Yeast E-Book
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- Herausgeber: Wiley-VCH
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Finally, a stand-alone, all-inclusive textbook on yeast biology.
Based on the feedback resulting from his highly successful monograph, Horst Feldmann has totally rewritten he contents to produce a comprehensive, student-friendly textbook on the topic. The scope has been widened, with almost double the content so as to include all aspects of yeast biology, from genetics via cell biology right up to biotechnology applications. The cell and molecular biology sections have been vastly expanded, while information on other yeast species has been added, with contributions from additional authors. Naturally, the illustrations are in full color throughout, and the book is backed by a complimentary website.
The resulting textbook caters to the needs of an increasing number of students in biomedical research, cell and molecular biology, microbiology and biotechnology who end up using yeast as an important tool or model organism.
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Seitenzahl: 1448
Veröffentlichungsjahr: 2012
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Contents
Cover
Related Titles
Title Page
Copyright
Preface
Authors
Chapter 1: Introduction
1.1 Historical Aspects
1.2 Yeast as a Eukaryotic Model System
Further Reading
Chapter 2: Yeast Cell Architecture and Functions
2.1 General Morphology
2.2 Cell Envelope
2.3 Cytoplasm and Cytoskeleton
2.4 Yeast Nucleus
2.5 Organellar Compartments
Further Reading
Chapter 3: Yeast Metabolism
3.1 Metabolic Pathways and Energy
3.2 Catabolism of Hexose Carbon Sources
3.3 Gluconeogenesis and Carbohydrate Biosynthesis
3.4 Fatty Acid and Lipid Metabolism
3.5 Nitrogen Metabolism
3.6 Nucleotide Metabolism
3.7 Phosphorus and Sulfur Metabolism
3.8 Vitamins and Cofactors
3.9 Transition Metals
Further Reading
Chapter 4: Yeast Molecular Techniques
4.1 Handling of Yeast Cells
4.2 Genetic Engineering and Reverse Genetics
4.3 More Genetic Tools from Yeast Cells
4.4 Techniques in Yeast Genome Analyses
Further Reading
Chapter 5: Yeast Genetic Structures and Functions
5.1 Yeast Chromosome Structure and Function
5.2 Yeast tRNAs, Genes, and Processing
5.3 Yeast Ribosomes: Components, Genes, and Maturation
5.4 Messenger RNAs
5.5 Extrachromosomal Elements
5.6 Yeast Mitochondrial Genome
Further Reading
Chapter 6: Gene Families Involved in Cellular Dynamics
6.1 ATP- and GTP-Binding Proteins
6.2 Regulatory ATPases: AAA and AAA+ Proteins
6.3 Protein Modification by Proteins and Programmed Protein Degradation
6.4 Yeast Protein Kinases and Phosphatases
6.5 Yeast Helicase Families
Further Reading
Chapter 7: Yeast Growth and the Yeast Cell Cycle
7.1 Modes of Propagation
7.2 Cell Cycle
7.3 Meiosis
Further Reading
Chapter 8: Yeast Transport
8.1 Intracellular Protein Sorting and Transport
8.2 Nuclear Traffic
8.3 Membrane Transporters in Yeast
Further Reading
Chapter 9: Yeast Gene Expression
9.1 Transcription and Transcription Factors
9.2 RNA Polymerases and Cofactors
9.3 Transcription and its Regulation
9.4 DNA Repair Connected to Transcription
9.5 Coupling Transcription to Pre-mRNA Processing
9.6 Yeast Translation Apparatus
9.7 Protein Splicing – Yeast Inteins
Further Reading
Chapter 10: Molecular Signaling Cascades and Gene Regulation
10.1 Ras–cAMP Signaling Pathway
10.2 MAP Kinase Pathways
10.3 General Control by Gene Repression
10.4 Gene Regulation by Nutrients
10.5 Stress Responses in Yeast
Further Reading
Chapter 11: Yeast Organellar Biogenesis and Function
11.1 Mitochondria
11.2 Peroxisomes
Further Reading
Chapter 12: Yeast Genome and Postgenomic Projects
12.1 Yeast Genome Sequencing Project
12.2 Yeast Functional Genomics
12.3 Yeast Systems Biology
12.4 Yeast Synthetic Biology
Further Reading
Chapter 13: Disease Genes in Yeast
13.1 General Aspects
13.2 Trinucleotide Repeats and Neurodegenerative Diseases
13.3 Aging and Age-Related Disorders
13.4 Mitochondrial Diseases
Further Reading
Chapter 14: Yeasts in Biotechnology
14.1 Introduction
14.2 Yeasts: Natural and Engineered Abilities
14.3 Biopharmaceuticals from Healthcare Industries
14.4 Biomedical Research
14.5 Environmental Technologies: Cell Surface Display
14.6 Physiological Basis for Process Design
Further Reading
Chapter 15: Hemiascomycetous Yeasts
15.1 Selection of Model Genomes for the Génolevures and Other Sequencing Projects
15.2 Ecology, Metabolic Specificities, and Scientific Interest of Selected Species
15.3 Differences in Architectural Features and Genetic Outfit
15.4 Molecular Evolution of Functions
Further Reading
Chapter 16: Yeast Evolutionary Genomics
16.1 Specificities of Yeast Populations and Species, and their Evolutionary Consequences
16.2 Gene Duplication Mechanisms and their Evolutionary Consequences
16.3 Other Mechanisms of Gene Formation and Acquisition of Novel Functions
Further Reading
Chapter 17: Epilog: The Future of Yeast Research
Appendix A: References
Appendix B: Glossary of Genetic and Taxonomic Nomenclature
Appendix C: Online Resources useful in Yeast Research
Appendix D: Selected Abbreviations
Index
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Preface
For the Second Edition
Until some 20 years back, there was no need to write a book on yeast molecular and cellular biology: the field was covered by “standard monographs” such as Broach, J.N., Pringle, J.R., and Jones, E.W. (eds) (1991) The Molecular and Cellular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY., and Guthrie, C. and Fink, G. (eds) (1991) Guide to Yeast Genetics and Molecular Biology, Academic Press, San Diego, CA. Unfortunately, these editions were not updated, so that any novel information after the Yeast Genome Sequencing Project had succeeded in 1996 was scarcely available in a comprehensive form.
When I discussed this drawback with my colleagues during the first years of the “postgenome” era, it was André Goffeau who suggested to me that we should at minimum publish a paper documenting the outstanding contributions that had involved Saccharomyces cerevisiae as a model system for eukaryotic molecular and cell biology for over half a century. Finally, however, my engagement in this subject ended in preparing a small volume describing all those achievements.
I had started working with yeast in 1962, so that I still retain reminiscences of things happening in the past 50 years. Over the years, I had kept a collection of papers documenting the achievements in various fields of yeast research. I also gained a lot of information from the weekly seminars that were arranged in the departments where I worked, and from lectures and courses that I had a chance to present. For teaching purposes, I kept a huge collection of tables and figures that I personally had designed. I gratefully remember the many fruitful discussions with my colleagues from all over the world – at congresses or privately – that helped broaden my background.
Unfortunately, the brochure, entitled “Contribution of Yeast to Molecular Biology: A Historical Review,” did not raise the interest of a publisher, by using the argument “... history does not sell ...” Nonetheless, they became interested in the subject itself after I had converted it into a “modern” textbook (which still might retain notes on historical background), because such an item was absolutely missing on the market. Thus, the first edition of Yeast: Molecular and Cell Biology appeared in November 2009.
