1

In mammalian cells mitochondrial carriers are involved in a number of disorders:

In mammalian cells mitochondrial carriers are involved in a number of disorders:

diseases, like several diseases, like several myopathiesmyopathies

obesityobesity

programmed cell death (apoptosis)programmed cell death (apoptosis)

2

3

Distinct features of the “carrier pathway” compared to “matrix targeting”:

Distinct features of the “carrier pathway” compared to “matrix targeting”:

1. Requirement for a chaperone function in the IMS1. Requirement for a chaperone function in the IMS

2. No ATP hydrolysis in the matrix is necessary2. No ATP hydrolysis in the matrix is necessary

3. No translocation motor in the matrix is required3. No translocation motor in the matrix is required

4. The electrochemical membrane potential across the 4. The electrochemical membrane potential across the IM is enough to complete insertion at the IMIM is enough to complete insertion at the IM

4

Properties of the “small Tim proteins” - 1Properties of the “small Tim proteins” - 1

Sequence characteristics:Sequence characteristics:

They are all homologousThey are all homologous

They are intrinsically soluble (No They are intrinsically soluble (No transmembranetransmembrane domains)domains)

They have a putative “zincThey have a putative “zinc--binding” motif, twin CX3C motifbinding” motif, twin CX3C motif

Found in all eukaryotic cellsFound in all eukaryotic cells

Human homologue of Tim8Human homologue of Tim8

involved in Mohrinvolved in Mohr--TranebjaergTranebjaerg syndromesyndrome

Organisation in assemblies:Organisation in assemblies:

Tim9/10 and Tim8/13 are found in 70 Tim9/10 and Tim8/13 are found in 70 kDakDa complexes in the IMScomplexes in the IMS

Tim12 is associated with the membraneTim12 is associated with the membrane--embedded TIM22 complex (IM)embedded TIM22 complex (IM)

CX3C

X15/16

CX3C

Zn2+H2N COOH

5

Properties of the “small Tim proteins” - 2Properties of the “small Tim proteins” - 2

Function:Function:Tim9/10 (Tim9/10 (essentialessential) function as chaperones in the IMS and facilitate ) function as chaperones in the IMS and facilitate targeting to the IMtargeting to the IM

Tim12 (Tim12 (essentialessential) facilitates insertion in the context of the TIM22 ) facilitates insertion in the context of the TIM22 complexcomplex

Tim8/13 are Tim8/13 are nonnon--essentialessential but also seem to function as chaperones but also seem to function as chaperones in the IMSin the IMS

6

The AAC substrate: targeting signals??

7

Which are the sorting and insertion signals of the carrier proteins?

Which are the sorting and insertion signals of the carrier proteins?

1 30 DHFR

1 50 DHFR

1 80 DHFR

40 80 DHFR

AAC

1

8050

300

IM

MATRIX

IMS

A B C

TM I II III IV V VI30

40

A. Deletion Analysis

B. Site-directed mutagenesis

Radiolabelled protein synthesised in a rabbit

reticulocyte lysate.

Purified mitochondria

OM

IMS

IM

Matrix

TOM

TIM23 TIM22

Cytosol

OM

IMS

IM

Matrix

TOM

TIM23 TIM22

Cytosol

ProteinaseK treatment

Import for 10min at

30°C

TIM10

TIM10

TIM10

AAC fragment DHFRN C

1TM

2TM

4TM

1Loop

AAC

N C

IM

MATRIX

IMS

A B C

2 3 4 5 6TM 1

Loop 1 Loop 2 Loop 3

AAC-DHFR CONSTRUCTS

LOCALISATION

S1

S2

OM

IMS

IM

Matrix

TOM

TIM23 TIM22

Cytosol

P TIM10

Mitoplast

1TM

1loop

2TM

4TM

10% tot S1 P S2

•Main localisation in IM

•Increase TM numbers IM and Matrix

Import into tim12-tsmitochondria

• No Tim12 • No Tim22

OM

IMS

IM

Matrix

TOM

TIM23 TIM22

Cytosol

TIM10

IS THE TIM22 COMPLEX REQUIRED FOR IMPORT?

