Saccharomyces cerevisiae (budding yeast)
Genome: 16 chromosomes per haploid cell, DNA content = 1.3 x 107 bp (3.5X E. coli)
Genome has been completely sequenced: link to yeast genome project databases: http://www.yeastgenome.org/
For a current “snapshot” of the reference genome, see
Saccharomyces cerevisiae is a very important model organism. It has numerous protein homologs to humans, short generation time, high accessibility and can easily be genetically altered. In the video link below, Professor Rhona Borts from the Department of Genetics at the University of Leicester (UK), discusses her research into the molecular basis of infertility and also the important role Saccharomyces cerevisiae plays as a model organism in her research.
Model eukaryotic cell, unicellular life style. Reproduces asexually by budding (asymmetric cell division).
There are three different possible cell types:
- haploid mating type a
- haploid mating type alpha
- diploid a/alpha cells
Each cell type expresses a specific set of genes. How do yeast cells achieve differential gene expression in the different cell types?
Haploid cells of opposite mating types, a and alpha, signal to each other through mating type-specific pheromones (cell-cell interaction, signal transduction), and fuse to form diploid a/alpha cells that undergo meiosis and sporulation. Spores germinate to form new haploid a cells and alpha cells. How do yeast cells sense and respond to cells of the opposite mating type?
In some yeast strains, cells switch mating type each generation, but switching is restricted only to mother cells. How is yeast mating type switching regulated?
Mating Pheromones and Signal Transduction:
- The mating pheromones (a factor and alpha factor) bind to serpentine transmembrane receptors.
- A heterotrimeric G protein is activated; GDP is exchanged for GTP and Galpha-GTP dissociates.
- The free Galpha subunits activate STE20 and STE5 protein kinases.
- STE20 and STE5 then activate a protein kinase cascade involving STE11, a MAP kinase kinase kinase homologous to mammalian RAF.
- STE11 activates STE7, a homologue of mammalian MEK (MAP kinase kinase).
- STE7 activates FUS3 and KSS1, homologues of mammalian MAP kinases.
- The STE12 transcription factor is phosphorylated and becomes active.
- Cell surface proteins are synthesized that mediate cell-cell adhesion.
- Proteins that mediate cell fusion are synthesized.
- The cell cycle is arrested in G1.
- Cells turn into shmoos (gametic cells with mating projections).
Sex Determination: What determines whether a yeast cell is mating type a or alpha?
a cells express a-specific genes: a peptide pheromone called a factor, and a cell surface receptor for alpha factor. a cells also express haploid-specific genes.
Alpha cells express alpha-specific genes: a peptide pheromone called alpha factor, and a cell surface receptor for a factor. Alpha cells also express haploid-specific genes.
Diploid a/alpha cells express diploid-specific genes and turn off a-specific, alpha-specific, and haploid-specific genes.
MAT locus: two alleles of this single genetic locus determine cell type.
In alpha cells:
- MAT alpha encodes two transcription factors, alpha-1 and alpha-2.
- Transcription factor MCM1 binds with alpha-1 to promoters of alpha-specific genes and activates their transcription.
- MCM1 and alpha-2 bind to promoters of a-specific genes and represses their transcription.
- MCM1 alone binds to promoters of haploid-specific genes and activates transcription.
In a cells:
- MAT a encodes the transcription factor a1.
- a1 protein does nothing.
- MCM1 by itself binds to promoters of a-specific genes and haploid-specific genes to activate transcription.
- alpha-specific genes are silent without alpha-1 protein.
In diploid a/alpha cells:
- MAT a expresses the a1 protein.
- MAT alpha expresses alpha-2 protein.
- A heterodimer of a1/alpha-2 represses transcription of alpha-1 from the MAT alpha locus, and all haploid- specific genes.
- MCM1/alpha-2 complex represses a-specific genes.
- Alpha-specific genes are silent without alpha-1 protein.
Mating Type Conversion: heterothallic strains of yeast switch sex each generation!
Silent copies of the MAT locus (HMLalpha & HMRa) reside on either side of MAT on chromosome III.
Gene conversion, a process involving DNA cleavage, copies the silent information from HMLalpha or HMRa to the MAT locus.
For in-depth information on how a cell selects either HMLα or HMRa as a donor, visit Determination of donor preference during mating-type switching (written by Sarah Wetherington).
HO endonuclease mediates switching, and is the master regulator of mating type switching.
HO gene transcription is regulated by cell lineage, ploidy and cell cycle:
- Only mother cells can switch; daughter cells cannot switch until they have budded off a daughter cell of their own.
- Only haploid cells can switch, and only during the G1 phase of the cell cycle (before replication of DNA).
Expression of HO determines whether a cell switches mating type. Mother cells express HO during G1, daughter cells do not express HO. Ectopic expression of HO in daughter cells causes them to switch. So what regulates HO expression?
Upstream Regulatory Sequences (URS):
The promoters of yeast genes and other eukaryotic genes are combinatorial, and have multiple binding sites for transcriptional activators and repressors.
The HO promoter has two major URSs: URS1 mediates mother/daughter control, and URS 2 mediates cell-cycle control.
Expression of HO in diploids is repressed by binding of a1/alpha2 factor at 10 sites located throughout URS1 and URS2.
Transcription of HO in the late G1 phase of the cell cycle is determined by binding of cell- cycle box factor (CCBF, composed of Swi4p and Swi6p) to the cell-cycle box sequences, a 12-base sequence motif that is repeated 10 times in URS2. CCBF is active only during G1.
Swi5p (product of swi5 gene) is a transcription factor with a zinc-finger type DNA binding domain.
- Transcribed in S, G2, and M, then , then rapidly degraded during G1 of next cell cycle.
