1. Sex and longevity
Or: why mating of eukaryotic single cell organisms may be less boring than you think

2. If it can find a suitable partner, yeast prefers to mate to become diploid rather than haploid

3. Processes required for mating to occur successfully

4. Processes required for dating to occur successfully
Sensing of the partner
Signaling to the partner
Orientation towards the partner
Synchronization of behavior

5. Processes required for mating to occur successfully cont’d
Synchronization of Cell cycle
Fusion (cell/nuclear)
Repression of Haploid specific genes after mating

7. Signalling to/Sensing of the partner
Haploid yeast cells produce pheromones:
a cells produce a- factor (12 aa peptide)
“ “ a- factor (13 aa peptide)
Haploid yeast cells produce pheromone receptors that localized to the cell membrane:
a cells produce a- factor-receptor Ste2p
“ “ a- factor receptor Ste3p
Intercellular signalling! Analogous/Homologous to cell-cell signalling in higher eukaryotes
Environmental signalling pathway

8. Selection of mutants unable to mate
Lee Hartwell, 1980
Seminal work on cell cycle regulation (Nobel Prize 2001 for Medicine/Physiology)
Interest in factors that regulate the cell cycle
S. cerevisiae cells arrest their cell cycle in G1 phase upon exposure to mating factor

9. Selection scheme
Mutants were selected for their ability to grow on media containing mating factor (unresponsive/
resistant to mating factor) at high temperature (34oC) but not at low temperature (22oC) (temperature sensitive “ts” mutants, “conditional mutant”)
These mutants can be tested for their ability to mate by mixing with cells of opposite mating type and selecting for diploids via unique markers present in the haploids
Mutants deficient for mating (sterile) at high temperature can be sorted into complementation groups
 STE genes

10. Cell-to-cell signal recognition and transduction

11. Ste2p/Ste3p 7-transmembrane receptor molecules
(highly conserved cell membrane signal transduction molecules; e.g. opiate receptors in humans)
- C-terminus involved in regulation of degradation of Ste2/3 (ubiquitination pathway), phosphorylation sites
Interact with the Heterotrimeric G-protein
Gpa1p = a subunit, Ste18p = b-subunit, Ste4p = g-subunit
Upon binding of pheromone release of b, g-subunits of heterotrimeric G-protein, initiation of MAP-kinase cascade

12. The mating signalling pathway is a MAP – kinase pathway
MAPK = mitogen activated protein kinase (FUS3)
MAPKK= mitogen activated protein kinase-kinase (STE7)
MAPKKK= ….. (STE11)
MAPKKKK= …..(STE20)
“MAP kinase cascade”
Activation of expression of genes required for changes in cells necessary for mating
Dependent on recruitment of “Scaffold protein” Ste5p to the plasma membrane

13. This slide was nicked from internet lecture notes of a course held at the Universität München

14. Conservation of Signal Transduction Mechanisms in Eukaryotes
Fig. 1. Scheme of distinct MAPK signalling pathways in mammals, yeast and plants. Note the general similarity in the organization of MAPK pathways in all three eukaryotic systems. MAPKKK, mitogen activated protein kinase kinase kinase; MAPKK, mitogen activated protein kinase kinase; MAPK, mitogen activated protein kinase. Scaffolding proteins (depicted in dark blue) are integrating signalling pathways.
Samaj J, Baluska F, Hirt H. From signal to cell polarity: mitogen-activated protein kinases as sensors and effectors of cytoskeleton dynamicity. J Exp Bot Jan;55(395): Epub 2003 Dec 12

15. Transcription Factors/Effectors
Far1
Ste12
Tec1
Ste12 ?
Multiple MAP kinase (MAPK) cascades using shared components regulate growth and differentiation in S. cerevisiae. Mating, invasive growth, pseudohyphal development, high-osmolarity/glycerol response and maintenance of cell wall integrity are each regulated by structurally similar but functionally distinct MAPK cascades that are activated by different upstream signals but have in common at least three kinds of kinases: a MAPKKK, a MAPKK (or MEK) and a MAPK. Yellow highlighting indicates the kinases that are shared by the different pathways. Note that for simplicity Ste50p is not shown in this figure, although it associates with Ste11p and is required for optimal signaling through all of the pathways shown. Details can be found elsewhere (Gustin et al., 1998; Elion, 2000; Pan et al., 2000). (Elion EA.The Ste5p scaffold.J Cell Sci Nov;114(Pt 22): Review).

