Systematically Ranking the Tightness of Membrane Association for Peripheral Membrane Proteins

Yingchun Wang

State Key laboratory of Molecular Developmental BiologyInstitute of Genetics and Developmental Biology, CAS

Membrane Proteins

Model: Synechocystis

Widely used model for photosynthesis and respiration. Many proteins are related with photosynthesis or respiration.

3.96 Mb, 3672 protein coding genes.

Easy to uptake foreign genes and integrate into its own genome through homologous recombination, easy to make mutations.

Can grow photoautotrophically or heterotrophically using glucose as the sole carbon source.

Generation of renewable energy and waster water treatment.

Contains a large fraction of thylakoid membrane.

Arch Microbiol (2006) 184: 259–270

Overview of Synechocystis Membrane Proteomics

N-terminal sequencing (Electrophoresis, 1997; 1999).

MALDI-TOF (integral and thylakoid fraction) (Electrophoresis, 2000; Proteomic science, 2009).

MALDI-TOF (Outer, plasma, and thylakoid membrane) (Mol. Cell. Proteomics, 2002; 2004).

2-DE

Proteomics, 2005; 2007;

J Proteome Res, 2006; 2007;

J Chromatography A, 2010;

LC-MS

M

S

WCL1185

69

95

202

228

Identification of membrane and soluble proteins

MCP, 2014, 13:204-19

How to decide a non-TM containing protein identified from isolated membranes is a peripheral membrane protein or just some carry-over contamination from soluble the fraction.

Are membranes the primary functional places for such a non-TM containing protein.

Questions

Rational and Experimental Design

Identification of Synechocystis Proteome

B

No

of

pro

tein

s

A

216 3481783

(157,115) (20,0)(291, 133)

Membrane soluble

No. of TM

0

100

200

300

400

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18

Total

Identified

Separation Efficiency of Membranes

Proteins Encoded by Chromosome- or Plasmid-Borne Genes

Summary

2347 proteins were identified with 2-peptide match (64% of the proteome).

Coverages of identification for TM-containing proteins and the total proteins are both the highest.

Separation of membranes from the soluble fractions in highly efficient.

Proteins encoded by chromosome-borne genes are move likely to be identified than those encoded by plasmid-borne gene.

Tightness of Membrane Association

A

0

1

2

3

-1

-2

-3

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

1 2

3 4

0 1 2 3 4-1-2-3-4-5 5 6

Log2(Spectra count ratio (M/S))

216 3481783

(157,115) (20,0)(291, 133)

Membrane soluble

Known periplasmic proteins or

proteins with predicted signal

peptide

Proteins with 1 predicted TM

(by Sosui) at N-terminus

Proteins without predicted

signal peptide or TMs

predicted by LipoP and Sosui,

respectively

0

-1

-2

-3

-4

-5

-6

-7

-8

-0.5-1.0-1.5-2.0-2.5-3.0-3.5-4.0

Log2(Spectra count ratio (M/S))

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

Proteins with 1 predicted TM

Tightness of Membrane Association: PSI Subunits

Eric Boudreau, et al, The EMBO Journal, 1997

ycf4psaC

psaA

psaE

psaF

psaB

ycf3

psaL

B

0

1

2

3

-1

-2

-3

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

0 1 2 3 4-1-2-3-4-5 5 6

Log2(Spectra count ratio (M/S))

psaD

psbEC

psbD2 psbC

psbB

psbA2

ycf48

psbU

psbP2

psbB28-2

psbB28

psbB27

psbV

psbO

0

1

2

3

-1

-2

-3

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

0 1 2 3 4-1-2-3-4-5 5 6

Log2(Spectra count ratio (M/S))

Tightness of Membrane Association: PSII Subunits

atpE (ε)

atpF (b)

atpG (b’)

atpB (β)

atpA (α)

atpD (δ)

atpC (γ)

D

0 1 2 3 4-1-2-3-4-5 5 6

Log2(Spectra count ratio (M/S))

0

1

2

3

-1

-2

-3

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

ATP synthases

cpcG2

cpcE

apcE

apcF

cpcF

cpcD

apcB

cpcC1

cpcC2

apcCcpcB

apcD

cpcG1

E

apcA

cpcA

0

1

2

3

-1

-2

-3

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

0 1 2 3 4-1-2-3-4-5 5 6

Log2(Spectra count ratio (M/S))

Phycobilisomal Proteins

A working model for the PBS-dependent state transition.

Kondo , Photosynth Res, 2009

Ribosomal Proteins

F

rpl10

0

1

2

3

-1

-2

-3

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

0 1 2 3 4-1-2-3-4-5 5 6

Log2(Spectra count ratio (M/S))

Signaling Proteins

G

cheW

slr0302

0

1

2

3

-1

-2

-3

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

0 1 2 3 4-1-2-3-4-5 5 6

Log2(Spectra count ratio (M/S))

2-component signal transduction systems

ABC Transporters

H

0

1

2

3

-1

-2

-3

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

0 1 2 3 4-1-2-3-4-5 5 6

Log2(Spectra count ratio (M/S))

Lipoproteins

I

0

1

2

3

-1

-2

-3

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

0 1 2 3 4-1-2-3-4-5 5 6

Log2(Spectra count ratio (M/S))

Validation of The Tightness Measurement

Enriched Functions of PMPs With Strong orWeak Membrane Association

0

1

2

3

-1

-2

-3

0 1 2 3 4-1-2-3-4-5 5 6

Log2(Spectra count ratio (M/S))

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

Tight membrane association： M/S ratios > 0 (Red)Less tight membrane association： M/S ratios < 0 (Blue)

Enriched Functions of PMPs With Strong orWeak Membrane Association

Tightly membrane-associated proteins

Less tightly membrane-associated proteins

Smad-FHA

(IPR008984)

0 1 2 3 4 5 6-1-2-3-4

log2(Spectra count ratio (M/S))

Radical SAM

(IPR007197)

0 1 2 3 4 5 6-1-2-3-4

Log2(Spectra count ratio (M/S))

Methyltransferase

FkbM (IPR006342)

0 1 2 3 4 5 6-1-2-3-4

log2(Spectra count ratio (M/S))

01

2-1

-20

12

-1-2

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

Lo

g2(

Inte

nsi

ty r

atio

(M

/S))

Rossmann-like

α/β/α sandwich fold

(IPR014729)Elongator protein 3/MiaB/NifB

(IPR006638) GAF (IPR003018)

A B C

D E F

Proteins Sharing the Same Domain Associate the Membranes with Similar Tightness

Conclusions

This tightness of membrane association for PMPs can be measured using asemiquantitative proteomics approach.

Different tightness of membrane association may be required for performingdifferent functions.

Proteins sharing the same domain tend to associate the membranes withsimilar tightness.

The method can be extended to all prokaryotic and eukaryotic organisms

This work provides a global view of the structural organization of themembrane proteome with respect to divergent functions, and built thefoundation for future investigation of the dynamic membrane proteomereorganization in response to different environmental or internal stimuli.

Acknowledgements

Institute of Botany, CASDr. Fang Huang

Institute of Hydrobiology, CASDr. Jindong Zhao

Dr. Xudong Xu

Wang Lab at IGDBHaitao GeDr. Kehui LiuXiahe HuangChunting ShenYajun XieYuanya ZhangChen BuXiaofei LiuYujian WuJinglong WangYu KangLongfa Fang

University of Louisiana, LafayetteDr. Wu Xu