) Saccharomyces cerevi-
crete, pebbles in a sand/cement matrix; secreted siae. showing scar tissue on the surface of S. cerevi-
mannoproteins represent pebbles, some 25-50% siae cells ( x 10000). BS, bud scar; BirS, birth scar.
of the wall, encased and bonded to the reinfor- Micrograph kindly provided by Professor Masako
cing fibrils by a matrix of amorphous P-glucan Osumi, Japan Women‘s University, Tokyo. (b) Zygo-
and chitin’. saccharomyces bailii. showing a bud scar on the
surface of 2.bailii NCYCI 766 cells ( x 55 000). Micro-
Bud scars are chitin-rich, convex, ringed pro- graph taken by Mark Kirkland and kindly provided by
trusions which remain on the mother cell surface Dr Malcolm Stratford, Unilever Research, Bedford, UK.
of budding yeasts following birth of daughter
cells (Figure 2.7).
The localization of bud sites on the cell wall of pombe, these fission scars or scar plugs are
S. cerevisiue is discussed in Chapter 4. Birth scars formed during cleavage of mother cells. During
are concave indentations which remain on the the next round of growth these scar plugs grow
daughter cell surface following budding. These out and a circular scar is retained between the
structures do not contain as much chitin as bud original cell and the new growth (see Chapter 4
scars and are less well visualized using fluorescent for a fuller description of fission in Sch. pombe).
dyes. With regard to the physiological function of
Fission yeasts similarly leave scar tissue on the yeast cell wall, it, should be stressed from the
their cell surfaces following division. In Sch. outset that the wall is not merely an inert exoske-
24 YEAST PHYSIOLOGY AND BIOTECHNOLOGY

leton whose only role is to protect the protoplast. cell wall assembly. Figure 2.8 shows cell wall
On the contrary, the yeast cell wall should be regeneration in spheroplasts of the fission yeast,
recognized as a living organelle whose functions Schizosacckaromyces pombe.
change during yeast growth and metabolism. In Inter- and intrageneric fusion of yeast spher-
S. cerevisiae, the availability of several cell wall oplasts is also an extremely valuable procedure
mutants have enabled physiological function for both basic genetic studies and for producing
studies to be undertaken. For example, mutants hybrids with biotechnological potential (see
have been isolated with defects in: killer toxin Mann and Jeffery, 1986; and Chapter 6).
reception, protein glycosylation, mannose bio- Several structural and functional aspects of the
synthesis, chitin deposition and glucan fibril yeast cell wall have practical implications in yeast
assembly. Such mutants have revealed that the biotechnology and in medicine (see Schreuder et
wall serves vital roles in yeast cell physiology al., 1996a,b; also Table 2.6). However, in addi-
(Table 2.5). tion to being a barrier for the yeast cell itself, the
In short, the yeast cell wall is a multifunctional wall is also a barrier for the yeast technologist.
organelle involved in cell protection, shape main- For example, in yeast recombinant DNA tech-
tenance, cellular interactions, reception, attach- nology, both the influx of foreign DNA and the
ment and specialized enzymatic activities (Fleet, efflux of expressed heterologous proteins are
1991). restricted by the presence of a thick cell wall.
Removal of the yeast cell wall with lytic There are several biochemical and genetic
enzymes in the presence of osmotic stabilizers approaches which can be employed to overcome
produces spheroplasts. The lytic enzymes in ques- this barrier. These include the use of lithium
tion are usually commercially available prepara- acetate to transform intact cells and the use of
tions such as ‘Helicase’ from snail digestive juice secretory signal sequences to direct protein
or ‘Zymolyase’, ‘Novozyme’ and ‘Lyticase’ from export. Several cell wall mutants of yeast may
microbial sources. Studies of cell wall regenera- also be of value in yeast biotechnology:
tion in yeast spheroplasts has provided valuable Fragile mutants are yeasts with defects in cell
information on the cytology and biochemistry of wall mannoprotein biosynthesis which are osmo-

Table 2.5. Physiological functions of the yeast cell wall.