1TM

1loop

2TM

4TM

10% M MP M MP

WT tim12-ts

Import into Mitoplasts

• No TIM10 complex

OM

IMS

IM

Matrix

TOM

TIM23 TIM22

Cytosol

TIM10

IS THE TIM10 COMPLEX REQUIRED FOR IMPORT?

1TM

1loop

2TM

4TM

10% M MP

Import into mitoplasts +

peptide competition

-Hsp60= presequence peptide

- Synb2= charged peptide

OM

IMS

IM

Matrix

TOM

TIM23 TIM22

Cytosol

TIM10

IS THE TIM23 COMPLEX REQUIRED FOR IMPORT?

1TM

1loop

2TM

4TM

10% M Ø 60 b2

MP

Sufficient targeting information within each construct, BUT:

•No interaction with TIM10 complex

•Therefore no targeting to TIM22

•Translocation via TIM23 as a default pathway

WORKING MODEL

OM

IMS

IM

Matrix

TOM

TIM23 TIM22

Cytosol

TIM10

15

Import Scheme for AAC

Blue native gel analysis= dimer formation

Sodium carbonate treatment= integral membrane proteins

Import25o C, ∆Ψ +

35 S-precursor,Rabbit reticulocyte lysate

IsolatedMitochondriaAAC,

or mutants

+

Mitoplasting +Proteinase K

16

Stage III of AAC import

70 20 2240 6 7 5

109 9 9

10 10

10129

2254

TOM COMPLEX

TIM 22 COMPLEX

OUTER MEMBRANE

INNER MEMBRANE

INTERMEMBRANE SPACE

18

STAGE III

∆Ψ-

AAC

17

Stage IV of AAC Import

70 20 2240 6 7 5

109 9 9

10 10

1012922

54

TOM COMPLEX

TIM 22 COMPLEX

OUTER MEMBRANE

INNER MEMBRANE

INTERMEMBRANE SPACE

18

STAGE III

STAGE IV(Insertion)

∆Ψ+

?

18

Stage V of AAC Import

70 20 2240 6 7 5

109 9 9

10 10

1012922

54

TOM COMPLEX

TIM 22 COMPLEX

OUTER MEMBRANE

INNER MEMBRANE

INTERMEMBRANE SPACE

18

STAGE IV STAGE V(Dimerisation)?

19

Inner Membrane

Intermembrane space

Matrix

NC

REPEAT 1 REPEAT 2 REPEAT 3

TM1

TM3

TM4

TM5

TM6

TM2

63

279

262

= Cysteine residue

20

Cys mutants affecting AAC insertion in the IM Cys mutants affecting AAC insertion in the IM

1 2 5 10 10 Time/min∆Ψ+ +++ -10%

input

+ +++ -1 2 5 10 10

wtAAC1 CS63

A

AACV

1 2 5 10 20 20 Time/min∆Ψ+ ++ ++ -10%

input

CS262

CS279

CS3X

B

AACV

AACV

21

Cys mutants affecting AAC insertion in the IM Cys mutants affecting AAC insertion in the IM

• Functionally impaired Cys mutants have a significantdefect also in insertion and dimerisation of AAC1

• CS63 (first loop) and the triple Cys mutant are abrogated in their efficiency to form a dimer in the membrane

CS63 CS262 CS279 CS3X wtAAC1

22

Can we reconstitute in vitro the TIM10 complex in

a functional form?

Can we reconstitute in vitro the TIM10 complex in

a functional form?

23

1. Co-expression of Tim9 and Tim10 in E. coli on a single plasmid in an operon

2. Expression and purification of the individual proteins separately

3. Purification of the authentic complex from yeast mitochondria

Tim9, Tim10 necessary and sufficient for assembly of TIM10 complex

Reconstituted complex indistinguishable from mitochondrial complex

YES!

Reconstitution of the complex from the individual subunits

Reconstitution of the complex from the individual subunits

24

Is the reconstituted complex functional?

