A possible project:

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A possible project
Building a synthetic Organelle
S. Peisajovich Lim Lab RT10
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Make modified endosome that 1- Doesnt 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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Fig4
Lsb6/Pik1, Mss4
Vsp34 Ymr
Inp52/53 cytoplasm
Fab1 Ymr
SacI Inp54
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PI3K ClassI
PtdIns3,4,5P3
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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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?-factor
Ste2
Pathway activation
Ste2
PIgtPIP4gtPIP4,5
Lsb6Pik1
Mss4
PIgtPIP3
Ste2
Vsp15
PIP3gtPIP3,5
Fab1
FYVE
Ste2
Vacuole
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1- Preventing merging with vacuole Recruit Ymr1,
Fig4, SacI to Ste2-containing endosomes (SacI
will not hydrolyze any PI with vicinal
phosphates, ideal?)
Block Vsp34 (recruiting YmrI?)
Block Fab1 (recruiting Fig4 or SacI?)
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?-factor
Ste2
Pathway activation
Ste2
PIgtPIP4gtPIP4,5
Lsb6Pik1
Mss4
PIgtPIP3
Ste2
Vsp15
PIP3gtPIP3,5
Fab1
FYVE
Ste2
Vacuole
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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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?-factor
Ste2
Pathway activation
Ste2
PIgtPIP4gtPIP4,5
Lsb6Pik1
Mss4
PIgtPIP3
Ste2
Vsp15
PIP4,5gtPIP3,4,5
PTEN
PI3K
PH
Is PIP4,5 preset in the early endosome? Or we
will need to recruit Lsb6/Mss4 also?
Vacuole
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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.