The necessity to update and publicize information on yeast was recently raised in an article (“Yeast: an experimental organism for 21st century biology”) by our American colleagues (Botstein and Fink, 2011). In the November 2011 issue of Genetics, the Genetics Society of America launched its YeastBook series – a comprehensive compendium of reviews that presents the current state of knowledge of the molecular biology, cellular biology, and genetics of S. cerevisiae.
This second edition of Yeast: Molecular and Cell Biology was started more than a year ago, and is aimed at presenting all aspects of modern yeast molecular and cellular biology, starting from the “early” discoveries and trying to cover the most recent developments in all relevant topics. The reader will find included chapters that reach out to yeast species other than S. cerevisiae, which have turned out (i) as interesting objects for large-scale genome comparisons, (ii) as ideal organisms to follow genomic evolution, and (iii) as appropriate “cell factories” in biotechnology. I think this will fulfill all of the requirements of a textbook for students and researchers interested in yeast biology.
I have tried to document the developments by including more than 3000 references. Whenever possible, these references are selected such that the reader can follow achievements made over the past decades to the present (in addition, a number of individual chapters include a list of references for recommended “Further reading”). Undoubtedly, this collection will not completely mirror the engagement of the numerous yeast laboratories. Wherever possible, I have cited original papers, but in many cases I have had to rely on review articles contributed during these years by competent researchers. Therefore, I apologize to all colleagues who might be disappointed that their original work has not been quoted adequately.
Foremost, I again wish to thank André Goffeau and Jean-Luc Souciet, who supported me in preparing this book. I am indebted to Danilo Porro and Paola Branduardi (Univerity of Milan Biococca), Claude Gaillardin (INRA, Thiverval-Grignon), and Bernard Dujon (Institut Pasteur and Institut Pasteur and University P. & M. Curie, Paris) for their excellent contributions of Chapters 14, 15 and 16, respectively. Not to forget the nice contacts with so many colleagues I found during the Yeast Genome Sequencing Project and the Génolevures Project; I am grateful for their suggestions and encouragement.
With great pleasure, I wish to acknowledge the care of the team of Wiley-Blackwell publishers at Weinheim (Germany) in editing and manufacturing this book: Dr Gregor Cicchetti (Senior Commissioning Editor, Life Sciences), who kindly invited me to consider a second edition with a considerable extension of the contents, and Dr Andreas Sendtko (Senior Project Editor) and his colleagues who took over production. Many thanks for their excellent and accurate handling of my manuscript and the pictures, so that I had little trouble with corrections.
Finally, but most importantly, I wholeheartedly thank my wife Hildegard for her patience and encouragement, who for many years found me toiling over my computer at home.
Horst FeldmannBergkirchenJune 2012
Authors
Paola BranduardiUniversity of Milano BicoccaDepartment of Biotechnology and BiosciencesPiazza della Scienza 220126 MilanItaly
Bernard DujonInstitut Pasteur and University P. & M. CurieDepartment of Genomes and Genetics25–28, Rue du Docteur Roux75724 Paris Cedex 15France
Horst FeldmannLudwig-Thoma-Strasse 22B85232 BergkirchenGermany
Claude GaillardinINRAAgroTechParisAvenue Lucien Brétignières, BP 0178850 Thiverval GrignonFrance
Danilo PorroUniversity of Milano BicoccaDepartment of Biotechnology and BiosciencesPiazza della Scienza 220126 MilanItaly
1
Introduction
1.1 Historical Aspects
In everyday language, yeast is synonymous for Saccharomyces cerevisiae – a name given to a yeast strain discovered in malt in 1837 (Meyen) – in connection with making beer. This notion immediately calls to mind that yeast probably is the oldest domesticated organism – it was used for beer brewing already in Sumeria and Babylonia around 6000 BC. In parallel, S. cerevisiae strains were employed in wine production in Georgia and for dough leavening in old Egypt. In Egypt, beer was a common refreshment, and gifts of beer were awarded to civil servants and workers for extraordinary services. The scientific name “Saccharomyces” is derived from a word meaning “sugar fungus” in Greek, while the root for cerevisiae stems from Ceres, the Roman God of the crops.
The French word for yeast, levure, goes back to Latin levare, and so is leaven, simultaneously used for dough and yeast as an organism able to anaerobically release carbon dioxide during the baking process. The English word yeast, like Dutch guist, or even the German Hefe, is derived from a west-Germanic expression, haf-jon, meaning the potential to leaven. The provenance of the words used for beer in western European languages (French “bière,” German “Bier,” and Italian “birra”) is not known, but in Roman languages, the expressions used for beer are directly related to the organism (cerevisiae), most obvious in the Spanish “cerveza” or in the Portuguese “cerveja.” The Greek zymi (ζυµι) is used simultaneously for yeast and dough, and occurs as a root in words related to beer or fermentation. Thus, the modern expression “enzymes” (en zymi = in yeast), originally coined by Kühne in 1877, designates the compounds derived from yeast that are able to ferment sugar.
We owe the description of the microscopic appearance of yeasts in 1680 to Antoni van Leeuwenhoek in Leiden, who also observed bacteria and other small organisms for the first time. The observation that yeast budding is associated with alcoholic fermentation dates back to Cagnaird-Latour in 1835. In his work carried out during his tenure at Strasbourg University, Louis Pasteur correlated fermentation with yeast metabolism (1857). Pasteur's famous “Études sur la bière” appeared in 1876. Sometime later, two technical applications were based on this notion. In the late 1880s, E. Buchner and H. Buchner used cell-free fermentation to produce alcohol and carbon dioxide, and in 1915, Karl Neuberg used “steered” yeast fermentations to produce glycerol (unfortunately as a convenient source to convert it into trinitroglycerol). The knowledge of yeast physiology, sexuality, and phylogeny was later reviewed in a book by A. Guilliermond (Guilliermond, 1920).
In the 1950s, when yeast research entered a novel era of biochemistry, researchers became aware that many useful compounds could be isolated from yeast cells. Among the first companies to produce biochemicals from yeast (nonengineered at that time and obtained from a local Bavarian brewery) for the biochemical and clinical laboratory was Boehringer Mannheim GmbH in Tutzing (Germany). In a “semi”-industrial procedure, a variety of compounds were manufactured and commercialized, dominated by the coenzyme nicotinamide adenine dinucleotide (NAD). In many enzymatic tests (also called optical tests), NAD was an obligatory ingredient, because the increase of NADH generated from NAD by an appropriate enzymatic reaction (or coupled reaction) could be used to follow the timecourse of that reaction by spectrophotometry. This was, for the time being, also a helpful technique to determine enzyme levels or metabolites in the clinical laboratory. The methodology had been collected by Hans Ulrich Bergmeyer, a representative of Boehringer Company, who edited a famous compendium (16 volumes) of Methods in Enzymatic Analysis (Wiley & Sons).
1.2 Yeast as a Eukaryotic Model System
The unique properties of the yeast, S. cerevisiae, among some 1500 yeast species (a subgroup from 700 000 different fungi, which still may expand to over 3000 different yeast species) and its enormous “hidden potential” that has been exploited for many thousands of years made it a suitable organism for research. In fact, yeast was introduced as an experimental organism in the mid-1930s by Hershel Roman (Roman, 1981) and has since received increasing attention. Many researchers realized that yeast is an ideal system in which cell architecture and fundamental cellular mechanisms can be successfully investigated.
