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Membrane Protein Insertion

Membrane Protein Insertion. Dr. Ross Dalbey dalbey@chemistry.ohio-state.edu Feb 3, 2012. Membrane Proteins. Significance: 25% of proteins in human cells are membrane proteins; membrane proteins make up more than 50% of the known drug targets. Membrane proteins play essential cell functions.

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Membrane Protein Insertion

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  1. Membrane Protein Insertion • Dr. Ross Dalbey • dalbey@chemistry.ohio-state.edu • Feb 3, 2012

  2. Membrane Proteins Significance: 25% of proteins in human cells are membrane proteins; membrane proteins make up more than 50% of the known drug targets

  3. Membrane proteins play essential cell functions • energy conversion ATPase • energy gain photosynthetic complex • signallingchemosensors • substrate transport permeases • protein translocation translocases ATPase F1Fo chemoreceptor Tar reaction center lactose permease LacY translocase SecYEG h. H+ Key Question: How do proteins insert into the membrane?

  4. Current Understanding of Membrane Protein Insertion Different Insertion Pathways: Spontaneous, SRP, Sec Translocase

  5. The Discovery of YidC • YidC mediates membrane protein insertion in bacteria • James C. Samuelson1, Minyong Chen1, Fenglei Jiang1, Ines Möller2, Martin Wiedmann2, Andreas Kuhn3, Gregory J. Phillips4 & Ross E. Dalbey1

  6. Significance • Our objective as scientists is to advance the knowledge of membrane protein insertion in humans. • Humans contain the essential mitochondrial YidC homolog Oxa1 • The essential bacterial YidC is a potential antibacterial target

  7. What we know about YidC and its mitochondrial homology Oxa1p

  8. E. coli is used as model system E. coli has 4485 genes compared to ~ 25,000 in humans

  9. How did we discover YidC is a membrane insertase? • In vivo: • Made a YidC depletion strain and showed insertion • was inhibited • In vitro: • i)Crosslink YidC to a membrane protein during membrane • insertion • ii) Reconstitute membrane protein insertion with lipid • vesicles containing only YidC

  10. Main Question • Does YidC catalyze membrane insertion?

  11. In vivo

  12. Construction of YidC depletion strain

  13. YidC depletion studies • Grow overnight E.coli culture with arabinose • Wash cells to remove residual arabinose • Back-dilute into LB containing arabinose or glucose • Grow until a significant growth defect is observed (2.5 – 3 hrs) • Switch to minimal media for protein labeling with 35S-methionine

  14. Assay for Membrane Protein Insertion IM IM Proteinase K Lysozyme OM Cyto Cyto Spheroplast Peri Bacteria PK PK Peri IM Cyto Inserted-Digested by PK Non-inserted-Resistant to PK SDS-PAGE Gel PK - + PK - + Uninserted Protein Inserted Protein

  15. Effect of YidC depletion on Membrane Insertion

  16. In Vitro Can YidC be crosslinked to an inserting membrane protein?

  17. Photocrosslinking Approach

  18. In vitro photocrosslinking studies • Synthesize radioactive truncated membrane protein • Membrane protein remains bound to ribosome since lacks stop codons • Generate insertion intermediate with inverted inner membrane vesicles • Introduce photoprobe • Shining UV light results in photocrosslinking

  19. YidC and Ffh (SRP) interact with Pf3 coat

  20. In vitro Reconstitute insertion of a membrane protein using purified YidC incorporated into lipid vesicles

  21. Reconstitution of YidC into large unilamellar vesicles (LUVs) (Serek et al, 2004, EMBO J. 23, 294-301) purified YidC E. coli lipids mix extruder proteo-LUVs 200 nm 

  22. YidC substrate determinants Function in inserting multispanning membrane proteins YidC function: as a foldase or insertase YidC Structure The Border region and TM organization

  23. Undergraduate Projects Defining the Aqueous/ Transmembrane border regions R447 Kelsey Kerton P510 E445 F509 I330 L340 Y465 N23 periplasm L535 L7 Y370 K480 M495 K416 Abdul Wasey L372 N6 E536 S482 K493 E415 YidC determinants ? Spontaneous YidC only Sec/YidC

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