Function Comments
~~

Physical protection As well as protecting the protoplast, the cell wall maintains the shape of yeast cells.
Osmotic stability Removal of cell walls results in protoplast lysis in the absence of osmotic stabilizers.
Permeability barriers Solutes larger than about 600 Da fail to permeate through the wall. The wall also
plays a role in controlling the entry of water into yeast.
Enzyme support Wall-softening enzymes (e.g. glucanases) and hydrolases (e.g. invertase) are
immobilized in the matrix of the cell wall.
Cation binding Several cations are known to be effectively sequestered by the cell wall, including
heavy metals.
Cellcell recognition Recognition sites for mating pheromones and killer toxins are located in the cell wall.
Cellcell adhesion Yeast-yeast sexual agglutination, flocculation and agglomeration are wall-related
phenomena. Yeast-human cell adhesion is relevant in pathogenicity of some species
(e.g. Candida albicans).
 YEAST CYTOLOGY 25

tically fragile. Such mutants have been described

in several species, including S. cerevisiae (Venkov
er al., 1974; Blagoeva er al., 1991) and Sch.
pombe (Belda and Zarate, 1996) and some have
been considered useful in both the uptake of
DNA (Venkov and Ivanov, 1982) and in the
recovery of recombinant proteins (Broker, 1994).
Autolytic mutants of S. cerevisiae, which are
thermosensitive cells with an osmotically unstable
cell wall (Cabib and DurPn, 1975), may be of
value in biotechnology to facilitate release of
intracellular proteins into the extracellular
medium (Alvarez er al., 1994) following tempera-
ture upshift.
Permeability mutants of S. cerevisiae, which
possess enhanced cellular permeability to macro-
molecules due to the presence of truncated man-
noprotein side chains in the cell wall, have been
shown to have practical potential in testing for
chemical mutagens in environmental samples
(Staleva et al., 1996).
The cell wall confers flocculant, buoyant or
adhesive properties on yeast which are of prac-
tical relevance to the fermentation industries and
also in medicine.
Flocculation is a phenomenon of particular
importance in brewing and physiological, bio-
chemical and genetic aspects of brewing yeast
flocculence have been widely discussed in the lit-
erature (e.g. Speers er al., 1992; Stratford, 1992;
Straver er al., 1993; Rose, 1993; Teunissen and
Steensma, 1995; Dengis and Rouxhet, 1997).
Yeast flocculation is the phenomenon of asexual
cellular aggregation when cells adhere, reversibly,
to one another to form macroscopic flocs which
sediment out of suspension. Historically, brewing
Figure 2.8. Cell wall regeneration in spheroplasts of yeast strains were distinguished as highly floccu-
Schizosaccharomyces pombe. (a) Transmission EM lant bottom yeasts (for lager fermentations) or
micrograph of a 3-h-reverting protoplast of Sch. pombe
( x 20000; bar = 1 km). Regeneration of new cell wall
weakly flocculant or buoyant top yeasts (for ale
b l u c a n from the cell surface with polarity is demon- fermentations). However, brewing strains of S.
strated (F). (b) Scanning EM micrograph of a 5-h-revert- cerevisiae are quite heterogeneous with respect to
ing protoplast of Sch. pombe ( x 18 0 0 0 bar = 1 km). flocculation and individual strains may be classed
The protoplast is covered with a network of Pglucan into several flocculence types based on their sedi-
microfibrils. The micrographs were kindly provided by
Professor Masako Osumi Japan Women's University,
mentary and flotation characteristics (Hough er
Tokyo. Further information on the formation of 'the al., 1982). These distinctions are primarily reflec-
glucan network in fission yeast can be found in Osumi tions of differences in cell wall composition
eta1 ( 1 995). between yeast strains. Yeast flocculation can be
26 YEAST PHYSIOLOGY AND BIOTECHNOLOGY

Table 2.6. Biotechnological and medical significance of the yeast cell wall.

