Protein Complexes, Networks, and Pathways

• components of protein complexes

• interactions between protein complexes

• functional pathways

α

γ

τ

δ

δ`

ε

Ssb

χψ

Pol III SubunitAccessory Proteins

TopB

RecQ

* *

*

*

*

χ-AP

θ-AP

ψ-AP

α-APε-A

PDNA Polymerase III Core Enzyme and Clamp Loader Complexes

• purification of a core enzyme subunit (αθε) yields sub- stoichiometric amounts of the clamp-loader complex (γδδ’χψ)

• purification of a clamp- loader subunit (γδδ’χψ) yields sub-stoichiometric amounts of core enzyme

• the clamp loader is also associated with Ssb, TopB, and RecQ

• TopB and RecQ are also associated with RNaseH1, which degrades RNA primers

Core Polymerase

DnaB helicase

Clamp loader complex

β Sliding clamp

ψχ

RecQTopoisomerase III

RNase HI

α

ε θ

PrimaseSsb

RNA primer

REPLICATION FORK IN E. coli

Interaction Network for DNA Polymerase III

DNApol III

THE MOST HIGHLY CONSERVED ESSENTIAL E. coli PROTEINSTARGETS FOR NEW BROAD-SPECTRUM ANTIBIOTICS

• The 189 most highly conserved non-ribosomal E. coli proteins known to be essential in at least one bacterial species were chosen for study

• 176 of these genes were successfully tagged with SPA or TAP tags.

• 168 (89 %) of the proteins were successfully purified.

• 188 distinct interacting proteins were identified, 46 of which represent hypothetical proteins or proteins of unknown function.

CURRENT STATUS: More than 1000 proteins tagged and purified

Predicting protein functions using microarray expression data(Wu et al. (2002), Nature Genetics)

Essential

Conserved

Protein translocation (2)Cell Wall (9)

DNA Repair (11)Vesicular transport (13)

Recombination (15)Cell cycle control (17)

Phosphate metabolism (19)Protein folding (29)

Nucleotide metabolism (38)Cell Stress (39)

Protein degradation (63)Pol II transcription (65)

Protein modification (66)Mating response (79)

Meiosis (97)Chromatin chromosome structure (100)

Lipid fatty-acid and sterol metabolism (139)Carbohydrate metabolism (179)Small molecule transport (192)

Amino-acid metabolism (249)RNA processing and modification (285)

Energy generation (292)Protein synthesis (370)

80%

70%

60%

50%

40%

30%

1,655 uncharacterized yeast ORFs

456

mic

roar

ray

expe

rimen

ts

Anticipatedaccuracy(based on

characterizedgenes)

RNA processing and modification (285 genes)

RNA Processing and Modification Genes

• 91 representative genes from literature and databases

• 88 from transcriptional co-regulation

• 28 homologous to human nucleolar proteins

The Program for RNA Processing and Modifying Proteins

Mutants(deletions, TET promoters)

TAP tagged strains

MALDI-TOF MS

Trypsin digestion

Purification

Phenol extraction

Cell pellets

DNase I

Labeling of RNA

SDS PAGE

Hybridization to Microarray

Gel Densitometry LC-MS/MS

Non-coding RNA Microarray

A0 A1 D A2 A3 B1 E C2 C1 B2

5' ETS and A2cleavage

(U3 processome-like)

3' UTRprocessing

defective(rnt1)

A2cleavage

(snoRNP-like)

D-A2 processing(RIO1-like)

A2-B1 processing(RNAseP-like)

E-C2(exosome/

pre-60S-like)

Mutated gene GO biological processA0 A1 D A2 A3 B1 E C2 C1 B2

<-0.3 -0.2 -0.1 0 0.1 0.2 >0.3

Log(ratio)