16. What confers specificity to kinase cascades?βγ
GDP α
GTP
Ste5
Ste11
Ste7
Ste20
Kss1
Ste12
Fus3
Tec1

17. Distinct “scaffolds” for distinct signaling pathways?
Cartoon of Ste5p, Pbs2p and Far1p scaffolds. Ste5p is required for activation of the mating MAPK cascade in response to mating pheromone and does not have an intrinsic kinase activity, whereas Pbs2p encodes the MAPKK of the high osmolarity/glycerol pathway that is activated by increased osmolarity. Far1p is required for oriented polarized growth in response to mating pheromone. Pbs2p and Far1p are postulated to be analogs of Ste5p on the basis of their ability to associate with multiple components of an individual signal transduction pathway (Posas and Saito, 1997; Butty et al., 1998; Nern and Arkowitz, 1998; Nern and Arkowitz, 1999; Rait et al., 2000), but it is not known whether they simultaneously bind to associated signaling components. Similarities between Ste5p, Pbs2p and Far1p include the ability to associate with an uppermost component of a pathway that is membrane associated and senses the external signal, as well as to downstream components that regulate the activity of effectors within a pathway. In addition, all three scaffolds link signaling components that also associate with a Rho-type G protein (Cdc42p). Ste5p and Far1p share two domains of homology (Leberer et al., 1992), one of which overlaps with the RING-H2 domain that is thought to associate with the Gß subunit Ste4p of the same heterotrimeric G protein. It is not known whether the RING-H2 domains have a function in ubiquitin-mediated proteolysis (Borden, 2001). Elion EA.The Ste5p scaffold. J Cell Sci Nov;114(Pt 22): Review

18. Mechanisms of cell polarization

19. Yeast cells undergo polarized cell growth during the mating reponse
In response to mating factor, yeast cells “grow” towards the highest concentration of mating factor forming a “shmoo”
identification of factors involved in shmooing allowed dissection of cell polarization mechanisms

20. Isolation of shmooing-deficient mutants
Ethyl-methane sulfonate mutagenesis of yeast cells (ade2 deficient, red)
Plating of mutagenized cells at low cell titer on YPD plates
Replica plating of mutagenized cells onto a lawn of inefficiently mating yeast strains and incubated for 6 hours to allow for mating
Mating plates were replica plated on selective media to select for diploids
Mutants unable to mate were identified by inability to grow on selective plate (but leaving a red “shadow” of dying cells due to ade2 mutation) while cells that were able to mate produced white colonies
(Also other screens to identify mutants e.g. in bud site selection etc)
mutations isolated in FAR1, CDC24, CDC42, BEM1 and other factors  investigation of mutant phenotypes

21. Cell polarization steps
Cell surface site determination using intrinsic (e.g.for cell budding) or extrinsic (e.g. for mating) cues
Marking of cell surface by ”Landmark protein” (Polarity cue)
Establishment of polarity by activation of small GTPase proteins in vicinity of the landmark
Cytoskeletal re-orientation/polarisation (actin and other polarized components)

22. Cell polarization
Model for the localization of Cdc24 during budding and mating. a, In the G1 phase of the cell cycle, Far1 localizes Cdc24 in the nucleus. b, Activation of the Cdc28–Cln kinase triggers degradation of Far1 in the nucleus, allowing rapid export of Cdc24 to the cytoplasm. Cytoplasmic Cdc24 is then recruited to the incipient bud site, possibly marked by Rsr1/Bud1-GTP. c, In the presence of pheromones, the Far1–Cdc24 complex is exported by Msn5 into the cytoplasm, where Far1 targets Cdc24 to Gβγ at the site of activated receptor. As a result, the actin cytoskeleton polarizes towards the position of the mating partner, signalled by the morphogenetic pheromone gradient (Shimada Y, Gulli MP, Peter M. Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating. Nat Cell Biol Feb;2(2): )

23. G1 phase α
Far1p βγ
Cdc24
Bud1
Cdc42
GDP

24. Budding α
Far1p βγ
Cdc24
Bud1
Cdc42
GTP
GDP

25. Reorientation of Actin networks during shmooing
Map kinase cascade α
Far1p βγ
Cdc24
Msn5
Bud1
Fus3
Bem1
Cdc42
Activates?
GTP
GDP

26. Polarity Pathways in Yeast
 Schematic representation of the molecular pathways leading to a polarized organization of the cytoskeleton in response to internal cues during budding and external signals during mating. Common to both pathways is the asymmetric activation of Cdc42p at the site determined by the landmarks. In turn, activated Cdc42p triggers the polarized assembly of the actin cytoskeleton by binding to various effectors. The mediators Bud1p and Far1p are thought to recruit and activate the GEF Cdc24p downstream of the specific landmark proteins. Far1p interacts directly with activated Gβγ at activated receptors, whereas Bud1p is regulated at landmarks by the GEF Bud5p and the GAP Bud2p. The mechanisms required to localize the landmark proteins Bud10p, Bud8p and Bud9p are not fully understood, but in the case of Bud9p and Bud10p require an intact septin structure. Genetic analysis has identified a number of novel proteins, which alter the budding pattern of diploid cells, and may thus be involved in localizing these landmark proteins. Chang F, Peter M.Yeasts make their mark. Nat Cell Biol Apr;5(4): Review.