Practical significance Comments/References

~ ~~~ ~~

Biosorption of metals Yeast cell walls have uses in bioremediation of heavy metals (e.g. Cd, Cu, Ag, Zn)
in industrial wastewaters (Volesky et al., 1993; Blackwell er al., 1995; Simmons
eta/., 1995).
Antigenicity properties May be exploited in yeast taxonomy and serological diagnosis of pathogenic
yeasts.
Yeast 'glycan' Non-nutritive food stabilizer (Hay, 1993).
Heterologous protein Hepatitis B virus surface antigens have been expressed in the outer mannoprotein
binding of S. cerevisiae which may have potential as a live oral vaccine (Schreuder et el.,
1996b).
Cell adhesion Flocculation and agglomeration of S. cerevisiae is important during fermentation
(see text), while adhesion of C.a/bicans is medically important (Calderone and
Braun, 1991).
Protein secretion barrier Fragile and other mutants overcome this (see text).
Killer toxin reception Killer yeasts have several applications in biotechnology (see Chapter 4, Table
4.33).
Immunornodulation, Wall components such as (1,3)+glucans may act as biological response modifiers
anti-tumour agent and anti-tumour agents (Bohn and BeMiller, 1995).

readily quantified (Soares and Mota, 1997) and Although far from being completely under-
in brewing fermentations, it is now possible to stood, it is now widely recognized that yeast floc-
measure on-line the intensity of yeast flocculation culation conforms to the lectin-like hypothesis
for process control purposes (Podgornik et al., originally proposed by Miki et al. (1982). Thus,
1997). interactions occur between calcium-activated
In general, the flocculation of brewing yeast is lectins (sugar-binding proteins) and a-mannan
associated with the onset of stationary phase, but receptors on neighbouring yeast cell walls (Figure
the timing and degree of flocculation is not pre- 2.9).
cisely regulated. This is an important considera- Cell wall lectins in yeast may be the FLO gene-
tion in brewing practice because if flocculation encoded flocculins which are surface glycopro-
occurs too early, fermentation will cease prema- teins capable of directly binding mannoproteins
turely, leaving residual sugar in the wort. This of adjacent cells (Teunissen and Steensma, 1995).
can cause microbiological stability problems and Cell surface hydrophobicity of yeast cell walls
can adversely affect the flavour characteristics of has also been implicated in playing a distinct or
the final product. Conversely, if it takes an complementary role with lectins to mediate cell-
unduly long time for cells to flocculate, this can cell flocculation (Straver et al., 1993). Brewing
cause downstream processing problems in beer yeast hydrophobicity may be readily assayed
clarification. For these and other reasons, the using a novel magnetic separation procedure
biochemical mechanisms and genetic basis of (Straver and Kijne, 1996). Smart and her co-
brewing yeast flocculation have been intensively workers (Smart et al., 1995) have studied cell
studied over the years. Flocculation also occurs surface hydrophobicity of brewing yeast strains
in other yeasts and has been investigated in Kluy- in relation to physiological stress and their influ-
veromyces, Zygosaccharomyces, Pichia, Candida ences on flocculation. Research has suggested
and Schizosaccharomyces species (see Stratford, that cell surface charge, rather than hydro-
1994 for references). phobicity, is the primary determinant of cell floc-
 YEAST CYTOLOGY 27

in influencing flocculation and flotation proper-

ties of brewer’s yeast, but also in adhesion of
yeasts. Yeast cells can adhere to:

0 inert surfaces (e.g. walls of bioreactors, sur-

faces of medical implants);
p mannose rrceptors on ceU wall
0 soft tissues (e.g. human epithelial cells); and
< mannmpecflc lrctin proteins (flocculins) 0 water-immiscible carbon sources (e.g. hydro-
carbon globules).
Figure 2.9. Lectin hypothesis for flocculation of yeast
cells. Surface proteins with lectin properties specifi- There is particular fundamental and practical
cally bind to mannose residues in the wall of neigh- interest in the hydrophobic nature of the surface
bouring cells. Calcium ions are required to maintain
the lectins in active conformations. Yeast flocs will be
of Candid0 albicans cells, due to the medical sig-
progressively built up by simultaneous inter-binding of nificance of this yeast as the causative agent of
many cells. human and animal mycoses (collectively referred
to as candidosis). The ability of C. albicans to
adhere to and colonize host cells and surgical
devices is an important determinant in the patho-
culence in brewing yeasts (Wilcocks and Smart, genicity of this yeast (Calderone and Braun,
1995). 1991). The hydrophobic adherence properties of
Yeast flocculation is genetically determined C. albicans have been studied using spectroscopic
and different flocculation phenotypes have been techniques by Hobden et al. (1995).
identified based on their sensitivities to sugar True flocculation in yeast cells is reversible and
inhibition and proteolytic enzymes (Stratford and can be distinguished from chain formation or
Assinder, 1991). These phenotypes, named Flo aggregation by the ability of the chelating agent
and NewFlo, also possess different sensitivities to EDTA to disperse flocs, but not chains or aggre-
yeast growth conditions, most notably culture pH gates. The phenomenon of agglomeration is
(Stratford, 1996), temperature (Soares et al., different from flocculation and may be regarded
1994) and glucose availability (Soares and Mota, as an extensive, non-reversible cell aggregation
1996). Lo and Dranginis (1996) have identified a process and is particularly relevant in baking
novel flocculin-encoding gene, FLO11, in S. cere- strains of S. cerevisiae. Some baker’s yeast strains
visiae which exhibited homology to the STA have a tendency to agglomerate into macroscopic
genes which encode secreted glucoamylase. The aggregates which do not resuspend when cells are
product of STAl was implicated in Ca-dependent mixed with water. The appearance of granular
flocculation activity. yeast material is often referred to by bakers as
The converse of flocculation in yeasts is flota- ‘grit’ or ‘sand’. Grittiness is detrimental to
tion which is associated with cell wall hydro- baker’s yeast quality since it results in inadequate
phobicity. The flotation characteristic relevant in mixing into bread dough leading to limited lea-
brewing is the ability of yeasts to trap CO2 vening ability. The formation of yeast grit is
bubbles in a fermenting liquid and form a yeast strain-dependent but is also influenced by the
‘head’ at the top of fermentation vessels. This ionic strength of the suspending solvent (Guinard
behaviour is typical of top-fermenting yeasts and Lewis, 1993). Therefore, although agglom-
employed in traditional ale breweries. Note that eration (or grit formation) is distinct from floccu-
unlike flocculant cells, S. cerevisiae strains prone lation, both phenomena are influenced by a
to flotation do not form cell-cell aggregates (see variety of genetic and physiological factors and
Chapter 4 and Figure 4.25). both are cell wall-mediated processes. The
Cell wall hydrophobicity is important not only mechanism of yeast agglomeration is unknown.
28 YEAST PHYSIOLOGY AND BIOTECHNOLOGY

Guinard and Lewis (1993) found no significant with that of vegetative cell walls of yeasts. Griffin
difference between gritty and non-gritty yeasts in (1994) has discussed physiological, biochemical
terms of cell wall mannoproteins, surface hydro- and genetic aspects of sporulation in S. cerevi-
phobicity or flocculence. Phosphorus and lipid siue. With respect to external signals involved in
concentrations did appear to be higher in non- sporulation, Suizu et ul. (1995) found an eleva-
gritty cells and Guinard and Lewis (1993) have tion in intracellular Ca2+ ions during spore for-
proposed a model for yeast agglomeration based mation and have discussed the possible
on Ca2+-activated protein-mannan binding regulatory role of Ca2 -dependent signalling for
+