Among all eukaryotic model organisms, S. cerevisiae combines several advantages. It is a unicellular organism that, unlike more complex eukaryotes, can be grown on defined media, giving the investigator complete control over environmental parameters. Yeast is tractable to classical genetic techniques. Both meiotic and mitotic approaches have been developed to map yeast genes (e.g., Mortimer and Schild, 1991). The first genetic map of S. cerevisiae was published by Lindegren in 1949 (Lindegren, 1949).
The life cycle of S. cerevisiae (Figure 1.1) normally alternates between diplophase and haplophase. Both ploidies can exist as stable cultures. In heterothallic strains, haploid cells are of two mating-types, a and α. Mating of a and α cells results iin a/α diploids that are unable to mate, but can undergo meiosis. The four haploid products derived from meiosis of a diploid cell are contained within the wall of the mother cell (the ascus). Digestion of the ascus and separation of the spores by micromanipulation yields the four haploid meiotic products. Analysis of the segregation patterns of different heterozygous markers among the four spores constitutes the “tetrad analysis” and reveals the linkage between two genes (or between a gene and its centromere). It was mainly Mortimer and his colleagues who undertook the considerable task of collecting and editing all of the genetic data accumulating in diverse laboratories (Mortimer and Hawthorne, 1966), up to the point when genetic maps could be replaced by physical maps. Prior to the start of the Yeast Genome Sequencing Project in 1989 (cf. Chapter 12), some 1200 genes had been mapped to the 16 yeast chromosomes, most of them attributable to particular gene functions and others to particular phenotypes only.
Figure 1.1 Life cycle of S. cerevisiae. Vegetative growth is indicated by the circles.
During molecular biology's infancy, around the late 1950s, yeast became a convenient organism to be used for the mass preparation of biological material in sufficient quantity or the mass production of other biological compounds. Yeast has a generation time of around 80 min and mass production of cells is easy. Simple procedures for the isolation of high-molecular-weight DNA, ribosomal DNA, mRNA, and tRNA were at hand. It was possible to isolate intact nuclei or cell organelles such as intact mitochondria (maintaining respiratory competence). Eventually, yeast also gained a leading position in basic molecular research. The possibility to apply genetics and molecular methods to an organism at the same time made yeast such a successful a model system. It was the technical breakthrough of yeast transformation (Beggs, 1978; Hinnen, Hicks, and Fink, 1978) that could be used in reverse genetics and for the characterization of many yeast genes that essentially fostered the enormous growth of yeast molecular biology.
The elegance of yeast genetics and the ease of manipulation of yeast substantially contributed to the fact that functions in yeast were studied in great detail using biochemical approaches. A large variety of protocols for genetic manipulation in yeast became available (e.g., Campbell and Duffus, 1988; Guthrie and Fink, 1991; Johnston, 1994). High-efficiency transformation of yeast cells was achieved, for example, by the lithium acetate procedure (Ito et al., 1983) or by electroporation. A large variety of vectors have been designed to introduce and to maintain or express recombinant DNA in yeast cells (e.g., Guthrie and Fink, 1991; Johnston, 1994). The ease of gene disruptions and single-step gene replacements is unique in S. cerevisiae, and offered an outstanding advantage for experimentation. Further, a large number of yeast strains carrying auxotrophic markers, drug resistance markers, or defined mutations became available. Culture collections are maintained, for example, at the Yeast Genetic Stock Center (YGSC) and the American Type Culture Collection (ATCC).
The wealth of information on metabolic pathways and the characterization of the enzymes involved in biochemical processes, such as carbon, nitrogen, or fatty acid metabolism, as well as the underlying regulatory circuits and signal transduction mechanisms (e.g., roles of cAMP, inositol phosphates, and protein kinases), has been gathered by numerous yeast researchers. For cytology, studies on yeast contributed to the knowledge of mechanisms in mitosis and meiosis, biogenesis of organelles (such as endosomes, Golgi apparatus, vacuoles, mitochondria, peroxisomes, or nuclear structures), as well as cytoskeletal structure and function. Major contributions came from investigations into nucleic acid and genome structure, protein traffic and secretory pathways, mating-type switching phenomena, mechanisms of recombination, control of the cell cycle, control of gene expression and the involvement of chromatin structure, functions of oncogenes, or stress phenomena. There is too little space here to describe all the achievements made through “classical” approaches and the reader is referred to detailed collections of articles in standard books (Strathern, Hicks, and Herskowitz, 1981; Broach, Pringle, and Jones, 1991; Guthrie and Fink, 1991).
The success of yeast as a model organism is also due to the fact, which was not fully anticipated earlier than some 20 years ago (Figure 1.2), that many basic biological structures and processes have been conserved from yeast to mammals and that corresponding genes can often complement each other. In fact, a large variety of examples provide evidence that substantial cellular functions are also highly conserved from yeast to mammals.
Figure 1.2 Yeast around the start of the Yeast Genome Sequencing Project.
It is not surprising, therefore, that in those years yeast had again reached the forefront in experimental molecular biology. When the sequence of the entire yeast genome became amenable to thorough analysis, the wealth of information obtained in this project (Goffeau et al., 1996; Goffeau et al., 1997) turned out to be useful as a reference against which sequences of human, animal, or plant genes and those of a multitude of unicellular organisms under study could be compared. Moreover, the ease of genetic manipulation in yeast still opens the possibility to functionally dissect gene products from other eukaryotes in this system.
As it is extremely difficult to follow the contributions of yeast to molecular biology in a strictly chronological sequence in toto, I prefer to select particular fields of interest in which the yeast system has served to arrive at fundamental observations valid for molecular and cell biology in general.
Further Reading
Goffeau, A., Barrell, B.G., Bussey, H. et al. (1996) Life with 6000 genes. Science, 274, 546, 563–567 (review).
Hartwell, L.H. (2002) Yeast and cancer. Nobel Lecture Bioscience Reports, 22, 373–394. http://nobelprize.org/nobel_prizes/medicine/laureates/2001/hartwell-lecture.html.
2
Yeast Cell Architecture and Functions
2.1 General Morphology
Cell structure and appearance. Yeast cells exhibit great diversity with respect to cell size, shape, and color. Even individual cells from a pure strain of a single species can display morphological heterogeneity. Additionally, profound alterations in individual cell morphology will be induced by changing the physical or chemical conditions at growth. Yeast cell size varies widely – some yeasts may be only 2–3 µm in length, while other species may reach lengths of 20–50 µm. Cell width is less variable at about 1–10 µm. Under a microscope, Saccharomyces cerevisiae cells appear as ovoid or ellipsoidal structures, surrounded by a rather thick cell wall (Figure 2.1). Mean values for the large diameter range between 5 and 10 µm, and for the small diameter between 1 and 7 µm. Cell size in brewing strains is usually bigger than that in laboratory strains. Mean cell size of S. cerevisiae also increases with age.
Figure 2.1 Cells of S. cerevisiae under the microscope. The white arrows point to dividing cells.
With regard to cell shape, many yeast species are ellipsoidal or ovoid. Some, like the Schizosaccharomyces, are cylindrical with hemispherical ends. Candida albicans and Yarrowia lipolytica, for example, are mostly filamentous (with pseudohyphae and septate hyphae). There are also spherical yeasts (like Debaryomyces species) or elongated forms (with many yeasts depending on growth conditions).