27. A model for cell specialization

28. Mating type regulation
Three yeast cell types:
haploid
diploid
a cells
a cells
a/a cells
Differ in expression from MAT (mating type) locus and subsequent gene activation/repression
Haploid cells are unable to sporulate but can mate to form diploid a/a cells

29. Homothallic life cycle of S. cerevisiae1 2 3
 Homothallic life cycle of Saccharomyces. A MAT a haploid cell that has divided can switch to the opposite mating-type and the original cell and its switched partner can conjugate to form a MAT a/MAT diploid cell. Meiosis and sporulation will regenerate haploid cells. Homothallic switching is confined to cells that have previously divided (mother cells) by the action of the Ash1 repressor protein that is localized to the newly formed bud that gives rise to a daughter cell. Ash1p represses a positive regulator of HO endonuclease expression, Swi5p. In germinating spores, it is most likely the absence of Swi5p itself that prevents MAT switching until the cells have budded and divided Haber JE.Mating-type gene switching in Saccharomyces cerevisiae. Annu Rev Genet. 1998;32: Review.

30. Independent of mating type, yeast cells contain the information for both a and a- specific regulators
All genes for determination of the expression of mating type are physically present in a and a cells
These genes are contained in so called mating type loci HMRa and HMLa which are silenced (no gene expression)
A third locus (MAT) carries the expressed information
In HO+ cells, mating type can be switched by homologous recombination of one of the silent cassettes into the active MAT locus
The switch is initiated by a double-strand cut of the HO endonuclease in the MAT locus

31. W X Z1 Z2 X Z1 W X Z1 Z2 W X Z1 Z2 X Z1 W X Z1 Z2
Fig. 1. Mating-type switching: switching from (a) MATa or (b) MATa. An HO endonuclease-induced double-strand break at MAT initiates gene conversion/replacement of the Ya region with Ya sequences copied from HMLa. MATa cells express Mata1p and Mata2p regulatory proteins, while MATa encodes Mata1p. HMLa and HMRa contain complete copies of mating-type genes but are not expressed because of the silencing imposed through the adjacent E and I silencer sequences that organize a repressed chromatin structure (indicated by hatched lines). HML shares more sequences (regions W, X, Z1 and Z2) with MAT than does HMR (regions X and Z1). MATa strains preferentially recombine with HMLa, even if HMR also contains Y a instead of Ya. Donor preference is dependent on a 244 bp cis-acting recombination enhancer (RE).
Trends in Genetics Volume 14, Issue 8, 1 December 1998, Pages
A locus control region regulates yeast recombination
James E. Haber*

32. Gene silencing

33. Silencing of mating type loci
Expression from HMRa and HMLα needs to be turned off tightly to ensure proper regulation of mating type genes
Turning off these loci ( as well as other regions, e.g. telomeres) is achieved by a process called silencing, which results in heterochromatin formation over the silcenced region
Heterochromatin is “a specialized chromatin structure that blocks expression of most genes within the silenced domain, irrespective of which activator or RNA polymerase is used”

34. Silencing is a process that is mechanistically different from repression
Silencing turns off the expression of existing genes permanently in an organism
Repression turns of genes temporarily, but can be relieved due to changing environment (nutrients, growth factors)
Silencing in yeast is present at the HML/HMR loci and at telomeres
Epigenetic phenomenon (change in expression without change in DNA sequence)
Can you think of a silencing event in humans?

35. Main components involved in silencing
Rap1p, Orc1p, Abf1p: DNA binding proteins with affinity to Sir proteins
Sir (silent information regulator) proteins:
- Sir1p: required for establishment of silencing
- Sir2p: histone deacetylase
- Sir3p: structural protein, interacts with deacetylated histones
- Sir4p:structural protein, interacts with Sir2p in a 1:1 complex; interacts with deacetylated histones

36. Cis-acting elements required for silencing in yeast
Sir1p is only required in Mating type loci silencing!
First: “Nucleation event”Sir1 (silent information regulator 1) protein required for establishment of silencing but not for maintenance
targeting of Sir1p to ORC is sufficient for establishment of silencing
Passing through S-phase in cell cycle is required for establishment of silencing of mating type loci

37. Hierarchy of establishment of silencing in yeast
Rap1p, Orc1p, Abf1p binding of silencer region
Recruitment of Sir1p
Sir4p recruited to silencer by binding to Sir1p and Rap1p
Sir2p joins the complex by physical interaction with Sir4p
Sir3p interacts via Sir4p, Rap1p and Abf1p
Deacetylation of Histones by Sir2p results in further recruitment of Sir3p and Sir4p, leading to chain reaction-like chromatin spreading