between adjoining cells which is facilitated by both meiosis and sporulation in S. cerevisiue.
reduced phosphorus and lipid concentrations on The genetics of S. cerevisiue meiosis and spor-
gritty yeast cell walls. ulation have been discussed, respectively, by
Malone (1990) and Dawes (1983). Several genes
of both the mating type control pathway and the
2.4.1.4 The Yeast Spore Wall nutritional control pathway are involved in reg-
ulating meiosis and sporulation in S. cerevisiue
The development of spores by yeast represents a (e.g. Kawaguchi et ul., 1992).
process of morphological, physiological and
biochemical differentiation of sexually reproduc-
tive cells. Sporulation involves meiosis and asco- 2.4.1.5 Fimbriae
pore development in cells and in S. cerevisiue can
be initiated by depriving diploid cells of nitrogen Fimbriae are long (variable from 0.1-10 pm),
and providing acetate as a respiratory carbon thin (5.7 nm diameter), proteinaceous protrusions
source. The cytology of meiosis, ascospore emanating from the surface of several basidiomy-
formation and spore germination in fission and cetous and ascomycetous yeast species (Gardiner
budding yeasts has been discussed by Robinow et ul., 1982). They appear to be mainly involved
and Johnson (1991) and by Williamson (1991). in cell-cell interactions before sexual conjuga-
Meiotic division in S. cerevisiue produces four- tion. However, fimbrial protein is also present
lobed nuclei from which four spores are formed. during vegetative growth in some yeasts which
The spindle pole body (SPB) in each nuclear lobe plays a different role from conjugal fimbriae. For
polarizes the meiotic spindles and forms thick- example, short fimbriae in S. cerevisiue are impli-
ened plaques in the outer nuclear membrane cated in flocculation. Smart et nl. (1995) have
which serve as the beginnings of spore wall for- illustrated fimbriae-like ‘hairy’ protrusions from
mation. Similar modifications of the outer mem- the surface of flocculant cells of brewing yeast.
brane plaque of meiotic SPBs occurs in Sch. By comparison, non-flocculant cells appeared
pombe (Hirata and Shimoda, 1994). These outer smooth.
plaques in lobed yeast nuclei initiate the develop-
ment of spore walls which eventually grow round
to envelope the lobes of the mitotic nucleus and 2.4. 1.6 Capsules
to delimit spores.
After meiosis is completed, yeast spore walls Golubev (1991) has reviewed the structure and
develop within the ascus from a structure known function of yeast capsules. These slimy extra-
as the forespore membrane. Robinow and mural layers are regarded as yeast orgunelles due
Johnson (1991) have discussed the biogenesis and to their distinctive structural and functional char-
elaboration of multilayered spore walls in S. cer- acteristics. Capsules are prevalent in basidiomy-
evisiue, Sch. pombe and other spore-forming cetous yeasts such as Cryptococctis, Rhodotorula
yeasts. Generally speaking, however, knowledge and Sporobolomyces species where they may
of spore wall synthesis is lacking in comparison serve in protecting cells from physical and biolo-
 YEAST CYTOLOGY 29

