AlleleSpecific Suppression

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Allele-Specific Suppression

• Luke, Mike and Nomalie
• April, 5th 2005

Overexpression of the yeast formin protein
Bni1p http//www.utoronto.ca/boonelab/index.htm
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(No Transcript)
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Actin and Fimbrin
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Actin and Fimbrin

• How would one go about identifying these
  interactions genetically?

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What types of mutants are best suited for
suppressor screens?(specifically for physical
interactions)
dominant/recessive missense/nonsense temperature
sensitive
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So you have a suppressor . . . . now what?

• Is it intragenic or extragenic?
• Does it suppress other alleles of the same
  gene?
• If not, is it allele-specific?

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Allele-Specific Suppression

• Suppression in which only particular alleles of
  one gene are suppressed by particular alleles of
  another gene.

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S. cerevisiae Act1p Bundling in vitro
Act1p w/o Sac6p
Act1p w/Sac6p
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Suppressible Act1p mutants
Subdomain 2
Subdomain 1
Actin mutations that show suppression with
fimbrin mutations identify a likely
fimbrin-binding site on actin.Honts JE, Sandrock
TS, Brower SM, O'Dell JL, Adams AE. J Cell Biol.
1994 Jul126(2)413-22.
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Lock and Key vs. Novel Contact
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Lock and Key vs. Novel Contact
Restoration
Novel Contact
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Results Based on Biochemical Assay
Unlike previous papers in class that derived
results by scoring or classifying phenotypes, the
results in this study rested primarily on a
biochemical assay.
Relevant issues to keep in mind
1) Does the in vitro analysis capture what is
happening in the cell?
A) Are the concentrations of salt used in the
this study (.125 to 1.0 M) within the range of
what would be encountered in a cell? Does it
matter? B) What other conditions may not match?
Temperature? Neighboring proteins? C) Can useful
data still be extracted even if conditions are
not equivalent?
2) Is there any corroborating phenotypic evidence
that may help alleviate concerns in 1 above?
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Binding test
Stronger binding (sac6p in pellet)

• Purified actin and Sac6p are mixed and
  centrifuged
• Recover actin filaments in pellet
• Sac6 (bound to actin) is recovered in pellet
• Unbound Sac6 remains in supernant
• Performed at different salt concentrations to
  probe strength of interaction

Weak/no binding (sac6p in supernatant)
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Bundling test
Bundling of actin due to sac6p

• Similar to binding assay except centrifuged at
  low speed to recover cross linked actin
  filaments.
• Additionally, bundling was assayed by electron
  microscopy before centrifugation to visualize
  bundles vs. individual elements.

Unbundled individual actin filaments In absence
of sac6p
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Results from Binding Test
Wildtype
Binds Stronger than WT
Strongest of all
Suppression
Suppression
Weak/No Binding
Wildtype Binding
Wildtype Binding
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Results from Bundling Test Overlaid
Wildtype
Binds Stronger than WT
Strongest of all
Suppression
Suppression
Weak/No Binding
Wildtype Binding
Wildtype Binding
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Results from Both Tests combined
Wildtype
Binds Stronger than WT
Strongest of all
Suppression
Suppression
Weak/No Binding
Wildtype Binding
Wildtype Binding
Is there any phenotypic evidence that
corroborates or contradicts the above results for
the interactions that have stronger binding than
WT?
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Results from Both Tests combined
Wildtype
Binds Stronger than WT
Strongest of all
Suppression
Suppression
Weak/No Binding
Wildtype Binding
Wildtype Binding
Is there any phenotypic evidence that (possibly)
corroborates the above results for the
interactions that have stronger binding than WT?
ACT1 sac6-19 mutants have a strong growth defect
while ACT1 sac6-5 strains do not.
Why could the strongest binding be detrimental?
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Results from Both Tests combined
Wildtype
Binds Stronger than WT
Strongest of all
Suppression
Suppression
Weak/No Binding
Wildtype Binding
Wildtype Binding
Do the above results demonstrate allele specific
interaction?
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Results from Both Tests combined
Wildtype
Binds Stronger than WT
Strongest of all
Suppression
Suppression
Weak/No Binding
Wildtype Binding
Wildtype Binding
Do the above results demonstrate allele specific
interaction?
The act1-120 allele can be suppressed by more
than one mutant Sac6 protein (other examples
exist). Thus the authors state We therefore
prefer the term allele-restricted suppression to
describe this phenomenon.
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Results from Both Tests combined
Wildtype
Binds Stronger than WT
Strongest of all
Suppression
Suppression
Weak/No Binding
Wildtype Binding
Wildtype Binding
Do these results tend to support a lock-and-key
model?
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Results from Both Tests combined
Wildtype
Binds Stronger than WT
Strongest of all
Suppression
Suppression
Weak/No Binding
Wildtype Binding
Wildtype Binding
Do these results support a lock-and-key model?
What model of protein interaction could explain
this (more to come)
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Structural studies of FimbrinDoes structure
support Genetics?

• Fimbrin one of the largest family of
  actin-crosslinking proteins is characterized by a
  conserved F-actin binding domain (ABDs).
• Unique property of Fimbrins is that they possess
  two tandem repeats of the ABD within a single
  polypeptide chain.
• Results in the formation of tightly bundled actin
  filaments that function in dynamic process (i.e.
  cytokinesis in yeast, intestinal microvilli in
  vertebrates).
• Recent Crystal structure analysis of the Fimbrin
  core from A. thaliana and S. pombe reveal unique
  features of the molecule supporting previous
  genetic studies.

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Interaction of the fimbrin molecule with actin to
form crosslinked actin polymers
The two ABD domains have nonidentical
interactions with F actin
Volkmann et al., 2001
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Structural plasticity of the CH domains
ABD1
ABD2
Klein et al, Structure, 2004
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Superpostition of allele specific suppressor
mutations on to the actin binding domain

• Suppressor mutants are not in a region that
  directly bind to actin
• How can they restore function?

ABD1
ABD2
Sac 6-19P A383E
Sac 6-5P D264Y
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Amino acid mutations involved in the two
suppressors
Sac 6-19P A383E
Mutation results in a switch in a hydrophobic
residue to a hydrophilic residue.
Sac 6-5P D264Y
Mutation introduces a bigger side chain and a
loss of a charge.
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APOG- Structure supports genetics

• In both ABDs, the participation of ABS1in F-actin
  binding require a conformational rearrangement.
• This corrobates the conclusion reached by the
  authors that the supression occurs via the
  formation of new sites and not through a lock and
  key mechanism.