Protein complexes and proteome organization from yeast to man

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Protein complexes and proteome organization from yeast to man Anne-Claude Gavin and Giulio Superti-Furgay Protein complexes may well be the most relevant molecular units of cellular function. The activities of protein complexes have to be regulated both in time and space to integrate within the overall cell programs. The cell can be compared to a factory orchestrating individual assembly lines into integrated networks ful®lling particular and superimposed tasks. Recent proteome-wide studies provide insight into the properties of cellular protein complexes, their modular nature, their interaction with other complexes and the resulting preliminary organization chart of the proteome. Addresses Cellzome AG, Meyerhofstrasse 1 69117 Heidelberg, Germany  e-mail: [email protected] y e-mail: [email protected]

Current Opinion in Chemical Biology 2003, 7:21±27 This review comes from a themed issue on Proteomics and genomics Edited by Matthew Bogyo and James Hurley 1367-5931/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1367-5931(02)00007-8

Introduction

Protein complexes beyond individual binary interactions

Proteome-wide characterizations of protein±protein interactions using an ex vivo genetic selection system, yeast two-hybrid, greatly extended our understanding on eukaryotic `interactomes' [2±6]. However, proteins inside the cell do not seem to interact through constant trial and error. In the crowded environment of a cell [7,8], protein associations need to be orchestrated and regulated very precisely to ensure the ef®cient execution of cellular programs. Similar to the genetic code that directs the translation of nucleic acid sequences into amino acid sequences and the principles governing the folding of the newly synthesized polypeptide, an as yet poorly elucidated protein assembly code converts the genome's information into a biologically functional third dimension (Figure 1) [9]. Over the past few decades, a wide variety of modular binding domains with speci®city for distinct sequence motifs have been mapped that contribute to the solution of the protein assembly puzzle [10]. While the chemical properties of interaction interfaces are emerging, the exact biophysical rules governing highly speci®c binding and exquisite precision of recognition remain elusive [11].

Our understanding of protein complexes has mainly come from the pioneering analysis of the molecular machines involved in transcription and translation. However, several recent studies demonstrate that protein complexes are ubiquitous and represent the molecular norm, rather than the exceptional functional units of proteomes. Complexes are composed of subunits that probably are the result of coordinated gene expression, concerted translation and assembly as well as transport, activity and degradation.

The association of more than two binding partners in a single protein complex introduces levels of complexity and regulation beyond binary interactions. One hallmark of protein complexes may be binding cooperativity. Loss or gain of binding capacity via cooperative interactions confers important contributions to the functionality of the resulting protein complex [12,13,14]. From a very simple thermodynamic perspective, multiplying the number of interaction surfaces will contribute to an exponential increase in the af®nity constant. Cooperative binding also involves allosteric events and conformational changes that might affect and in¯uence the subsequent association with other factors [15] (Figure 2). Recent studies on the assembly of the ribosomes [16] and the proteasome [17,18] illustrate particularly well that the assembly of protein complexes is a dynamic process, involving posttranslational modi®cations and ATP-driven conformational changes through chaperone assistance. It requires sequential recruitment of speci®c proteins and non-peptidic components in speci®c subcellular locations.

This review describes some general features of protein complexes based on a few detailed examples and discusses insights gained from the systematic analysis of protein complexes and the functional organization of eukaryotic proteomes.

The composition of protein complexes is not easily inferred by studying pair-wise interactions on a large scale. Accordingly, recent large-scale analyses of protein complexes [19,20] show rather poor overlap with data generated by two-hybrids screens [21,22,23,24]. More

Proteins do not act alone. Instead, they usually interact with one another to exert their cellular function in concert. The resulting protein complexes are highly ordered dynamic structures that assemble, store and transduce biological information [1]. De®ning the relationships between the components of a complex and indeed between complexes themselves is essential to understanding the molecular essence of the cell.

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Current Opinion in Chemical Biology 2003, 7:21±27

22 Proteomics and genomics

Figure 1

1st Position (5' end)

2rd Position

U

C

U

Phe Phe Leu Leu

C

3rd Position (3' end)

A

G

Ser Ser Ser Ser

Tyr Tyr STOP STOP

Cys Cys STOP TRP

U C A G

Leu Leu Leu Leu

Pro Pro Pro Pro

His His Gln Gln

Arg Arg Arg Arg

U C A G

A

IIe IIe Leu Met

Thr Thr Thr Thr

Asn Asn Lys Lys

Ser Ser Arg Arg

U C A G

G

Val Val Val Val

Ala Ala Ala Ala

Asp Asp Glu Glu

Gly Gly Gly Gly

U C A G

Genetic code

Protein folding ‘code’

Protein interaction/assembly ‘code’ Current Opinion in Chemical Biology

The biological codes from amino acid sequence to protein assembly. The sequence of the genes determines the amino acid sequence of the proteins. The genetic code that translates the genetic information into amino acid sequences represents the universal `Central Code' of biology, bridging two different classes of molecules: nucleic acids and proteins. The amino acid sequence of a protein governs its dynamic folding into secondary and tertiary structures essential to the protein function. The DNA and protein sequences, however, also determines when and where the protein will be expressed, how it can interact with other polypeptides to form large complexes, where the protein will reside and when it will be degraded. Signals embedded in the sequences are used by the cell to place the protein in its proper context or molecular environment. It is likely that the rules governing this poorly understood protein assembly code involve coordinated gene expression, concerted translation, assembly, transport and degradation.

