Membrane Protein Insertion Dr. Ross Dalbey 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 ATPase F 1 F o reaction center H+H+ chemoreceptor Tar translocase SecYEG lactose permease LacY energy conversion ATPase energy gain photosynthetic complex signallingchemosensors substrate transport permeases protein translocation translocases Membrane proteins play essential cell functions h. Key Question: How do proteins insert into the membrane? Current Understanding of Membrane Protein Insertion Different Insertion Pathways: Spontaneous, SRP, Sec Translocase The Discovery of YidC YidC mediates membrane protein insertion in bacteria James C. Samuelson 1, Minyong Chen 1, Fenglei Jiang 1, Ines Mller 2, Martin Wiedmann 2, Andreas Kuhn 3, Gregory J. Phillips 4 & Ross E. Dalbey 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 What we know about YidC and its mitochondrial homology Oxa1p E. coli has 4485 genes compared to ~ 25,000 in humans E. coli is used as model system How did we discover YidC is a membrane insertase? a)In vivo: Made a YidC depletion strain and showed insertion was inhibited b)In vitro: i) Crosslink YidC to a membrane protein during membrane insertion ii) Reconstitute membrane protein insertion with lipid vesicles containing only YidC Main Question Does YidC catalyze membrane insertion? In vivo Construction of YidC depletion strain 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 35 S-methionine Lysozyme Proteinase K Spheroplast Bacteria Inserted-Digested by PK Non-inserted- Resistant to PK Inserted Protein Uninserted Protein PK Peri IM Cyto IM Cyto Peri OM IM PK - + SDS-PAGE Gel Assay for Membrane Protein Insertion Effect of YidC depletion on Membrane Insertion In Vitro Can YidC be crosslinked to an inserting membrane protein? Photocrosslinking Approach 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 YidC and Ffh (SRP) interact with Pf3 coat In vitro Reconstitute insertion of a membrane protein using purified YidC incorporated into lipid vesicles Reconstitution of YidC into large unilamellar vesicles (LUVs) (Serek et al, 2004, EMBO J. 23, ) purified YidC E. coli lipids extruder proteo-LUVs mix 200 nm YidC YidC substrate determinants YidC function: as a foldase or insertase Structure The Border region and TM organization Function in inserting multispanning membrane proteins periplasm L7 N6 N23 I330 L340 Y370 L372 E415 K416 E445 R447 K480 S482 Y465 K493 M495 F509 P510 E536 L535 Defining the Aqueous/ Transmembrane border regions Undergraduate Projects Kelsey Kerton Abdul Wasey SpontaneousYidC onlySec/YidC YidC determinants ?