38. Sir2p Sir1p Sir4p Sir4p Sir2p Sir4p Sir2p Sir4p Sir2p Orc1p Rap1p
Abf1p Ac Ac Ac Ac
TATA
TATA
TATA
TATA

39. Grunstein M. Yeast heterochromatin: regulation of its assembly and inheritance by histones. Cell May 1;93(3): Review

40. Limiting of Chromating spreading
Uncontrolled spreading of chromatin would be detrimental to the cell
Spreading is limited by several mechanisms:
boundary elements (e.g pol III transcription complex on tRNA promoter proximal to HMR)
anti-silencing modifications on nucleosomes (acetylation of specific histone residues)
Intrinsic limitations of the spreading mechanism due to rapid turnover and limited availability of Sir proteins

41. Epigenetic Inheritance of Silenced chromatin
Epigenetic Inheritance: “Heritability of two different states in otherwise identical cells”
Ability of a specialized chromatin structure (or protein structure) to template its own reformation
Model for epigenetic inheritance:
- during DNA replication, epigenetically marked (acetylated or methylated) histones (H3 and H4) are distributed randomly between the sister DNA strands
- presence of epigenetically marked histones/ nucleosomes on DNA strands causes propagation of silenced chromatin

42. Yeast as a model for aging processes

43. Sir2p and longevity
Unlike E. coli cells, S. cerevisiae cells are not immortal – the replicative life span (i.e. how often a cell can make buds) of yeast is finite = yeast cells die of old age!
Replicative life span is assessed by counting bud scars on yeast cells
Life span is strain dependent (18 – 100 budding events)
Model for research on human longevity?
Bud scar

44. Screen for genes involved in aging
Guarente group (Harvard)
Prolonged life span is a complex trait (several genes involved)
Life span for individual cells not easy to score
Higher stress resistance (e.g resistance starvation) is associated with longevity
Mutagenesis of a short-lived, starvation sensitive yeast strains and selection for starvation resistant clones led to the isolation of yeast mutants with an increased life span

45. Silent information regulator genes affect life span
One long-lived mutant (uth2-42) had additional phenotypes that co-segregated with longevity: poor mating and bipolar budding pattern (like diploid cells)
Cloning of the mutation by library complementation, selecting for mating-competent transformants

46. Mating-competent clones carried the complementing plasmid
Grow cells of opposite mating type
Grow mutant cells
High frequency transformation with genomic library
Spread on Petri dish to make a lawn of cells
Replica plate to allow for mating
Select diploids
Mating-competent clones carried the complementing plasmid
The uth2-42 mutant was complemented by SIR4

47. SIR genes affect longevity
The uth2-42 mutation was a gain of function mutation that redirected the Sir2p/Sir3p/Sir4p complex away from silenced loci (telomeres and mating type loci) to the nucleolus
Deletion mutations of SIR2, SIR3 and SIR4 caused shortened life span
Overexpression of yeast SIR2 increases life span
SIR2 mediates the beneficial effects of calorie restriction (CR) on longevity in yeast (at least in some strain backgrounds)

48. Sirtuins and aging
Role in silencing and suppression of recombination at the ribosomal DNA locus (which can give rise to toxic extrachromosomal rDNA circles - ERCs)
The yeast rDNA locus on chromosome XII is known to give rise to 3 μm circular forms of rDNA, composed of a single rDNA repeat and containing an ARS
The accumulation of ERCs is such that after 15 divisions, they can reach levels equal to the DNA content of the yeast genome (Sinclair et al., TIGS 23, Vol4, 1998)

49. DNA - damage induced relocalization of SIR complex results in aging
As a result of yeast senescence, ERCs accumulate in yeast cells, and the Sir protein complex disassociates from silenced loci and moves to the nucleolus to inactivate ERCs
“A redistribution of chromatin modifying factors results in epigenetic changes that promote aging phenotypes”
Sir proteins are also required for the repair of DNA double strand breaks
DNA - damage induced relocalization of SIR complex results in aging
Some aspects of these mechanisms are conserved in humans
Fig. 2. Characteristics of yeast aging. The replicative life span of yeast is defined as the number of daughter cells produced by a mother cell before cessation of cell division and senescence. Yeast cells undergo many age-related changes, including an increase in cell size, a slowing of the cell cycle, enlargement and fragmentation of the nucleolus (red), and the redistribution of the Sir3 silencing protein (green) from telomeres and HM loci to the nucleolus (Sinclair et al., TIGS 23, Vol 4, 1998).

50. Sirtuins in higher eukaryotes
SIR2 is conserved in eukaryotes
Overexpression of SIR2.1 in worms increases life span*
Mammals with 7 Sirtuin variants
Beneficial effects of calorie restriction in mammals mediated in part by SIRT1
*This may not be true!!
Chen D and Guarente L. TIMM Vol. 13 No.2

51. Mammalian Sir homologues (Sirtuins) are major regulators of aging in mammals