A possible project:

Building a synthetic Organelle

S. PeisajovichLim LabRT10

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?

Lsb6/Pik1, Mss4

Vsp34 Ymr

Fab1 Ymr

Fig4

SacI

Inp54

Inp52/53cytoplasm

PI3K ClassI

PtdIns[3,4,5]P3

Starting point:

Endocytosis of Ste2

Add C-t fusions to Ste2 for protein recruitment to specific endosomes.

Ste2

-factor

Pathway activationSte2

Ste2

Ste2

Vacuole

Vsp34

Fab1

PI>PIP4>PIP4,5

PI>PIP3

PIP3>PIP3,5

FYVE

Vsp15

Lsb6Pik1

Mss4

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?)

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

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?)

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?

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?)

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?)

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.