Membrane Protein Insertion

Membrane Protein Insertion

Dr. Ross Dalbey

dalbey@chemistry.ohio-state.edu

Feb 3, 2012

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

ATPaseF1Fo

reactioncenter

H+

chemoreceptorTar translocase

SecYEG

lactosepermease

LacY

• energy conversion ATPase• 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?

Current Understanding of Membrane Protein Insertion

Different Insertion Pathways: Spontaneous, SRP, Sec Translocase

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

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

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

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 35S-methionine

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

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 YidCincorporated into lipid vesicles

Reconstitution of YidC into large unilamellar vesicles (LUVs) (Serek et al, 2004, EMBO J. 23, 294-301)

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

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

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