gical stresses encountered in their natural habi- she, the mean cytosolic pH of exponential-
tats. Golubev (1991) has discussed the sugges- phase cells suspended in water was calculated at
tions that yeast capsules act as buffers to prevent 5.25 (Cimprich et al., 1995).
water loss from cells and to enhance acquisition Freely-suspended yeast ribosomes, as opposed
of trace levels of nutrients in oligotrophic envir- to endoplasmic reticulum-associated or mito-
onments. These views are supported by the fact chondrial ribosomes, consist of large 60s and
that capsule-forming yeasts predominate in poor small 40s ribonucleoprotein subunits (as in other
habitats such as polar soils. eukaryotic cells) and exist as single ribosomes
Most biochemical and physiological informa- and as multiple mRNA-linked aggregates called
tion on yeast capsules has come from studies polysomes (see Lee, 1991). The latter are the sites
with the pathogenic yeast, Cryptococcus neofor- of protein biosynthesis. Unlike membrane-delim-
mans. Capsules in this yeast may be important ited organelles, cytoplasmic ribosomes are syn-
virulence determinants. Other yeasts, for thesized de novo (Warner, 1989; Planta et al.,
example, C . laurentii and Hansenula capsulata, 1995).
produce extracellular polysaccharide materials Lipid particles (or ‘sphaerosomes’) function as
exhibiting plastic rheological characteristics storage vesicles which may serve as lipid reser-
which have potential biotechnological applica- voirs for yeast membrane biosynthesis (Clausen
tions (Slodki and Cadmus, 1978; Sutherland and et al., 1974). They contain mainly sterol esters,
Elwood, 1979). Growth conditions will greatly but not triglycerides, and small amounts of phos-
influence the amount and type of capsule pro- pholipid, protein and unsaturated free fatty acids.
duced by yeasts. Nevertheless, Phaff et al. (1978) The content of the latter are known to increase
have noted that the principal extracellular/cap- in baker’s yeast at the end of the growth phase in
sular compounds produced by yeasts are: phos- batch culture.
phomannans (e.g. in Hansenula, Pichia and Microbodies in yeast cells include peroxisomes
Pachysolen spp.); P-linked mannans (e.g. Rhodo- and glyoxysomes which are single membrane-
torula spp.); heteropolysaccharides (e.g. Crypto- delimited organelles distinct from endoplasmic
coccus, Lipomyces, Candida and Trichosporon reticulum-derived vesicles. Characteristic features
spp.) and sphingolipid-type compounds (e.g. of microbodies are: the presence of catalase,
Hansenula spp.). the occasional presence of crystalloids in their
matrix and their osmotic fragility (Carson and
Cooney, 1990; Veenhuis and Harder, 1991).
2.4.2 The Cytoplasm and Cytoskeleton The appearance of these organelles is largely
determined by yeast growth conditions, in
The yeast cytoplasm is an aqueous acidic particular, the sources of available carbon and
colloidal fluid containing low and intermediate nitrogen.
molecular weight compounds, dissolved Peroxisomes perform a variety of metabolic
proteins, glycogen and other soluble macro- functions in eukaryotic cells. In yeasts, peroxi-
molecules. Also suspended in the cytosol are somes are ubiquitous organelles which contain, in
membrane-delimited microbodies and macro- addition to catalase, several oxidases which are
molecular aggregations such as ribosomes, involved in the oxidative utilization of specific
proteasomes and lipid particles. The cytoskeletal carbon and nitrogen sources. For example,
network providing structural organization to the Hansenula polymorpha and Pichia pastoris can
yeast cytoplasm comprises microtubules and utilize methanol using peroxisomal enzymes such
microfilaments. The cytosolic (non-organellar) as alcohol oxidase; Yarrowia lipolytica and
enzymes of yeasts include glycolytic enzymes, Candida tropicalis can utilize n-alkanes using
the fatty acid synthetase complex and the several oxidases and Candida utilis can utilize
enzymes of protein biosynthesis. In S. cerevi- D-alanine and uric acid using, respectively,
30 YEAST PHYSIOLOGY AND BIOTECHNOLOGY

peroxisomal D-alanine oxidase and uricase. When

methylotrophic yeasts such as H.polymorpha are
transferred from a glucose-containing medium to
one containing methanol as sole carbon and
energy source, there is a rapid increase in the size
and crystalline ultrastructure of peroxisomes
(Figure 2.10).
The organelles develop from the small
peroxisomes originally present in glucose-grown
cells mainly as a result of rapid synthesis of
enzymes such as alcohol oxidase and catalase. A
more detailed discussion of peroxisome bio-
genesis has been provided by Waterham and
Cregg (1997). Several gene (PEX) products
which are required for peroxisome biogenesis
have now been identified in several yeasts. Mole-
cular biological work on yeast pex mutants is
relevant to our understanding of Zellweger
syndrome, a congenital cerebro-hepato-renal dis-
order in humans which is characterized by an
absence or deficiency of peroxisomes in cells
from Zellweger patients (Waterham and Cregg,
1997).
In methanol-limited chemostats, H. polymorpha
peroxisomes exhibit a completely crystalline sub-
structure and the organelles can reach a cell
volume fraction as high as 80%. In addition to
offering an ideal system in which to study the
biochemistry and genetics of organelle biogenesis
and function (Titorenko et al., 1993; Sudbery,
1994), peroxisome-producing yeasts also have
biotechnological applications - for example, in
the use of alcohol oxidase-promoted gene expres-
sion and intracellular packaging of heterologous
proteins. The gene for this peroxisomal enzyme is
one of the most powerful promoters known and
has stimulated great interest in the use of methy-
lotrophic yeasts such as H. polymorpha and P .
Figure 2.10. Yeast peroxisomes. (a) Candida fropicalis. pastoris in recombinant DNA technology (see
The micrograph shows the ultrastructure of a C. fropi-
calis protoplast of n-alkaneqrown cells. N, nucleus; P, Chapter 6).
peroxisomes; M, mitochondria; CM, cell membrane. Glyoxysomes in yeast contain, in addition to
Bar = 1 pm. Micrograph kindly provided by Professor catalase, enzymes of the glyoxylate cycle (see
Masako Osumi, Japan Women's University, Tokyo. (b) Chapter 5 ) and amine metabolism. The orga-
Hansenula polymorpha. The micrograph shows the
ultrastructure of H. polymorpha cells grown in the pre
nelles become especially prominent in ethanol-
sence of methanol. Bar= 1 pm. Kindly provided by Dr grown cells of H. polymorplia and C . utilis.
Marten Veenhuis, University of Groningen, The Neth- Veenhuis and Harder (1991) have discussed the
erlands. possibility that glyoxysomes and peroxisomes
 YEAST CYTOLOGY 31