Is the reconstituted complex functional?

Functionality Assays:

1. Restoration of AAC import into TIM10-depleted mitochondria2. Binding in vitro to AAC3. Chaperone activity in vitro

25

WT Tim9ts Tim9ts Tim9tsTim9ts Tim10ts

+Tim9 +Tim9+Tim10

+Tim10+Tim9

+Tim9

T P S T P S T P S T P S T P S T P S

Reconstitution in Tim9ts Mitochondria

AAC import into Tim9ts Mitochondriais restored after Import of Tim9 and/or Tim10

26

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60 70

Time (minutes)

Luci

fera

seac

tivity

(p

erce

nt o

f nat

ive

The TIM10 complex chaperones luciferaserefolding

The TIM10 complex chaperones luciferaserefolding

DnaK/J/E

TIM10 complex

9 or 10 alone

27

What is the structural basis for the assembly of

the TIM10 complex?

What is the structural basis for the assembly of

the TIM10 complex?

28

190 200 210 220 230 240 250 260-30

-20

-10

0

10

20

30

Wavelength (nm)

+6M GuHCl+1mMDTT

+6M GuHCl

+1mM DTT Tim10

Tim10

θ*1

0-3

(deg

cm

2dm

ol-1

res-

1 )

260190 200 210 220 230 240 250-30

-20

-10

0

10

20

30

40

Wavelength (nm)

+6M GuHCl+1mMDTT

+6M GuHCl

+1mM DTT Tim9

Tim9θ

*10-

3(d

eg c

m 2

dmol

-1re

s-1 )

CD Analysis of individual Tim9 and Tim10 CD Analysis of individual Tim9 and Tim10

49%39%12%redTim9

38%8%53%oxTim9

59%33%8%red Tim10

35%-65%oxTim10

restβ-sheetα-helix

29

14

7TCEP - - + - - +AMS - + + - + +

M Tim10 Tim9

GuHCl-DTT GuHCl+DTTTim9 0 4.2 ± 0.5Tim10 0 3.6 ± 0.3

DTNB assay

Thiol-trapping and accessibility by AMS and DTNB

30

-0.2

-0.1

0.0

0.10 50 100 150 200

Time (min)

µcal

/sec

0.0 0.5 1.0 1.5 2.0 2.5-12

-10

-8

-6

-4

-2

0

2

Molar Ratio (Zinc/Tim9)

kcal

/mol

e

0.0 0.5 1.0 1.5 2.0-10

-8

-6

-4

-2

0

2

Tim10Tim9

Molar Ratio (Zinc/Protein)kc

al/m

ole

N= 0.71Ka= 6x106 ±3x106 M-1

∆H= -8500cal/mol

N= 1.1Ka= 9x106 ±3x106 M-1

∆H= -7700cal/mol

Zinc binds to reduced Tim9 and reduced Tim10, not the oxidised states

31

Oxidised Tim9 and oxidised Tim10 form the complex

7 8 9 10 11 12 13 14 15

0

100

200

300

400

500 Oxi Red+EDTA

Red+Zn2+O

D21

5 (m

AU)

Volume (ml)

32

ITC study of the interaction between Tim9 and Tim10

-0.2

-0.1

0.0

0 20 40 60 80 100 120 140Time (min)

µcal

/sec

0.0 0.5 1.0 1.5 2.0-15

-10

-5

0

Molar Ratio

kcal

/mol

e N= 0.9 (Tim10/Tim9)Ka= 5x106 M-1

∆H= -13kcal/mol

Reduced Tim9/Tim10

Oxidised Tim9/Tim10

33

CD analysis of the TIM10 complex

190 200 210 220 230 240 250 260

-20

-10

0

10

20

30

40

Wavelength (nm)

θ x1

0-3(d

eg c

m2

dmol

-1re

s-1)

buffer only+1mM DTT+6M GuHCl+6M GuHCl+1mMDTT

34

DTT concentration dependence

0.0 0.5 1.0 1.5 2.00

20

40

60

80

100 TIM10 complex

Tim9

Tim10

Rel

ativ

e C

D s

igna

l at 2

22nm

(%)

