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A possible project:

A possible project:. Building a synthetic Organelle. S. Peisajovich Lim Lab RT10. 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?. Fig4. Lsb6/Pik1, Mss4.

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A possible project:

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  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?

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

  4. PI3K ClassI PtdIns[3,4,5]P3

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

  6. Vsp34 -factor Ste2 Pathway activation PI>PIP4>PIP4,5 Ste2 Lsb6Pik1 Mss4 PI>PIP3 Ste2 Vsp15 PIP3>PIP3,5 Fab1 FYVE Ste2 Vacuole

  7. 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?)

  8. X X 3-Phosphatase 3-Phosphatase Vsp34 -factor Ste2 Pathway activation PI>PIP4>PIP4,5 Ste2 Lsb6Pik1 Mss4 PI>PIP3 Ste2 Vsp15 PIP3>PIP3,5 Fab1 FYVE Ste2 Vacuole

  9. 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?)

  10. GFP PH PI3K PH X 3-Phosphatase Vsp34 -factor Ste2 Pathway activation PI>PIP4>PIP4,5 Ste2 Lsb6Pik1 Mss4 PI>PIP3 PIP4,5>PIP3,4,5 Ste2 Vsp15 PTEN PI3K PH Is PIP4,5 preset in the early endosome? Or we will need to recruit Lsb6/Mss4 also? Vacuole

  11. 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?)

  12. 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?)

  13. 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.

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