Missing data= grey

5’ETS 18S ITS1 5.8S ITS2 25S 3’ETS

RNT1 35S primary transcript processingYGR251W*** biological_process unknownECM16 processing of 20S pre-rRNAHCA4 35S primary transcript processingUTP20 biological_process unknownUTP22 biological_process unknownUTP19 biological_process unknownSAS10 establishment and/or maintenance of chromatin YOR287C biological_process unknownUTP11 processing of 20S pre-rRNAYGR272C*** biological_process unknownUTP6 processing of 20S pre-rRNAFYV7*** biological_process unknownRRP9 35S primary transcript processingRPS4A protein biosynthesisUTP4 processing of 20S pre-rRNANOP7 ribosomal large subunit biogenesisUTP9 processing of 20S pre-rRNAYHR040W biological_process unknownYOR004W biological_process unknownNAN1 processing of 20S pre-rRNACBF5 35S primary transcript processingMRD1 35S primary transcript processingBUD22 bud site selectionNSR1*** biological_process unknownBUD21 processing of 20S pre-rRNARPS1B protein biosynthesisPOL5 DNA dependent DNA replicationDBP6 35S primary transcript processingNOP4 rRNA processingPXR1 biological_process unknownYLR022C biological_process unknownMAK16 ribosomal large subunit biogenesisRNA1 rRNA-nucleus exportRRP43 35S primary transcript processingNSR1 rRNA processingDBP8 35S primary transcript processingFYV5 biological_process unknownBRX1 ribosomal large subunit assembly and maintenanceTIF6 processing of 27S pre-rRNARPL17A protein biosynthesisNOP15 ribosomal large subunit biogenesisNOC3 DNA replication initiationNUP82 NLS-bearing substrate-nucleus importYOL022C biological_process unknownPRP42 mRNA splicingYNL313C biological_process unknownLSM5 mRNA splicingSSU72 transcription initiation from Pol II promoterGLC7 glycogen metabolismYDR365C biological_process unknownCDC33 regulation of cell cyclePRP5 lariat formation, 5'-splice site cleavagePRP39 mRNA splicingRTS2 biological_process unknownKRE28 biological_process unknownYMR269W biological_process unknownEPL1 histone acetylationRRP12 processing of 20S pre-rRNARPS18B protein biosynthesisLOC1 ribosomal large subunit biogenesisYOR006C biological_process unknownRIO1 S phase of mitotic cell cycleYLR435W*** biological_process unknownFAP7*** response to oxidative stressTSR1 ribosome biogenesis and assemblyDRE3 cell growth and/or maintenanceLSM6 mRNA splicingRPS0A protein biosynthesisNOC2 biological_process unknownPTA1 mRNA cleavageRPP1 rRNA processingPOP1 rRNA processingSNM1 rRNA processingLSM1 deadenylation-dependent decappingGRC3*** cell growth and/or maintenanceMRT4 ribosomal large subunit biogenesisIPI3 biological_process unknownYDR412W*** biological_process unknownNOG2 mRNA splicingMAK5 rRNA processingSDA1 actin cytoskeleton organization and biogenesisIPI1 biological_process unknownMDN1 protein complex assemblyIPI2 biological_process unknownNUG1 biological_process unknownYDL062W biological_process unknownYDL063C biological_process unknownRAI1 RNA catabolismRRP6 35S primary transcript processingLRP1*** biological_process unknownTIF4631 translational initiationYNL227C endocytosisMPE1 mRNA cleavageRRP46 35S primary transcript processing

MICROARRAY ANALYSIS OF RNA PROCESSING DEFECTS

Ipi1

-TA

P

Ipi2

Ipi3

Ipi1-TAP

45

66

97

kDa

No

tag 35S

27S

20S

U2

25S

18S

U2

U1

7S

5.8SL

5.8SS

WT

TET-

IPI1

TET-

IPI2

TET-

IPI3

WT

C

5’ETS 18S ITS1 5.8S ITS2 25S 3’ETS 5S

IPI (Involved in Processing ITS2) Complex

BA

rRNA

TET-IPI1

TET-IPI2

TET-IPI3

Protein Complexes in RNA Processing

• 317 proteins “clustered” into complexes (~stoichiometric)

• 56 complexes (2-17 subunits)

• 36 singleton proteins

• 172 additional sub-stoichiometric proteins (possible interactions between complexes)

Diagonal-gram goes here

TAP Tagged Proteins

Pu

rifi

ed P

rote

ins

Exosome

RNA Polymerase III

RNA Polymerase II

RNA Polymerase I

TREX

RSC

MediatorTFIIIC

U1 snRNP

Arx1

mRNA Cap-Binding/eIF4F

U6 Specific snRNP

Sit4 Complex

Nop58/Sik1

Ydr117c/Rps4b Complex

U4/U6.U5 tri-snRNPNoc ComplexMdm20/Nat3 Mtr4 Complex

Dbp7/Rrp5

Gar1/Cbf5Dbp3 C

Met-Glut tRNA Synthetase

Pwp1/Nop12/Brx1Nap1UTP “C”