DTT (mM)

190 200 210 220 230 240 250

-15

-10

-5

0

5

10

15

20

CD

(m

deg)

Wavelength (nm)

[DTT]

35

AMS and DTNB assay for TIM10 complex

TCEP - - + - - + - - + AMS - + + - + + - + +

Tim10 Tim9 TIM10

DTNB assay of the complex:GuHCl-DTT: 0GuHCl+DTT: 8.2±0.5 per Tim9/10

36

The TIM10 complex is more stable against trypsin digestion than the individual proteins

αTim9

αTim10

Tim9 or Tim10 Complex

Time /min 0 5 10 20 30 60 0 5 10 20 30 60

37

αTim10

αTim9

Fraction 10 11 12 13 10 11 12 13

2921

12.5

6.5

2921

12.5

6.5

+DTT -DTT

Western Blotting

7 8 9 10 11 12 13 14 15 16 17

0

200

400

600

800

OD 21

5nm

(mA

U)

Volume (ml)

Inter- vs. intra- molecular disulfides: Misfolding & complex formation

38

Dynamics and size analysis by multi-angle Light scattering

8 9 10 11 12 13 14 15

1000

10000

100000

Molar M

ass (g/mol)

MolarMass

Volume (ml)

0.0

0.1

0.2

0.3

0.4

0.5 LS.AUX

LS, A

UX

(vol

ts)

TIM10 complex

9 10 11 12 13 14

0.0

0.1

0.2

0.3

0.4

LS, A

UX

(vol

ts)

Volume (ml)

1000

10000

Molar M

ass (g/mol)

Tim9

1000

10000

100000Molar M

ass (g/mol)

9 10 11 12 13 14

0.0

0.2

0.4

0.6

0.8

LS, A

UX

(vol

ts)

Volume (ml)

Tim10

17K

16K

20K

Individual proteins

Tim9

39

T10 . T10 T10 T10 –T10

intra-molecular S-Snon-covalentproductive

inter-molecular S-Scovalentabortive

T9 . T9 T9 T9 –T9

40

Compartment-specific redox regulation of TIM10

assembly?

41

Prior Oxidation inhibits import

µM ZnCl2

10%Control

OxidisedTim10

Reduced Tim10

0 5 50 100

Additional data:1. NEM alkylation2. Cys mutants

43

Oxidative folding locks the assembly

of the TIM10 complex in the intermembrane space

Cytosol Mitochondrion

OM IM

TOMoxidativefolding

oxidativefolding

IMS Matrix

2 reduced

4

3 oxidised

1 reduced

1 reduced 2 reduced

assembly

SHSH

SHSH

S-SS-S

S-SS-S

TOM

TOM

SHSH

SHSHTim9

Tim10

SH

45

Are there distinct functional domainsin the small Tims?

46

3D structure?

47

0.001

0.01

0.1

1

0 0.1 0.2 0.3 0.4 0.5 0.6

Tim10Tim9

complex

Inte

nsity

[a.

u.]

q = 4π sin Θ / λ [Å-1]

0

0.005

0.01

0.015

0.02

0.025

0.03

0 20 40 60 80

p(r)

r [Å]

SAXS Analysis

48

Tim9-Tim10complex

Tim9

Tim10

15 Å

49

Acknowledgments Acknowledgments

University of Manchester

Present: Stephanie Agius

Felicity Alcock

Scott Allen

Hui Lu

Maïlys Vergnolle

Past: Sabrina Dyall (UCLA, USA)

Pierre Luciano (CNRS,Marseilles France)

Peter Savory (Scotland Diagnostics)

Sarah Vial (Vertex Pharmaceuticals, Oxford UK)

CollaboratorsDaresbury Laboratory

Günter Grossmann

FundingThe Lister Institute, The Welcome Trust, MRC, BBSRC, EU, The Leverhulme Trust, The Royal Society

UMIST

Alexander Golovanov, Lu-Yun Lian

IMBB-FORTHCatherine Baud