In principle, the status of S. cerevisiae as a eukaryotic cell is reflected by the fact that similar macromolecular constituents are assembled into the structural components of the cell (Table 2.1). There are, however, some compounds that do not occur in mammalian cells or in cells of other higher eukaryotes, such as those building the rigid cell wall or storage compounds in yeast.
Table 2.1 Classes of macromolecules in S. cerevisiae.
ClassCategoryMajor compoundsProteinsstructuralactin, tubulin (cytoskeleton)histones (H2A, H2B, H3, H4, H1)ribosomal proteinshormonespheromones α and afunctionalenzymes and factorstransporterssignaling receptorsmotor proteins (myosins, kinesins, dynein)Glycoproteinscell wall componentsmannoproteinsenzymesmany functional enzymes (e.g., invertase)Polysaccharidescell wall componentsglucan, mannan, chitincapsular componentsglucan, mannan, chitinstorageglycogen, trehalosePolyphosphatesstoragepolyphosphate in vacuoleLipidsstructuralfree sterols in membranesstoragelipid particles (sterol esters and triglycerides)functionalphosphoglyceride derivatives, free fatty acidsNucleic acidsDNAgenomic DNA (80%), mitochondrial DNA (10–20%)RNArRNA (80%), mRNA (5% cytosolic, ER, mitochondria), tRNAs, snRNAs, snoRNAsFor a better understanding of what I will discuss in the following sections, Figure 2.2 presents a micrograph of a dividing yeast cell, indicating some of its major components and organelles. We will deal with the yeast envelope, the cytoplasm, and the cell skeleton, and briefly touch upon the nucleus. The major genetic material distributed throughout the 16 chromosomes residing within the nucleus and other genetic elements, such as the nucleic acids, the retrotransposons, and some extrachromosomal elements, are considered later in Chapter 5. Section 2.5 presents an overview of other yeast cellular structures.
Figure 2.2 Micrograph of a dividing yeast cell.
Preparations to view cells. Unstained yeast cells can only be visualized poorly by light microscopy. At 1000-fold magnification, it may be possible to see the yeast vacuole and cytosolic inclusion bodies. By using phase-contrast microscopy, together with appropriate staining techniques, several cellular structures become distinguishable. Fluorochromic dyes (cf.Table 2.2) can be used with fluorescence microscopy to highlight features within the cells as well as on the cell surface (Pringle et al., 1991).
Table 2.2 Some structure-specific dyes for yeast cells.
DyeStructures visualizedCommentsMethylene bluewhole cellsnonviable cells stain blueAminoacridinecell wallsindicator of surface potentialF-C ConAcell wallsbinds specifically to mannanCalcofluor whitebud scarschitin in scar fluorescesDAPInucleiDNA fluorescesDAPImitochondriamitochondria fluoresce pink-whiteNeutral redvacuolesvacuoles stain red-purpleIodineglycogen depositsglycogen stained red-brownRhodaminemitochondriaDAPI, 4,6-diamidino-2-phenylindole.The range of cellular features visualized is greatly increased, when monospecific antibodies raised against structural proteins are coupled to fluorescent dyes, such as fluorescein isothiocyanate (FITC) or Rhodamine B.
Flow cytometry has several applications in yeast studies (Davey and Kell, 1996). For example, fluorescence-activated cell sorting (FACS) can monitor yeast cell cycle progression, when cell walls are labeled with concanavalin A conjugated to FITC and cell protein with tetramethylrhodamine isothiocyanate (TRITC). These tags enable us to collect quantitative information on the growth properties of individual yeast cells as they progress through their cell cycle.
A very convenient tool to localize and even to follow the movement of particular proteins within yeast cells is the use of the Green Fluorescent Protein (GFP) from the jellyfish (Aequorea victoria) as a reporter molecule (Prasher et al., 1992), as well as several derivatives of GFP with fluorescence spectra shifted to other wavelengths (Heim et al., 1994; Heim, Cubitt, and Tsien, 1995). Fusions of genes of interest with the fluorescent protein gene (N- or C-terminal) also allow us to follow the expression and destiny of the fusion proteins followed by fluorescence microscopy (Niedenthal et al., 1996; Wach et al., 1997; Hoepfner et al., 2000; see also Chapter 4).
Organelle ultrastructure and macromolecular architecture can only be obtained with the aid of electron microscopy, which in scanning procedures is useful for studying cell topology, while ultrathin sections are essential in transmission electron microscopy to visualize intracellular fine structure (Streiblova, 1988). Atomic force microscopy can be applied to uncoated, unfixed cells for imaging the cell surfaces of different yeast strains or of cells under different growth conditions (De Souza Pereira et al., 1996).
A most convenient method to mark specific cellular structures or compartments is to check for particular marker enzymes that occur in those structures (Table 2.3).
Table 2.3 Marker enzymes for isolated yeast organelles.
OrganelleCompartmentsMarker enzymeCell wallperiplasminvertasesecretory pathwayacid phosphatasePlasma membranevanadate-sensitive ATPaseCytosolglucose-6-phosphate dehydrogenaseNucleusnucleoplasmRNA polymerasenuclear envelopetransmission electron microscopyERlight microsomalfractionNADPH: cytochrome coxidoreductaseVacuolemembraneα-mannosidasesapprotease A and BGolgiapparatusβ-glucan synthase,mannosyltransferaseMitochondrionmatrixaconitase, fumaraseintermembranespacecytochrome c peroxidaseinner membranecytochrome c oxidaseouter membranekynurenine hydroxylasePeroxisomecatalase, isocitrate lyase,flavin oxidase2.2 Cell Envelope
In S. cerevisiae, the cell envelope occupies about 15% of the total cell volume and plays a major role in controlling the osmotic and permeability properties of the cell. Looking from the inside out, the yeast cytosol is surrounded by the plasma membrane, the periplasmic space, and the cell wall. Structural and functional aspects of the yeast cell envelope have attracted early interest (Phaff, 1963) because – like the cell envelope of fungi in general – it differs from bacterial envelopes and from those of mammalian cells. A peculiarity of yeast is that once the cell has been depleted of its cell wall, protoplasts are generated that are able to completely regenerate the wall (Necas, 1971).
2.2.1 Cell Wall
Yeast cell wall. The outer shell is a rigid structure about 100–200 nm thick and constituting about 25% of the total dry mass of the cell (Figure 2.3). The cell wall is composed of only four classes of macromolecules: highly glycosylated glycoproteins (“mannoproteins”), two types of β-glucans, and chitin. The composition of the cell wall is subject to considerable variation according to growth conditions, and the biosynthesis of the single compounds is highly controlled both in space and in time. The literature that has accumulated on these issues has grown so voluminous that reference is given here to only a few review articles (Klis, 1994; Lipke and Ovalle, 1998; Cabib et al., 2001). Details of cell wall synthesis during yeast growth and budding, as well as septum formation (Cid et al., 1995; Cabib et al., 1997; Cabib et al., 2001; Smits, van denEnde, and Klis, 2001), are considered below.
Figure 2.3 Schematic representation of the yeast cell wall.