develop from each other in response to specific including mitosis and meiosis, organelle motility,
growth conditions and may not in fact be dis- and septation (see Heath, 1995). For example,
tinct organelles. The term glyoxy-peroxisome has the actin cytoskeleton in S. cerevisiae is inti-
been suggested to describe these yeast micro- mately involved in bud-site selection (Yang et al.,
bodies. 1997), and in Sch. pombe, microtubules play a
Proteasomes are large multisubunit protease key role in polarized growth and fission (see
enzyme complexes found in the cytoplasm and Chapter 4). The role of microtubules in govern-
nucleoplasm of yeast cells with no apparent ing the motility and positioning of mitochondria
association with intracellular structures. In S. in Sch. pombe has been studied by Yaffe et al.
cerevisiae, 20s and 26s proteasomes exist (1996). Cytoplasmic microtubules appear directly
whose function in regulating protein levels is apposed to the SPBs (Spindle Pole Bodies) on the
essential for cell viability. The 26s particle is nuclear envelope. Their role in maintaining cell
assembled from the 20s units which are cylind- shape of yeasts is evident when, for example, Sch.
rical particles composed of four stacked rings. pombe cells are treated with inhibitors of micro-
The 26s proteasome acts as an ATP-dependent tubule biogenesis (e.g. thiabendazole) they
protease in the ubiquitin pathway which is develop abnormal filamentous cell morphologies
responsible for the rapid degradation of short- (Walker, 1982).
lived, abnormal proteins that are detrimental
to the cell. Specific yeast physiological func-
tions in which proteasomes are involved 2.4.3 The Nucleus and Extrachromosomal
include cell cycle control, signal transduction, Elements
mating and adaptive stress responses (reviewed
by Hilt and Wolf, 1995). These multifunctional Comprehensive information on structural and
roles of proteasomes have led Hilt and Wolf functional aspects of yeast nuclei and extra-
(1996) to consider these complexes as ‘func- chromosomal elements is available from recent
tionally sophisticated counterparts of the ribo- published literature. For example, detailed
some’. coverage has been provided on: the nucleus by
The cytoskeleton of yeast cells comprises Williamson (199 1); karyogamy (nuclear fusion)
microtubules and microfilaments. These are by Rose (1996); nuclear pore complexes by
dynamic structures which perform mechanical Bucci and Wente (1997); genome structure by
work in the cell through assembly and dis- Kaback (1995); chromosome replication by
assembly of individual protein subunits. Thus, a Theis and Newlon (1996); chromatin by Perez-
and tubulin monomers polymerize as hetero- Ortin et al. (1989); histones by Ushinsky et al.
dimers into 25 nm-thick microtubules, while G- (1997); the nucleolus by Lkger-Silvestre et al.
actin globular monomers polymerize into 7 nm- (1997), centromeres and kinetochores by Hyman
thick double-stranded filaments of F-actin. and Sorger (1995) and Lechner and Ortiz (1996);
Cytoskeletal structures are assembled in an telomeres by Louis (1995) and Zakian (1996);
energy-dependent manner and the process is spindles by McDonald et al. (1996); spindle pole
regulated by an extensive system of associated bodies by Kahana et al. (1995) and Sobel
structural proteins and enzymes (Dustin, 1978; (1997); 2 pm plasmids by Wickner (1995); linear
Ayscough and Drubin, 1996). Ayscough and DNA plasmids by Fukuhara (1995); RNA
Drubin (1996) have discussed the value of S. cer- viruses by Wickner (1996); prions by Tuite and
evisiae in molecular biological studies of the Lindquist (1996) and Wickner et al. (1996) and
structure and function of the eukaryotic actin retrotransposons by Kingsman and Kingsman
cytoskeleton. (1988a). The following represents a very brief
Yeast microtubules and microfilaments are account of the structure and function of the
involved in several aspects of yeast physiology yeast nucleus and genetic material.
 Next Page

