a possible project: building a synthetic organelle s. peisajovich lim lab rt10
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A possible project:
Building a synthetic Organelle
S. PeisajovichLim LabRT10
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Make modified endosome that:
1- Doesn’t merge with vacuole.2- Has unique lipid/protein identity.3- Is distinctly localized.Long term:Add specific function?Can we do that now?
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Lsb6/Pik1, Mss4
Vsp34 Ymr
Fab1 Ymr
Fig4
SacI
Inp54
Inp52/53cytoplasm
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PI3K ClassI
PtdIns[3,4,5]P3
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Starting point:
Endocytosis of Ste2
Add C-t fusions to Ste2 for protein recruitment to specific endosomes.
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Ste2
-factor
Pathway activationSte2
Ste2
Ste2
Vacuole
Vsp34
Fab1
PI>PIP4>PIP4,5
PI>PIP3
PIP3>PIP3,5
FYVE
Vsp15
Lsb6Pik1
Mss4
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Block Vsp34(recruiting YmrI?)
Block Fab1(recruiting Fig4 or SacI?)
1- Preventing merging with vacuole
Recruit Ymr1, Fig4, SacI to Ste2-containing endosomes(SacI will not hydrolyze any PI with vicinal phosphates, ideal?)
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Ste2
-factor
Pathway activationSte2
Ste2
Ste2
Vacuole
Vsp34
Fab1
PI>PIP4>PIP4,5
PI>PIP3
PIP3>PIP3,5
FYVE
Vsp15
Lsb6Pik1
Mss4
3-Phosphatase
X
3-Phosphatase
X
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2- Unique lipid/protein composition
Recruit PI3K (positive feedback by fusing PI3K to Akt PH domain)
Global expression of PTEN (3-specific phosphatase)
Note that yeast expression of PI3K is lethal, but this is solved by co-expression of PTEN.
Recruit other factors (colors to identify new organelle?)
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Ste2
-factor
Pathway activationSte2
Ste2
Vacuole
Vsp34
PI>PIP4>PIP4,5
PI>PIP3
Vsp15
Lsb6Pik1
Mss4
3-Phosphatase
X
PI3K
PIP4,5>PIP3,4,5
PH
PI3KPH
GFPPH
PTEN
Is PIP4,5 preset in the early endosome?Or we will need to recruit Lsb6/Mss4 also?
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3- Distinct localization
Add tags to:
-Ste2-or other factors that are recruited later (PH containing proteins)
So that they will localize to specific sites (cytoskeleton? Other ideas?)
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Some tools needed:
-ability to follow Ste2 internalization in normal cells (add fluorescent tag to Ste2 in wt strain?)
-Can we activate expression of Vsp34 and/or Fab1 corresponding phosphatases (SacI?), as well as PI3K, using mating promoters (or Ste2 processing is too fast for that? -PTEN might need to be constitutively expressed)
-Ideally one would like to have specific colors fused to localization markers (say GFP-FYVE domain -that is PIP3 binding-, RFP-PH -that is PIP3,4,5 binding, some other for PIP 4,5, yellow, cyan???What is the maximum number of colors we could use?)
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Steps:
- Define specific proteins to be used and build alternative constructs (constitutive vs induced expression?)
-Ste2 with terminal tags (color to follow localization in wt strain, recruitment motifs -zippers?)
-Phosphatases (YmrI, Fig4, SacI?? Others coming from other sources -not yeast?) with recruitment motifs (target to Ste2-early endosome).
-PI3K-PH with recruitment motif also (target to Ste2-early endosome).
-PTEN constitutive expression.
-Color markers fused to domains binding to different PIPs.