Spt16Casein Kinase II

Ykl088w

19S Proteosome

eIF3

tIF2Rli1

Prp19mRNA Cleavge/Polyadenylation

Imd

UTP “B”Eft2/Hgh1

Trm7 Translation Release Factor

RNase PRcl1/Bms1

Kap104/Nab2Ipi

Ccr4 Bcp1

20S Proteosome

Gir

Cbc2/Sto1 Nop7 ComplexRrb1/Rpl3

Kap95/Srp1

SRPTrm1/Nsa1

CLUSTERINGTO

ORGANIZE PROTEIN

COMPLEXES

A Network Diagram Using Protein Complexes

RRP9 - Rrp9 (1)UTP13 - UTP B (6)UTP18 - UTP B (6)UTP9 - UTP A (8)UTP10 - UTP A (8)UTP8 - UTP A (8)RRP5 - Dbp7/Rrp5 (4)PRT1 - eIF3 (7)TIF35 - eIF3 (7)NIP1 - eIF3 (7)DBP3 - Dbp3/Bmh1/Nsr1 (3)HCA4 - Hca4 (1)NOP58 - Nop58/Sik1 (3)NPL3 - Npl3 (1)GAR1 - Gar1/Cbf5 (6)YGR283C - Gar1/Cbf5 (6)RRP8 - Rrp8 (1)YDR365C - Ydr365c (1)NOC2 - Noc (3)ERB1 - Nop7/Erb1/Ytm1 (3)NOP7 - Nop7/Erb1/Ytm1 (3)NOP12 - Pwp1/Brx1/Nop12 (3)PWP1 - Pwp1/Brx1/Nop12 (3)RRP45 - Exosome (12)RRP46 - Exosome (12)RRP6 - Exosome (12)IPI1 - Ipi (3)PRP19 - Prp19 (4)PUF6 - Puf6 (1)RNT1 - Rnt1 (1)YBR025C - Ybr025c (1)RPN6 - 19S Proteosome (17)PRE8 - 20S Proteosome (13)PUS1 - Pus1 (1)TRM1 - Trm1/Nsa1 (2)CBC2 - Cbc2/Sto1 (2)STO1 - Cbc2/Sto1 (2)LHP1 - SRP (3)THS1 - Ths1 (1)TSR2 - Tsr2 (1)BRR2 - U4/U6.U5 tri-snRNP (2)PRP3 - U6-specific snRNP core (14)LSM3 - U6-specific snRNP core (14)LSM5 - U6-specific snRNP core (14)KEM1 - U6-specific snRNP core (14)UTP22 - UTP22/RRP7 (2)

18S 5.8S 25S 5S

Tyr2 Ile2

Trp2

Thr3

M-Il

e

snR7

8sn

R77

snR7

6sn

R75

snR7

4sn

R73

snR7

2

RPR1

MRP

TL

C1Sc

R1

YDR0

64w

EF-1

RPL3

0

snR1

90 U14 U3

snR3

6sn

R43

snR3

8 U1 U2 U4 U5 U6

mRNAmisc.snRNAtRNAsnoRNArRNA

AB

C D

I

KJ

M

O

R

P

UTP “B”

5’ ETS ITS1 ITS2 3’ ETS

UTP “A”

eIF3

Nop58/Sik1

Nop7 Complex

Exosome

19S Proteasome20S Proteasome

SRP

U6-Specific snRNP

E F

GH

L

N

Q

Association of RNA-Processing Complexes with Specific RNAs

purified proteins

tag

ge

d p

rote

ins

Steps in the RNA Polymerase II Transcription Cycle

InitiationInitiation Elongation Elongation

Termination Termination

General General transcription transcription factorsfactors

MediatorMediator

CTD kinasesCTD kinases

Elongation Elongation factorsfactors

Cleavage, Cleavage, polyadenylation, polyadenylation, and termination and termination factorsfactors

chromatin-modifying enzymes

Elp3-TAPElp3-TAP

Elp1Elp1

Elp2Elp2

Elp4Elp4

Elp6Elp6

Elp5Elp5

Elp3-TAPElp3-TAP

Spt6-TAPSpt6-TAP

Spt6Spt6

Iws1Iws1

Chd1Chd1Spt16-TAPSpt16-TAP

Ctr9Ctr9

Pob3Pob3

Cdc73Cdc73CkaICkaI

CkaIICkaII

CkbIICkbII

CkbICkbI

Psh1Psh1

Histones Histones

Rtf1Rtf1

Paf1Paf1

Leo1Leo1

Spt16-TAPSpt16-TAP

TAP Purification of Various Elongation Factors

Elongator Spt6/Iws1FACT

-4

-3

-2

-1

0

1

2

3

-4 -3 -2 -1 0 1 2 3 4

“Old” and “New” Elongator Gene Deletions Have Similar Effects on Gene Expression

Wild type /elp1 deletion

Wil

d ty

pe /e

lp6

dele

tion

RNA Polymerase IIRNA Polymerase II Elongator (Elp1, 2, 3, 4, 5, 6)

TFIIS

TFIIF

Spt5

Spt6

Iws1

Paf1

Cdc73

Rtf1

Leo1

Spt16/Pob3 Spt16/Pob3 (FACT)(FACT)

Psh1Ctr9

Histones

Chd1

(Tfg1, Tfg2, Tfg3)

Protein Interactions Involved in Transcriptional Elongation (2002)

Casein Kinase II

Spt4

Phosphorylation?