32 YEAST PHYSIOLOGY AND BIOTECHNOLOGY

2.4.3.1 The Nucleus The nucleolar organizer in the nucleolus con-

tains ribosomal DNA repeats and is the site of
The nucleus in yeasts is a round-lobate organelle rRNA transcription and processing. It is also
of around 1.5 pm diameter which is located in involved in pre-mRNA processing and in the
the centre of the cell or excentrically. The assembly of ribosomal subunits.
nucleoplasm is separated from the cytoplasm by The nucleoplasm contains DNA, RNA, basic
a double membrane containing pores 50-100 nm proteins (protamines and histones) and non-
in diameter. Figure 2.11 shows a diagrammatic histone proteins. Some extrachromosomal ele-
representation of the yeast nucleus. The precise ments (e.g. 2 pm circles) may also be present.
size and number of nuclear membrane pores in The condensed basic nucleoprotein material, con-
yeast cells varies with growth conditions and sisting of double-helical DNA-histone complexes,
with phases of t.he cell cycle. Bucci and Wente is chromatin which is organized in structures
(1997) have studied the in vivo assembly dynam- called chromosomes. During S phase (DNA syn-
ics of nuclear pore complexes in S. cerevisiae. In thetic phase) of the cell cycle, each chromosome
S. cerevisiae, nuclear membranes are occasionally is replicated and during M phase (mitosis) the
contiguous with and have similar chemical duplicated chromosomes are segregated to
composition to the endoplasmic reticulum daughter cells. Asexual mitotic nuclear division in
(Zinser et al., 1991). Unlike most eukaryotic yeasts occurs within the confines of intact nuclear
cells, the yeast nuclear membrane does not break membranes, unlike mammalian cells in which the
down during mitosis. Within the nucleus there is nuclear membrane breaks down during mitosis.
a dense crescent-shaped region corresponding to Detailed cytological descriptions of the stages of
the nucleolus which disappears during mitosis mitosis (prophase, metaphase, anaphase and
and reappears in interphase. telophase) and of meiosis in various yeast species
are provided by Kockovh-Kratochvilova (1990),
Williamson (1991) and Robinow and Johnson
SFJB DCT (1991). The regulation of meiosis in budding and
fission yeasts has been discussed by Malone
(1990) and Yamamoto (1996), respectively.
CT Several general statements can be made about
yeast chromosomes:
P
Genome sizes are relatively constant and
generally range from 10-15 Mb and encode
between 5000 and 10000 genes.
Haploid chromosome numbers are variable.
For example, from 2 (Guilliernzondella selenos-
CHR CYT pora) to 16 ( S . cerevisiae). Most yeasts have
fewer, larger chromosomes compared with S.
Figure 2.1 1. Diagram of the yeast nucleus. The figure
is a diagrammatic cut-away view of a (budding) yeast cerevisiae.
nucleus in the late S or early G2 phase of the cell Individual yeast chromosomes vary in size by
cycle. The spindle is complete, but still 'short', and the more than an order of magnitude from
nucleus has not yet started to move into the bud. For approx. 0.2 to 6 Mb.
clarity only a few microtubules are shown. SPB,
spindle pole body; DCT, discontinuous microtubules;
CT, continuous microtubules; CYT, cytoplasmic micro- Yeast chromosomes can be analysed by pulse-
tubules; NUC, nucleolus; CHR, chromatin; P, pores. field electrophoretic techniques. Johnston (1994)
Reproduced with permission from Williamson ( 199 1 ) has described electrophoretic karyotyping methods
and Academic Press. for Succhuromyces and Zimmerman and Fournier