By treatment with lytic enzymes in the presence of osmotic stabilizers, the yeast cell wall can be removed without harming viability or other cellular functions. These “naked” cells are called spheroplasts. The cell wall will regenerate and this process has been used to study aspects of cell wall biosynthesis. Spheroplasts are amenable to intergeneric and intrageneric cell fusions; such hybrids are valuable instruments in genetic studies and possess a valuable biotechnological potential. A cell wall protein that contains a putative glycosylphosphatidylinositol (GPI)-attachment site, Pst1p, is secreted by regenerating protoplasts. It is upregulated by activation of the cell integrity pathway, as mediated by Rlm1p, as well as upregulated by cell wall damage via disruption of the FKS1 gene, representing the catalytic subunit of glucan synthase (cf. Chapter 3).
Yeast cell aggregation. A phenomenon of particular importance in brewing is flocculation. It is based on asexual cellular aggregation when cells adhere, reversibly, to one another, which leads to the formation of macroscopic flocs sedimenting out of suspension. Traditionally, brewing yeast strains are distinguished as highly flocculent bottom yeasts (used for lager or Pilsner fermentations) or weakly flocculent top yeasts (used for ale fermentations or, in Germany, to prepare “top-fermented” beers). Although flocculation is far from being completely understood, it appears that the phenomenon is due to specific cell wall lectins in yeast (so-called flocculins) – surface glycoproteins capable of directly binding mannoproteins of adjacent cells. Yeast flocculation is genetically determined by the presence of different FLO genes. One such protein is Flo1p, a lectin-like cell-surface protein that aggregates cells into “flocs” by binding to mannose sugar chains on the surfaces of other cells. Both the phenotypic characterization of FLO5 strains and the sequence similarity between Flo1p and Flo5p suggest that Flo5p is also a mannose-binding lectin-like cell surface protein.
As the yeast cell wall is involved in sexual agglutination, some attention has been given to this particular aspect (Lipke and Kurjan, 1992). a- and α-cells can be distinguished by their agglutinin proteins. The anchorage subunit of a-agglutinin, Aga1p, is a highly O-glycosylated protein with an N-terminal secretion signal and a C-terminal signal for the addition of a GPI anchor (cf. Section 3.4.3.2). Linked to the anchoring subunit by two disulfide bonds is the adhesion subunit Aga2p. The α-agglutinin of α-cells is Sag1p. It binds to Aga1p during agglutination; its N-terminus is homologous to members of the immunoglobulin superfamily, containing binding sites for a-agglutinin, while the C-terminus is highly glycosylated and harbors GPI anchor sites.
The cell wall as a target for the defeat of mycoses. Similarly, several peculiarities of fungal cell wall synthesis such as the occurrence of ergosterol have led to the development of strategies for their inhibition as a means to defeat severe mycoses (Gozalbo et al., 1993). A more recent brief account is given in an article by Levin (2005) describing cell wall integrity regulation in S. cerevisiae, which is considered a good model for the development of safe and effective antifungal agents. At present, effective antifungal therapy is very limited and dominated by the azole class of ergosterol biosynthesis inhibitors. Members of this class of antifungals are cytostatic rather than cytotoxic and therefore require long therapeutic regimens. The antifungal drugs can be applied to the major opportunistic human pathogens (Candida species, Aspergillus fumigatus, and Cryptococcus neoformans) causing systemic infections among immunocompromised patients. As this population has grown over the past three decades due to HIV infection, cancer chemotherapy, and organ transplants, and the number of life-threatening systemic fungal infections has increased accordingly, there is a need to develop safe, cytotoxic antifungal drugs (cf. Chapter 14).
2.2.2 Plasma Membrane
Like other biological membranes, the surface plasma membrane of yeast can be described as a lipid bilayer, which harbors proteins serving as cytoskeletal anchors, and enzymes for cell wall synthesis, signal transduction, and transport. The S. cerevisiae plasma membrane is about 7.5 nm thick, with occasional invaginations protruding into the cytoplasm. The lipid components comprise mainly phospholipids (phosphatidylcholine, phosphatidylethanolamine, and minor proportions of phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol) as well as sterols (principally ergosterol and zymosterol). Like the cell wall, the plasma membrane changes both structurally and functionally depending on the conditions of growth.
The primary functions of the yeast plasma membrane are:
A comprehensive coverage of the lipids and the yeast plasma membrane, as well as on the biogenesis of the cell wall, can be found in a book by Dickinson and Schweitzer (2004).
The periplasmic space (Arnold, 1991) is a thin (35–45 Å), cell wall-associated region external to the plasma membrane. It comprises mainly secreted proteins that are unable to permeate the cell wall, such as invertase and phosphatase, which catabolize substrates that do not cross the plasma membrane. The unique properties of invertase have inspired its commercial preparation for the confectionary industry. The signal sequences of invertase (SUC2) and phosphatase (PHO5) have been used in recombinant DNA technology to generate heterologous proteins that can be secreted (Hadfield et al., 1993). Most frequently used for secretion of heterologous proteins is the prepro-α-factor (MFα1) (Brake, 1989) (cf. Section 4.2.2.3).
2.3 Cytoplasm and Cytoskeleton
2.3.1 Yeast Cytoplasm
Like in all other cellular organisms, the yeast cytoplasm is the site for many cellular activities and the space for intracellular traffic. In yeast, it is an aqueous, slightly acidic (pH 5.2) colloidal fluid that contains low- and intermediate-molecular-weight weight compounds, such as proteins, glycogen, and other soluble macromolecules. Larger macromolecular entities like ribosomes, proteasomes, or lipid particles are suspended in the cytoplasm. The cytosolic (nonorganellar) enzymes include the glycolytic enzymes, the fatty acid synthase complex, and the components and enzymes for protein biosynthesis. Many functions essential for cellular integrity are localized to the cytoplasm (e.g., the components that form and control the cytoskeletal scaffold).
2.3.2 Yeast Cytoskeleton
The cytoskeleton of yeast cells, most intensely and successfully studied from early on by D. Botstein's and J. Pringle's groups, comprises microtubules and microfilaments (Botstein, 1986; Schatz et al., 1986; Huffacker, Hoyt, and Botstein, 1987). These are dynamic structures that perform mechanical work in the cell through assembly and disassembly of individual protein subunits. Yeast microtubules and microfilaments are involved in several aspects of yeast physiology, including mitosis and meiosis, organelle motility, and septation. It is noteworthy that the skeleton in yeast cells exhibits a marked asymmetry, which becomes evident in the way it divides during vegetative growth (cf. Section 7.1.1).
2.3.2.1 Microtubules
Microtubules are conserved cytoskeletal elements. They are formed by polymerization of polymerization-competent α- and β-tubulin heterodimers (Figure 2.4). Yeast cells are unusual among other eukaryotes in that they possess very few cytoplasmic microtubules, thus explaining that most aspects of cell polarity largely reside in the actin skeleton (Pruyne and Bretscher, 2000; Schott, Huffaker, and Bretscher, 2002).
Figure 2.4 Yeast microtubules and actin filaments (not to scale). Note that the actin monomers are differently colored only for better visualization.