Ctk1

Fcp1

55 Polypeptides55 Polypeptides

Iwr1

COMPASS(8 polypeptides)

Set2

Paf1-C

Theoretical Pathways in S. cerevisiae

A

B

C

X

Y

Z

P

X

X X

= synthetic lethality

xxxset2 XMating

MAT MATa

a/wild-type

Sporulation

MATa Haploid Selection(MFA1pr-HIS3)

Double Mutant Selection

Qu

ery

Gen

es

Hits

Two Dimensional Hierachical Clustering Identifies Similar Patterns of Genetic Interactions:

Clusters of Query Genes Y axisClusters of Interacting Genes (Hits) X axis

MMS4MUS81RTT107YBR094WHST3TOP1CDC8RAD50RAD52CDC45CDC7DBF4CSM3MRC1TOF1ELG1POL32RAD27ORC2ORC5RAD9RAD53ESC2SGS1RAD24ARP1DYN1PAC1NBP2NUM1BIK1TUB3CHL1MAD2CHL4YDR332WCTF18CTF8CTF4DCC1BIM1KAR3GIM3YKE2GIM4PAC10TUB2CLB4KAR9KIP3ARP6GOD1HTZ1SET2ARC40ARP2BNI1CLA4KRE1SLT2SHS1SMY1BNI4PHO85BBC1CDC42-118YDR437WCNB1CNB1/1ERG11RIC1YPT6HST1YJR070CAPP1RAS2

IgG

INPUT

1

TATAA

PMA1

-304 -47

5

2018 2290

6

3287 3500

1(ATG) 2757(STOP)

2

168 376

3

584 807

4

1010 1250

2823 3277

ChIP Distinguishes Localization in Various Regions of a Gene ChIP Distinguishes Localization in Various Regions of a Gene

Hpr1Hpr1

1 2 3 4 5 6

Coding RegionsCoding Regions

Rna14Rna14

1 2 3 4 5 6

3‘ Untranslated 3‘ Untranslated

Tfg2Tfg2

2 3 4 5

PromoterPromoter

1 6

Spt16

1 2 3 4 5 6

All ThreeAll Three

TFIIF TREX FACTCFIA

MR

kDa

97

66

45

31

Swr1-TAP

Vps72-TAP

Swc1-TAP

No Tag

Aor1-TAP

Vps71-TAP

Act1

Vps71

Vps71-TAPAor1

Arp4/God1Rvb1/Rvb2/Arp6Aor1-TAP

Bdf1Swc1

Swc1-TAP

Swr1Swr1-TAP

Vps72Vps72-TAP

Purification of the SWR1 Complex

Yaf9Translocated

to MLL in Acute

Myeloid Leukemia

Swr1-TAP Swc1-TAP Vps71-TAP Aor1-TAP Vps72-TAP

Swr1

Swc1

Vps72

Aor1

Vps71

God1

Arp6

Act1

Arp4

Yaf9

Rvb1

Rvb2

Bdf1

ORGANIZING PROTEINS INTO PATHWAYS

• Microarray analysis

• Synthetic genetic array analysis

Concept: proteins with the same function should have similar effects on gene expression and similar genetic interactions

RX

T2

DE

P1

SA

P3

0

RA

D6

BR

E1

SD

C1

RT

F1

PA

F1

BR

E5

SW

C4

SW

C2

SW

R1

LE

O1

SIF

2

HO

S2

SE

T3

RXTC RAD6C PAF1C SWR1C SET3C

Clustering of Microarray Data Groups Proteins into Complexes and Pathways

Histone Trans-Modification Pathway

Silent Heterochromatin

H4-Ac

Bdf1SWR1C

Step 1

Step 2 Step 3Htz1

The Case of ER to Golgi Transport

• ~400 non-essential proteins localize to ER and Golgi

• purify these proteins and organize into protein complexes

• use SGA to organize the proteins and protein complexes into functional pathways

Extending Pathway Analysis to Multiple Systems

Structural Genomics of Protein Complexes

• Production of protein complexes - co-overexpression - purification from a natural source - synthesis in vitro

• Structure determination - high resolution by x-ray crystallography - low resolution by electron microscopy

Acknowledgments

Nevan Krogan Joyce Li Guaqing Zhong Grace GuoAtanas Lalev Nira DattaRobin Haw

Andrew Emili Charlie Boone Tim HughesGerard Cagney Amy Tong Mark Robinson Gareth Butland Huiming Ding WenTao Peng Owen Ryan

Greenblatt Laboratory

University of Toronto

Affinium PharmaceuticalsDawn Richards

Veronica CanadienBryan BeattieKamal Boura

Harvard UniversitySteve Buratowski

Minkyu KimMichael Keough

UCSFErin O’Shea

Jonathan Weissman