- Binding of Swi5p to URS1 is required for expression of HO in mother cells.
- Constitutive Swi5 expression leads to daughter cell switching.
- Stabilized mutations (causes Swi5p not to be degraded) also lead to daughter cell switching.
- However, Swi5p is localized to both cells in late anaphase.
- Activates several other genes in early G1 both mother and daughter cells, but not HO (needs CCBF).
- So some other regulatory factor required.
Sin3 mutations allow HO expression without Swi5, lead to daughter cell switching. But again Sin3p is found in both mother and daughtercells.
Ash1p: a negative regulator of HO expression in daughter cells
3 different mutant screening strategies all found same gene:
- Sil & Herskowitz (1996) looked for microcolonies that have four schmoos in special mutant background: ste3 deletion mutant has no a factor pheromone receptor, mother cells continue to switch mating types back and forth without forming diploids. In medium with alpha-factor, wild type cells form microcolonies containing both budding cells and shmoos (a cells receive alpha-factor, stop dividing and form shmoos, but alpha cells cannot receive a factor, continue budding. Mutant cells where both mother and daughter cells switch to a type will arrest and form shmoos at 4-cell stage.
- Bobola et al (1996) engineered cells carrying CDC6 gene (essential for cell division) fused to HO promoter, so only mother cells can divide. No exponential growth, thus no colonies. But mutants where daughter cells activate HO promoter will form colonies.
- Bobola et al (1996) also looked for mutations that restore HO expression in a she1 mutant background. SHE1 (Swi5p-dependent HO expresson) is essential for HO expression in mother cells, but encodes an unconventional myosin, therefore may be involved in localization of negative regulator (repressor) of HO expression. Then loss of negative regulator would restore HO expression in she1 mutants.
In mother cells, Swi5 and CCBF (Swi4/6) turn on HO transcription; in daugher cells, Ash1 blocks the action of Swi5.
ASH1 deletions cause switching in daughter cells.
ASH1 overexpression reduces switching in mother cells.
Ash1p preferentially accumulates in daughter cell nuclei during G1, degraded and absent during S, G2, M.
Ash1p has zinc-finger domain.
Ash1p antagonizes Swi5p: no daughter cell switching in double mutants deleted for both ASH1 and SWI5.
Ash1 mRNA localization to the bud
This video clip from Beach & Bloom (2001) shows the localization of GFP-tagged Ash1 mRNA to the distal tip of the bud in live yeast cells:
Mutants in other genes cause defects in Ash1 mRNA localization to the bud:
SHE 1-5 genes (Jansen et al. 1996) required for asymmetric distribution of Ash1p.
SHE1 encodes a myosin, SHE5 is the same as BNI1, involved in cytokenesis.
She1p and She3p are cytoplasmic, accumulate in bud.
The SHE genes are part of an extensive mRNA localization system in eukaryotes, coupled to translational control (Vasquez-Pianzola and Suter, 2012).
PAU genes: Largest multi-gene family in yeast
The complete function of PAU genes is unknown but research done by Martinez, Barre, and Blondin and Luo and van Vuuren makes predictions about the function based on data suggesting the regulation and expression of PAU genes. Click here to read more about the research about the regulation and expression of PAU genes.
The Role of Doa1 in Budding Yeast
Saccharomyces cerevisiae controls DNA damage with the help of the the protein Doa1. Doa1 has been seen to regulate ubiquitin, which is needed in the membrane protein degradation pathway. Doa1 binds to the Ub molecule and transports the proteins to the proteasome for removal. To view more about this process visit here.
Ste5 protein controls a switch-like mating decision. here.
And if you’ve had enough of budding yeast:
Alberts et al., 1994. Molecular Biology of the Cell, 3rd ed. Garland Publishing, Inc. Chapter 9, pp. 441-442.
Amon, Angelika, 1996. Mother and daughter are doing fine: asymmetric cell division in yeast, Cell 84:651-654. http://dx.doi.org/10.1016/S0092-8674(00)81041-7
Beach, DL and K Bloom 2001 ASH1 mRNA localization in three acts, Mol Biol Cell 12:2567-2577. http://www.molbiolcell.org/cgi/content/full/12/9/2567
Bobola et al., 1996. Asymmetric accumulation of Ash1p in postanaphase nuclei depends on a myosin and restricts yeast mating-type switching to mother cells, Cell 84:699-709.
Cosma, JP, 2004. Daughter-specific repression of Saccharomyces cerevisiae HO: Ash1 is the commander, EMBO reports 5, 10, 953–957 doi:10.1038/sj.embor.7400251
Herskowitz, I., 1989. A regulatory hierarchy for cell specialization in yeast, Nature 342:749- 757.
Jansen et al., 1996. Mother cell-specific HO expression in budding yeast depends on the unconventional myosin Myo4p and other cytoplasmic proteins, Cell 84:687-697.
Lodish et al., 1995. Molecular Cell Biology, 3rd ed. Scientific American Books. Chapter 9, pp. 339-341; Chapter 13, pp. 550-556; Chapter 20, pp. 897-899.
Long, R.M., R.H. Singer, X. Meng, I. Gonzalez, K. Nasmyth and R.-P. Jansen, 1997. Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA, Science 277: 383-387.
Sil, A. and I. Herskowitz, 1996. Identification of an asymmetrically localized determinant, Ash1p, required for lineage-specific transcription of the yeast HO gene, Cell 84:711-722.
Vasquez-Pianzola, P. and B. Suter, 2012. Conservation of the RNA transport machineries and their coupling to translational control across eukaryotes, Comparative and Functional Genomics 2012. doi:10.1155/2012/287852