Yeast has two α-tubulins, Tub1p and Tub3p, and one β-tubulin, Tub2p. During biogenesis, the tubulins are protected by a specific chaperonin ring complex, CCT, which contains several subunits, Cct2p–Cct8p. (Note that the CCT complex is also needed in actin assembly.) Competence means that α-tubulin and β-tubulin need be properly folded, a reaction that requires specific cofactors for the folding of α- and β-tubulin (Alf1p/cofactor B for α-tubulin; Cin1p/cofactor D and Cin2p/cofactor C for β-tubulin). Homologs of these cofactors have been found in numerous organisms. An effector in this heterodimer formation is Pac2p (cofactor E) that binds to α-tubulin. One of the players in tubulin formation is Cin4p, a small GTPase in the ADP ribosylation factor (ARF) subfamily (cf. Section 6.1.2); it genetically interacts with several of the yeast tubulin cofactors, such as Pac2p, Cin1p, and Cin2p (the GTPase-activating protein (GAP) for Cin4p). As it appears (from analogy with the human homolog, Arl2), Cin4p is involved in regulating the yeast activity of the postchaperonin tubulin folding pathway, in part by decreasing the affinity of Cin1p/cofactor D for native tubulin. Yeast CIN4 was isolated in a genetic screen for mutants displaying supersensitivity to benomyl, a microtubule-depolymerizing drug; it was independently isolated in a genetic screen for elevated chromosome loss. Δcin4 mutants are cold-sensitive, show synthetic phenotypes in combination with tubulin mutants, and have defects in nuclear migration and nuclear fusion. Rbl2p, the homolog of mammalian cofactor A, participates in the morphogenesis of tubulin in that it protects the cell from excess of free β-tubulin, which would be lethal as it leads to disassembly of tubulin.
Tub4p, the γ-tubulin, is a conserved component of microtubule organizing centers (MTOCs) and is essential for microtubule nucleation in the spindle pole bodies (SPBs). Tub4p localizes to both nuclear (inner plaque) and cytoplasmic (outer plaque) faces of the SPB, and is essential for nucleating microtubules from both faces (see Section 7.1.1).
2.3.2.2 Actin Structures
Actin-based transport. Unlike animal cells, which rely primarily on microtubule-based transport to establish and maintain cell polarity, yeast cells utilize actin-based transport along cables to direct polarized cell growth and to segregate organelles prior to cell division. In budding, actin cable assembly is initiated from the bud, leading to reorientation of actin cables, and thus targeting of growth and secretion to the future bud tip (cf. Section 7.1). Polarized growth towards the bud tip (or cap) continues through a medium-budded stage, and depends on actin cables emanating from the bud tip and neck. These cables serve as polarized tracks for type V myosin-dependent delivery of cargos needed to build the daughter cell.
Types of actin filaments. Actin is an ATP-binding protein that exists both in monomeric (G-actin) and filamentous (F-actin) forms. Actin is encoded in yeast by the single gene ACT1 (Ng and Abelson, 1980). Actin filaments are assembled by the reversible polymerization of monomers and have an intrinsic polarity; the fast-growing end is called the barbed end and the slow-growing end is called the pointed end (Figure 2.4). Yeast cells contain three types of filamentous actin structures: (i) actin cables, (ii) an actin-myosin contractile ring (Bi et al., 1998), and (iii) actin cortical patches, all of which are subjected to extensive reorganization throughout the cell cycle. Actin cables serve as tracks for polarized secretion, organelle and mRNA transport, and mitotic spindle alignment. The actin–myosin contractile ring forms transiently at the mother–daughter neck and is important for cytokinesis. Cortical patches are branched actin filaments involved in endocytosis and membrane growth and polarity. Genetic screens and biochemical purifications have been fruitful in identifying numerous factors that regulate actin cytoskeleton dynamics, organization, and function (review: Moseley and Goode, 2006).
Assembly of actin filaments. The S. cerevisiae genome en-codes two genes, BNI1 and BNR1, that are members of the formin family assembling linear actin cables in the bud and bud neck, respectively. Formins constitute a well-conserved family of proteins that promote the assembly of actin filaments, which are necessary in remodeling of the actin cytoskeleton during such processes as budding, mating, cytokinesis, or endocytosis (and in higher cells, cell adhesion and migration). The formin proteins are characterized by the presence of two highly conserved FH (formin homology) domains: the FH1 domain, containing polyproline motifs that mediate binding to profilin (actin- and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-binding protein, Pfy1p), which in turn binds actin monomers, and the FH2 domain, which nucleates actin assembly. The FH2 domains of Bni1p and Bnr1p are distinct from those of the metazoan groups, containing a yeast-specific insert that is not found in other organisms. In addition to FH1 and FH2 domains, formins contain a regulatory Rho-binding domain (RBD) and a Dia-autoregulatory domain (DAD).
A model for formin-mediated actin assembly has suggested the following sequence of events. Activated Rho protein binds to the formin RBD domain and releases the formin from a conformation in which it is autoinhibited (due to an interaction between its N- and C-termini) to adopt a conformation that exposes the FH1 and FH2 domains. The FH1 domain then interacts with profilin-bound actin monomers, handing them over to the FH2 domain, a dimeric structure that may interact with two actin monomers to stabilize a dimeric actin form, prior to polymerization, whereby actin cables are formed. The FH2 domain remains associated with the growing end of the filament to protect it from interaction with capping proteins (a FH2 function termed “processive capping”).
Consistent with this model, Bni1p has been identified as a downstream target of Rho1p, which regulates reorganization of the actin cytoskeleton, and hence the process of bud formation (cf. Section 7.1.1). Additionally, Bni1p activation is regulated by the small GTPases Rho3p and Rho4p, which affect the inhibitory interaction between the RBD and the DAD domains in the formin, while the Rho protein Cdc42p is needed for proper cable assembly during initiation of bud growth. Bni1p autoinhibition (as mentioned before) can also be aborted by phosphorylation of Bni1p affected by Prk1p kinase. Support for the model also comes from crystal structure studies of the Bni1p FH2 domain complexed with actin.
Actin filament assembly. Long actin filament bundles are formed by Crn1p (coronin) (Rybakin and Clemen, 2005), which binds actin filaments (F-actin) and cross-links them. Crn1p also regulates the actin filament nucleation and the formation of branched actin filaments as found in cortical patches. Crn1p is composed of five N-terminal WD repeats, forming a β-propeller structure, a microtubule binding domain, and a C-terminal α-helical coiled-coil structure, whereby the β-propeller and coiled-coil domains are required for recruitment of Crn1p to cortical patches.
The highly conserved actin nucleation center required for the motility and integrity of actin patches, involved in endocytosis and membrane growth, is the Arp2/3 complex. In yeast, the complex consists of seven proteins, two of which (Arp2p and Arp3p) are actin-related, while five components (Arc15p, Arc18p, Arc19p, Arc35p, and Arc40p) are non-actin-related proteins (Winter et al., 1997; Evangelista et al., 2002). The Arp2/3 complex nucleates the formation of branched actin filaments by binding to the side of an existing (mother) filament and nucleating the formation of a new (daughter) actin filament at a 70̊ angle (Figure 2.4). Arp2p and Arp3p serve as the first two subunits of the daughter filament, likely mimicking actin monomers due to their structural similarity to actin. However, the Arp2/3 complex does not play a role in the formation of actin cables (unbranched actin structures). To achieve optimal actin nucleation activity, the Arp2/3 complex is assisted by an assembly protein, such as Las17p (also Bee1p, of the SCAR/WASP family), myosin I, Abp1p (Olazabal and Machesky, 2001), or Pan1p.
