Except in Every Detail: Comparing and Contrasting

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• Except in Every Detail Comparing and Contrasting
  
• G-Protein
• Signaling in Saccharomyces cerevisiae
• and
• Schizosaccharomyces pombe
• Charles S. Hoffman
• Biology Department, Boston College, Chestnut
  Hill, Massachusetts
• Eukaryotic Cell, March 2005, p. 495-503, Vol. 4,
  No. 3
• Presented in BIOL 566 byKaren Ambrose

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INTRODUCTION

• G-Proteins
• - Alfred Gilman and Martin Rodbells discovery
  in 1994.
• - Guanine nucleotide-binding proteins.
• - Family of proteins second messenger
  cascades.
• - Signaling mechanism exchange of GDP for GTP
  as a switch to allow or inhibit biochemical
  reactions.
• - Belong to the larger grouping of GTPases.

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• Refers to the membrane-associated heterotrimeric
  G-proteins "large" G-proteins.
• Made up of alpha (a), beta (ß) and gamma (?)
  subunits.
• Activated by G-Protein Coupled Receptors (GPCR)
• Small G proteins or GTPases (e.g. ras)
  monomeric not membrane-associated, but bind GTP
  and GDP.
• Important signal transducing molecules in cells.
  Diseases (e.g. diabetes and certain forms of
  cancer) have root in the malfunction of
  G-proteins.

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• Structure of a heterotrimeric G-protein that
  consists of a at/ai subunit (blue) and the ß?
  complex (red, green)

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Saccharomyces cerevisiae

• A species of yeast.
• The most intensively studied eukaryotic model
  organism in molecular and cellular biology.
• Reproduces by budding.
• Easy to culture, and as a eukaryote, it shares
  the internal cell structure of plants and
  animals.
• Many proteins important in human biology were
  first discovered by studying their homologs in
  yeast.

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• S. cerevisiae (large globes), surrounded by
  E.coli (small rods)

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Schizosaccharomyces pombe

• Fission yeast," is another species of yeast.
• It is used as a model organism in biology.
• Unicellular eukaryote having rod-shaped cells.
• Cells maintain shape by growing exclusively
  through the cell tips and divide by medial
  fission to produce two daughter cells of equal
  sizes.

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• The cells shown in Panel A of the figure are wild
  type cells, viewed under the microscope and
  treated with a fluorescent dye that stains the
  DNA in the nucleus. The nuclei are the bright
  blobs in the center of the cells. At the bottom
  of the panel, a cell that has divided is shown,
  with two nuclei and a medial septum.
• Panel B If the cells have a mutation that
  prevents the normal progression of the cell
  cycle, they become very elongated. This occurs
  because the cells can continue to grow but are
  unable to divide. These mutants are called cell
  division cycle (cdc) mutants.

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G-protein mediated signaling

• Mechanisms where extracellular stimuli sensing is
  converted into intracellular signals.
• Regulates transcriptional activators and
  repressors to control cell function and
  development.
• GPCRs and associated G proteins.
• Critical to human development.
• The majority of all pharmaceuticals act on GPCRs.
• Both fungal human pathogens and fungal plant
  pathogens utilize G-protein signaling pathways to
  control processes required for virulence.

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• The G-protein activation-inactivation cycle.

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• Cycle completion and the RGS proteins (regulators
  of G-protein signaling)

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• Widely accepted but two aspects are questioned
• (1) Although widely supported, the model in which
  G-protein activation results in Ga dissociation
  from the Gß? dimer, is challenged.
• The ability to chemically cross-link Ga subunits
  to Gß subunits is not altered by activation of
  the Ga, and Ga subunits within chemically
  cross-linked G proteins can be charged with GTP
  and convert to the activated conformation. In
  yeast, a translational fusion of the Ga and Gß of
  the pheromone pathway continues to function
  normally, although it is unable to fully
  dissociate.
• (2) A receptor-G-protein-effector preactivation
  complex may exist prior to signaling, and
  G-protein activation could simply alter the
  nature of the interactions among the various
  components of the signaling pathway.

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Exactly the same

• Both yeasts posses two known G-protein mediated
  signaling pathways.
• (1) detect extracellular pheromones activate a
  mitogen-activated protein kinase (MAPK) cascade.
• (2) detect glucose activate adenylate cyclase
  to produce a cyclic AMP (cAMP) signal.
• The S. cerevisiae pheromone pathways Ste2
  a-factor Ste3 a-factor receptors are homologous
  to S. pombe Mam2 P-factor Map3 M-factor
  receptors.
• In both
• The pheromone signals activate MAPK and
  p21-activated kinase (PAK) pathways comprised of
  related kinases.
• Use similar types of G-protein subunits with
  distinct Ga subunits

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Except in every detail
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• G-protein mediated signaling pathways of S.
  cerevisiae and S. pombe.
• Schematics identify the key receptors, G-protein
  subunits, and effectors, as well as some
  additional relevant proteins for (A) the S.
  cerevisiae pheromone pathway,and (B) the S. pombe
  pheromone pathway.

