1. A possible project: Building a synthetic Organelle S. Peisajovich Lim Lab RT10

2. 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?

6. Lsb6/Pik1, Mss4 Vsp34 Ymr Fab1 Ymr Fig4 SacI Inp54 Inp52/53 cytoplasm

7. PI3K ClassI PtdIns[3,4,5]P 3

8. Starting point: Endocytosis of Ste2 Add C-t fusions to Ste2 for protein recruitment to specific endosomes.

9. Ste2  -factor Pathway activation Ste2 Vacuole Vsp34 Fab1 PI>PIP4>PIP4,5 PI>PIP3 PIP3>PIP3,5 FYVE Vsp15 Lsb6 Pik1 Mss4

10. 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?)

11. Ste2  -factor Pathway activation Ste2 Vacuole Vsp34 Fab1 PI>PIP4>PIP4,5 PI>PIP3 PIP3>PIP3,5 FYVE Vsp15 Lsb6 Pik1 Mss4 3-Phosphatase X X

12. 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?)

13. Ste2  -factor Pathway activation Ste2 Vacuole Vsp34 PI>PIP4>PIP4,5 PI>PIP3 Vsp15 Lsb6 Pik1 Mss4 3-Phosphatase X PI3K PIP4,5>PIP3,4,5 PH PI3K PH GFP PH PTEN Is PIP4,5 preset in the early endosome? Or we will need to recruit Lsb6/Mss4 also?

14. 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?)

15. 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?)

16. 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.