Las17p/Bee1p as an activator of the Arp2/3 protein complex is the only S. cerevisiae homolog of the human Wiskott–Aldrich syndrome protein (WASP), which itself is a member of the larger WASP/SCAR/WAVE protein family. Las17p was identified biochemically as an essential nucleation factor in the reconstitution of cortical actin patches. Las17p localizes with the Arp2/3 complex to actin patches; disruption of LAS17 leads to the loss of actin patches and a block in endocytosis. In the physical interaction between Las17p and the Arp2/3 complex, the C-terminal WA (WH2 (WASP homology 2) and A (acidic)) domain of Las17p are required as are the two subunits of the Arp2/3 complex, Arc15p and Arc19p. The WA domain is sufficient for Arp2/3 complex binding and activation; it shares sequence similarity with an acidic domain in myosin type I (Myo3p and Myo5p in S. cerevisiae), which also interacts with the Arp2/3 complex. Genetic and biochemical studies have identified numerous proteins that physically interact with Las17p. The WH1 domain of Las17p binds strongly to verprolin (Vrp1p/End5p (Thanabalu and Munn, 2001)), the yeast homolog of human WIP (WASP-interacting protein), which is involved in Las17p localization. The proline-rich region of Las17p binds to SH3 domain-containing proteins, including Sla1p (an actin patch protein with a role in endocytosis) and many others that may regulate the activity of Las17p.
Two other proteins involved in formation and stabilization of actin bundles in cables and patches are Sac6p (fimbrin) and Scp1p (calponin/transgelin), which work together. The stabilization of actin filaments in patches also strictly depends on capping of the “barbed” ends by small capping proteins, Cap1p and Cap2p.
Actin filament disassembly. Debranching of the actin filaments in cortical patches by the Arp2/3 complex is induced by Gmf2p/Aim7p, which also inhibits further actin nucleation (Gandhi et al., 2010). The protein has similarity to yeast Cof1p (cofilin) and to the human glia maturation factor (GMF). Cofilin, Cof1p, promotes actin filament depolarization in a pH-dependent manner. It binds both actin monomers and filaments; its main task is to sever filaments (Moon et al., 1993; Theriot, 1997). Cofilin is regulated by phosphorylation at Ser4; homologs are ubiquitous and essential in eukaryotes. Aip1p promotes filament disassembly by enhancing cofilin severing and protecting severed filaments by capping.
Scd5p is an essential protein that colocalizes with cortical actin and as an adapter protein functionally links cortical actin organization with endocytosis. Scd5p and the clathrin heavy and light chains (Chc1p and Clc1p, respectively) physically associate with Sla2p (Wesp et al., 1997), a transmembrane actin-binding protein involved in membrane cytoskeleton assembly and cell polarization, which is also a homolog of the mammalian huntingtin-interacting protein HIP1 and the related HIP1R. Both Scd5p and clathrin are required for Sla2p localization at the cell cortex. Scd5p activity appears to be regulated by phosphorylation/dephosphorylation. Phosphorylation of Scd5p by protein kinase Prk1p results in its negative regulation, whereas dephosphorylation by the Glc7p type 1 protein phosphatase relieves this inhibition. Mutations in GLC7 that abolish Glc7p interactions with Scd5p result in defects in endocytosis and actin organization. Loss of function scd5 mutants suffer from defects in receptor-mediated endocytosis and normal actin organization. They exhibit larger and depolarized cortical actin patches and a prevalence of G-actin bars.
2.3.2.3 Motor Proteins
Myosins, kinesins, and dynein are three classes of motor proteins that are highly conserved throughout evolution; several members of these proteins occur in yeast (Figure 2.5). Remarkably, myosins and kinesins are proteins that are able to bind to polarized cytoskeletal filaments and use the energy derived from repeated cycles of ATP hydrolysis to move along them. By unidirectional movement, these molecules can carry cargo from one point to a distant location within the cell; other motor proteins may cause filaments to slide against each other, so that the generated force drives processes like nuclear migration and cell division (Hoyt, Hyman, and Bähler, 1997; Moore and Cooper, 2010).
Figure 2.5 Motor proteins in yeast. The chains in myosin and kinesin are identical; distinction by color is only for better perception. Kin1p is a “plus”-end motor; Kar3p is a “minus”-end motor.
2.3.2.3.1 Myosins
Myosins are rod-like, extended structures (around 2 nm wide and greater than 150 nm long) normally consisting of two heavy and four light chains, whereby the heavy chains wrap around each other to form a coiled-coil of two α-helices (called the tail), while the light chains are part of motor domains at the N-terminus (called the head); between the head and tail are so-called IQ domains. Of the 14 different types of myosins in the myosin superfamily, S. cerevisiae has members of type I, type II, and type V (Brown, 1997). Type I members are characterized by the occurrence of only one head per molecule, whereas type II members carry two heads, and type V members have two extended head regions.
Type II myosins. The only type II myosin in yeast is Myo1p; it fulfills a specialized function as part of the ring-shaped actomyosin complex that (early in the cell cycle) localizes to the presumptive bud site and remains at the mother–bud neck until cytokinesis is completed (VerPlank and Li, 2005). Formation, but not maintenance, of this contractile ring requires the intact septin collar at the bud neck (cf. Section 7.2). Late in anaphase, F-actin Act1p and the IQGAP-related protein, Iqg1p (Epp and Chant, 1997), also accumulate in the neck ring, whereby incorporation of F-actin depends on Myo1p, and Iqg1p determines the localization of axial markers Bud4p and Cdc12p. At the end of anaphase, the actinomyosin ring begins to contract. Myo1p is regulated by two light chains, an essential light chain (ELC), Mlc1p, and a regulatory light chain (RLC), Mlc2p, which displays significant sequence homology to calmodulin or myosin light chain related proteins. Like other light chains, Mlc2p contains an EF hand and a phosphorylatable serine residue, both close to the N-terminus. Mlc1p interacts with one of the two motifs (IQ1), which, however, does not play a major role in regulating Myo1p; instead, this interaction regulates actin ring formation and targeted secretion through further interactions with Myo1p, Iqg1p, and Myo2p. Mlc2p interacts with the IQ2 motif and most likely plays a role in the disassembly of the Myo1p ring. The human counterpart to Myo1p, MYH11, may give rise to leukemia or familial aortic aneurysm.
Type V myosin subfamily. Myo2p and Myo4p belong to the type V myosin subfamily. Myo2p promotes polarized growth by orienting the mitotic spindle and by taking over the vectorial transport of organelles along actin cables to sites such as the growing bud during vegetative growth, the bud neck during cytokinesis, and the shmoo tip during mating. Even organelles, including secretory vesicles, vacuoles, peroxisomes, and late Golgi elements, are transported into the growing bud (Johnston, Prendergast, and Singer, 1991). These tasks afford cargo-specific myosin receptors making contact between the cargo and the myosin tail. For example, there are specific receptors on vacuoles (Vac8p–Vac17p) or on peroxisomes (Inp2p). Sec4p, a vesicle-bound Rab protein, associates with Myo2p, and along with Sec2p and Smy1p, is critical for vesicle transport (Figure 2.6). Myo2p participates in spindle orientation by actively transporting Kar9p/Bim1p-decorated microtubule ends into the bud. Myo2p together with the Rab protein Ypt11p are required for distribution and retention of newly inherited mitochondria in the bud (Ito et al., 2002). Myo4p has the main function of moving mRNAs within the cell (Haarer et al., 1994).
Figure 2.6 Vesicle (and organelle) transport in yeast.
