membrane protein insertion dr. ross dalbey 580 bioscience [email protected] nov 16,...
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![Page 1: Membrane Protein Insertion Dr. Ross Dalbey 580 Bioscience dalbey@chemistry.ohio-state.edu Nov 16, 2012](https://reader035.vdocuments.mx/reader035/viewer/2022081519/56649f125503460f94c264b9/html5/thumbnails/1.jpg)
Membrane Protein Insertion
Dr. Ross Dalbey
580 Bioscience
Nov 16, 2012
![Page 2: Membrane Protein Insertion Dr. Ross Dalbey 580 Bioscience dalbey@chemistry.ohio-state.edu Nov 16, 2012](https://reader035.vdocuments.mx/reader035/viewer/2022081519/56649f125503460f94c264b9/html5/thumbnails/2.jpg)
Membrane ProteinsSignificance: 25% of proteins in human cells are membrane proteins; membrane proteins make up more than 50% of the known drug targets
![Page 3: Membrane Protein Insertion Dr. Ross Dalbey 580 Bioscience dalbey@chemistry.ohio-state.edu Nov 16, 2012](https://reader035.vdocuments.mx/reader035/viewer/2022081519/56649f125503460f94c264b9/html5/thumbnails/3.jpg)
ATP synthase F1Fo
reactioncenter
H+
chemoreceptorTar translocase
SecYEG
lactosepermease
LacY
• energy conversion ATP synthase• energy gain photosynthetic complex• signalling chemosensors• substrate transport permeases• protein translocation translocases
Membrane proteins play essential cell functions
h.
Key Question: How do proteins insert into the membrane?
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Current Understanding of Membrane Protein Insertion
Different Insertion Pathways: Spontaneous, SRP, Sec Translocase
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The Discovery of YidC
YidC mediates membrane protein insertion in bacteriaJames C. Samuelson1, Minyong Chen1, Fenglei Jiang1, Ines Möller2, Martin Wiedmann2, Andreas Kuhn3, Gregory J. Phillips4 & Ross E. Dalbey1
![Page 6: Membrane Protein Insertion Dr. Ross Dalbey 580 Bioscience dalbey@chemistry.ohio-state.edu Nov 16, 2012](https://reader035.vdocuments.mx/reader035/viewer/2022081519/56649f125503460f94c264b9/html5/thumbnails/6.jpg)
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
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N C
periplasm
cytoplasm
innermembrane
+11 +2+7+1
+1 0
YidC
+5 +1
-2 0
Oxa1pN
intermembrane space
innermembrane
mitochondrialmatrix
-7
C
+15
What we know about YidC and its mitochondrial homology Oxa1p
![Page 8: Membrane Protein Insertion Dr. Ross Dalbey 580 Bioscience dalbey@chemistry.ohio-state.edu Nov 16, 2012](https://reader035.vdocuments.mx/reader035/viewer/2022081519/56649f125503460f94c264b9/html5/thumbnails/8.jpg)
E. coli has 4485 genes compared to ~ 25,000 in humans
E. coli is used as model system
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How did we discover YidC is a membrane insertase?
In vivo: Made a YidC depletion strain and showed insertion was inhibited
![Page 10: Membrane Protein Insertion Dr. Ross Dalbey 580 Bioscience dalbey@chemistry.ohio-state.edu Nov 16, 2012](https://reader035.vdocuments.mx/reader035/viewer/2022081519/56649f125503460f94c264b9/html5/thumbnails/10.jpg)
Main Question
Does YidC catalyze membrane insertion?
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In vivo
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Construction of YidC depletion strain
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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
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Lysozyme Proteinase K
SpheroplastBacteria
Inserted-Digested by PK
Non-inserted-Resistant to PK
Inserted Protein Uninserted Protein
PK PKPeri
IM
Cyto
IM
CytoCyto
Peri
OM
IM
PK - + PK - +SDS-PAGEGel
Assay for Membrane Protein Insertion
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Effect of YidC depletion on Membrane Insertion
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YidC
YidC substrate
determinants
YidC function: as a foldase or
insertase
Structure
The Border region and TM organization
Function in inserting
multispanning membrane
proteins
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periplasm
L7
N6
N23I330
L340
Y370
L372
E415
K416
E445R447
K480
S482
Y465
K493
M495
F509P510
E536
L535
Defining the Aqueous/ Transmembrane border regions
Undergraduate Projects
Kelsey Kerton
Abdul Wasey
Spontaneous YidC only Sec/YidC
YidC determinants
?