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S. cerevisiae pheromone MAPK pathway

• Ste2 or Ste3 GPCR, which detects the a-factor or
  a-factor pheromones.
• Heterotrimeric G protein Gpa1/Scg1 Ga , Ste4
  Gß, and Ste18 G?.
• Gß? dimer activates a MAPK pathway composed of
  the Ste11 MAPK kinase kinase (MAPKKK), Ste7 MAPK
  kinase (MAPKK), and Fus3 MAPK by binding the Ste5
  scaffold protein.
• The Ste4-Ste18 Gß? dimer also binds Ste20, a PAK
  family member, also known as a MAP4K (MAPKKK
  kinase), which appears to phosphorylate and
  activate the Ste11 MAPKKK.
• By targeting both Ste5 and Ste20, the Ste4-Ste18
  Gß? dimer activates the Fus3 MAPK pathway at two
  levels.
• Gpa1 Ga subunit plays three roles
• In the absence of pheromone signaling, Gpa1 binds
  to the Ste4-Ste18 Gß? dimer to prevent activation
  of the MAPK pathway.
• Upon activation, Gpa1 binds the Fus3 MAPK to
  positively regulate cell growth and cellular
  fusion.
• Positive role in signaling through an interaction
  with Scp160.
• - RGS protein Sst2 accelerates Gpa1 GTPase
  activity and desensitize the cells to the
  pheromone present in the growth environment.

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S. pombe pheromone MAPK pathway

• Mam2 P factor receptor and the Map3 M-factor
  receptor.
• Coupled to Gpa1, a monomeric Ga subunit.
• Activation of a MAPK pathway Byr2 MAPKKK, the
  Byr1 MAPKK, and the Spk1 MAPK.
• Gpa1 and Ras1, the only Ras homolog in S. pombe,
  functionally converge on the Byr2 MAPKKK of the
  pathway. Ras1 directly binds Byr2. Ras1 is also
  part of a protein complex involving the Shk1
  PAK/MAP4K family member, which is similar to the
  S. cerevisiae Ste20 MAP4K.
• Activation of Ras1 in response to pheromone
  signaling appears to be due to pheromone-induced
  expression of the Ste6 GEF Ras1 is not directly
  activated by the pheromone receptors.

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Disregarding the G-protein signaling rules

• Activation of homologous MAPK pathways involves
  targeting of the core MAPK cassette and the
  MAP4K, the S. pombe MAPK pathway is not activated
  by a Gß? dimer. Possible answer S. pombe MAPK
  pathway does not possess an Ste5-like scaffold
  protein and therefore may involve different
  protein-protein interactions.
• Regulation and role of Gpa1 in S. pombe pheromone
  signaling? Like S. cerevisiae Gpa1, S. pombe
  Gpa1 is negatively regulated by an RGS protein.
• S. pombe Gpa1 Ga of the pheromone pathway couples
  with a GPCR in the absence of a Gß? partner.
• Gß? dimer in receiving signals from the GPCR
  Facilitate the interaction between the Ga
  subunit and the receptor.
• The assumed 111 stoichiometry of heterotrimeric
  G-protein subunits is not necessary for proper
  regulation of traditional G-protein signaling
  pathways.
• S. cerevisiae proteins indicates an apparent
  ratio of the G-protein subunits in the pheromone
  pathway of 5 (Gpa1 Ga )3 (Ste18 G? )1 (Ste4
  Gß).
• Similar imbalances of G-protein subunits in other
  signaling pathways or whether GPCR-mediated
  monomeric Ga signaling pathways in mammals?

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SUMMARY

• Both yeasts utilize G proteins to respond to
  ligand binding by receptors that detect
  pheromones in the environment.
• The specific use of the G-protein subunits in the
  pheromone pathways are not similar.
• The discoveries of signaling by monomeric Ga
  subunits in the S. pombe pheromone pathway the
  yeast possesses only a single Gß gene.
• Harder to identify monomeric Ga -signaling
  pathways in humans.
• Unlikely that the ability of monomeric Ga
  subunits to mediate G-protein signaling pathways
  is restricted to fungal systems.
• Both organisms provide valuable examples of
  alternative strategies for ways in which a
  eukaryote may utilize a similar G-protein-related
  tool set to accomplish similar goals.

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Acknowledgement

• American Society for Microbiology
• Eukaryotic Cell, March 2005, p. 495-503, Vol. 4,
  No. 3
• http//www.wikipedia.com
• The Forsburg Lab pombe Pages
• http//www-rcf.usc.edu/forsburg/
• GTP-binding G proteins online lecture notes
• http//bioweb.wku.edu/courses/biol566/L14GProtein
  s.html
• Dr. Claire Rinehart
• Fellow course-mates of BIOL 566 Advanced
  Molecular Genetics