Type V myosins have a particular domain architecture and distinct modes of regulation. Myo2p and Myo4p, in addition to the N-terminal actin-binding motor domain, have a globular C-terminal domain at the tail of the coiled-coil dimerization domain. Adjacent to the motor domain, there is a neck region that contains six IQ motifs that can bind calmodulin (Cmd1p). Through this interaction, calmodulin participates in polarized growth of yeast cells and inheritance of the vacuole by daughter cells. Calmodulin may also interact with the heavy chain of Myo4p. Through interactions with both the unconventional type I myosin (Myo5p) and Arc35p, a component of the Arp2/3 complex, calmodulin is also involved in receptor-mediated endocytosis.
Type V myosins are typically regulated by interactions with light chains. Mlc1p physically interacts with and regulates Myo2p. The binding of the Myo2p tail by the kinesin-like protein Smy1p promotes the polarized localization of Myo2p. The light chain(s) that regulate Myo4p are yet to be defined, but a novel motor-binding protein, She4p, may modulate Myo4p activity. While Myo2p predominantly moves organellar compounds, Myo4p moves mRNAs and acts as part of the mRNA localization machinery (see below).
Type I myosins. There are two yeast type I myosins represented by Myo3p and Myo5p that localize to actin cortical patches. Physical interaction between Myo5p and calmodulin (Cmd1p) has been detected, and was found to be required for endocytosis. Myo5p also interacts physically with verprolin (Vrp1p), a proline-rich protein. Deletion of the gene VRP1 causes delocalization of Myo5p-containing patches.
Tropomyosin. In addition to the myosins, yeast harbors two isoforms of tropomyosin. Tmp1p is the major isoform that binds to and stabilizes actin cables and filaments, which direct polarized cell growth and the distribution of several organelles. The protein is acetylated by the NatB complex; the acetylated form will bind actin more efficiently. Tmp2p, the minor isoform, largely has functions overlapping with those of Tmp1p.
2.3.2.3.2 Kinesins
Both kinesins and kinesin-related proteins are motor proteins remarkably similar to type V myosins. They generally function in mitotic spindle assembly and organization (see also Section 7.2.2.2), although each one takes over specialized functions. Cin8p, a kinesin motor protein, has an additional role in chromosome segregation. Functionally redundant with Cin8p is the kinesin-related motor protein Kip1(Cin9p), which, however, has an additional role in partitioning the 2 µm plasmid. The kinesin-related motor protein Kip2p stabilizes microtubules by targeting Bik1p, a microtubule-associated protein and component of the interface between microtubules and kinetochore (Berlin, Styles, and Fink, 1990; Moore and Cooper, 2010), to the plus end; Kip2p levels are controlled during the cell cycle. Kip3p is a further kinesin-related protein involved in spindle positioning. Cik1p is a kinesin-associated protein that stably and specifically targets the karyogamy protein Kar3p, a minus-end-directed microtubule motor that functions in mitosis and meiosis, localizes to the SPB, and is required for nuclear fusion during mating. Smy1p, a protein whose N-terminal domain is related to the motor domain of kinesins and that interacts with Myo2p, has already been mentioned; it may be required for exocytosis.
2.3.2.3.3 Dynein
Cytoplasmic dynein, Dyn1p (Pac6p), is the largest motor protein in yeast and a “minus”-end motor of microtubules. Dyn1p is active in the movement of the mitotic spindle that must move into the narrow neck between the mother cell and the bud in order to segregate duplicated chromosomes accurately. The process begins with the dynactin complex, directing spindle orientation and nuclear migration. This complex is composed of the actin-related protein Arp1p, together with Jnm1p (Pac3p) and Nip100p (Pac13p).
The movement of the spindle occurs in two main steps as part of nuclear migration into the neck region. (i) The nucleus moves to a position adjacent to the neck, a process involving cytoplasmic microtubules, the motor protein Kip3p, and Kar9p, a karyogamy protein required for correct positioning of the mitotic spindle and for orienting cytoplasmic microtubules; Kar9p localizes to the shmoo tip in mating cells and to the tip of the growing bud. (ii) The mitotic spindle is moved into the neck, which requires cytoplasmic microtubules from the SPB sliding along the bud cortex, and pulling the nucleus and the elongating spindle. Sliding depends on the heavy chain of cytoplasmic dynein (Dyn1p), the dynactin complex, and the regulators Num1p (Pac12p) and Ndl1p. In the second step, Pac1p functions in aiding the recruitment of dynein to the “plus” ends of microtubules. In this function, Pac1p is regulated by Ndl1p, a homolog of nuclear distribution factor NudE that interacts with Pac1p (Li, Lee, and Cooper, 2005). Cortical Num1p brings together the dynein intermediate chain Pac11p and the cytoplasmic microtubules (Farkasovsky and Kuntzel, 2001). Finally, Bim1p, a microtubule-binding protein, also known as Yeb1p (EB1, microtubule plus-end binding) together with Kar9p serves as the cortical microtubule capture site. In case the spindle is oriented abnormally, Bim1p will delay the exit from mitosis (Schwartz, Richards, and Botstein, 1997; Miller, Cheng, and Rose, 2000; Moore, Stuchell-Brereton, and Cooper, 2009).
2.3.2.4 Other Cytoskeletal Factors
2.3.2.4.1 Proteins Interacting with the Cytoskeleton
Other proteins that have been implicated in actin cytoskeleton reorganization and establishment of cell polarity are the proteins Boi1p and its functionally redundant homolog Boi2p. Both Boi1p and Boi2p contain SH3, pleckstrin homology (PH), and proline-rich domains. Several structure–function and genetic analysis experiments have tried to determine which domains are important for interactions with other proteins involved in the above processes. These studies showed that the Boi proteins interact physically and/or genetically with Bem1p, another SH3 domain protein, as well as three Rho-type GTPases – Cdc42p, Rho3p and the Rho3-related Rho4p (cf. Section 7.1.1).
Stt4p, the phosphatidylinositol-4-kinase involved in sphingolipid biosynthesis and in regulation of the intracellular transport of aminophospholipid phosphatidylserine from the endoplasmic reticulum (ER) to the Golgi, is required for actin cytoskeleton organization as well. Stt4p binds to the plasma membrane via the protein Sfk1p, thus promoting cell wall synthesis, actin cytoskeleton organization, and the Rho1/Pkc1-mediated mitogen-activated protein (MAP) kinase cascade (cf. Section 10.2). STT4 is an essential gene in some backgrounds, but not in others. Δstt4 mutants lack most of the phosphatidylinositol-4-kinase activity that is detected in the wild-type and are arrested in the G2/M phase of the cell cycle. Inactivation of Stt4p results in mislocalization of the Rho-GTPase guanine nucleotide exchange factor (GEF) Rom2p and also in the rapid attenuation of translation initiation.
2.3.2.4.2 Transport of Organellar Components
Of importance for the proper transfer of organellar components to the bud or, on the contrary, to restrict certain compounds to be accumulated in the bud is a specific mRNA localization machinery that becomes active during budding. In particular, mating-type switching should occur only in mother cells, meaning that HO transcription in daughter cells has to be prevented (cf. Chapter 7). This effect is brought about by Ash1p, a protein specifically localized to daughter nuclei late in the cell cycle, where it is poised to inhibit HO transcription in the following G1 phase. This asymmetric localization is achieved by the delivery of ASH1 mRNA to daughter cells by the products of the SHE genes. She2p and Loc1p bind to ASH1