Figure 2

(a) Interaction induces allosteric changes

(b) Energy-driven conformational changes ATP/GTP

ADP/GDP

(c) Chaperone-assisted assembly ATP/GTP

ADP/GDP

Current Opinion in Chemical Biology

Protein complex assembly involves cooperative binding. The assembly of protein complexes is a very dynamic and interactive process that cannot be easily mapped by the simple in vitro analysis of binary interactions. (a) Cooperative binding involves allosteric events and conformational changes that may affect and influence the subsequent association with other factors. This includes disorder to order transitions, large movements of the main chain and changes in the relative position of some domains. (b) Conformational changes required for the local assembly of protein complexes are very often energy-driven through nucleotide triphosphate hydrolysis and/or post-translational modifications (i.e. phosphorylation). (c) The building of protein complexes may also require assembly factors or chaperones that are recycled for assembling of other proteins. Current Opinion in Chemical Biology 2003, 7:21±27

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Protein complexes and proteome organization Gavin and Superti-Furga 23

generally, binary interactions characterized through classical biochemical methods (GST pulldowns and far western blotting) were found to map interaction surfaces observed in the crystal structure of protein complexes only poorly [25,26]. The conclusion of studies on the evaluation of different methods, emphasizes the inherent problems of comparing methods that monitor different properties of proteins, namely the ability to transiently interact and the ability to stably integrate and assemble in higher-order structures. Rather, the methods provide complementary information, leading to a more comprehensive view, when integrated [22,23,25].

Molecular machines

The study of protein complexes requires their isolation under physiological conditions and the identi®cation of often minute amounts of their components. Recent advances in the integration of analytical tools have led to a signi®cant increase in the molecular understanding of protein complexes. Some of the most prominent examples are summarized in Table 1. Protein complexes are more than the sum of their parts. They resemble molecular machines with different functional compartments and a modular structure that contribute to one superimposed cellular task (Table 1). The initiation of DNA replication, for example, involves the sequential activity of two enzymes: the DNA primase that starts the reaction with the synthesis of a short RNA primer and the DNA polymerase a that synthesizes DNA from the RNA primer. The coordination of these two activities is ensured by two scaffolding proteins that dock the complex at the site of replication, bind ssDNA, bridge the two enzymes, `count' the length of the RNA primer and couple the DNA polymerase a to the remaining replication machinery [27,28]. Other protein complexes show an even higher level of complication in the partition of labour. A good example is RNA polymerase II, responsible for the synthesis of the entire mRNA repertoire of eukaryotic cells. A set of ®ve proteins contributes to the building of the structures called jaw, clamp, cleft, wall and funnel that are necessary for attachment to DNA, the maintenance of the RNA±DNA duplex, the access of nucleotide to the catalytic site and the translocation of the polymerase along the template DNA [29,30]. Cell signaling also proceeds through protein complexes. Protein kinases, for example, are very often found in complex with regulatory subunits that are required for their activity, localization and substrate speci®city (for example cyclin/cdk complexes [31]), or with scaffolding proteins, that tether signaling components to each other and ensure speci®city of signaling [32,33,34]. Protein complexes have been very often found to interact with one another (Table 1). Connections between protein machines represent a higher level of organization. They are thought to ensure an ef®cient rate and speci®city of www.current-opinion.com

enzymatic reactions as well as a precise orchestration of various cellular activities. For example, the RNA polymerase II is extensively coupled to a panel of upstream protein complexes that regulates/orchestrates gene expression through the binding of promoters and upstream regions [35]. Through its C-terminal domain, the RNA polymerase II also interacts with a variety of proteins and complexes that couples transcription to down-stream events involved in mRNA processing: namely, mRNA capping, splicing, nonsense-mediated RNA decay, polyadenylation, and mRNA export [36]. Similarly, the ISWI-chromatin remodeling complex loads components of the sister chromatin cohesion machinery, the cohesin complex, on chromosomes [37] and the PolA2 subunit of the DNA pola-primase complex tethers the complex to the replication fork and couples of the initiation DNA duplication to the remaining of the replication machineries [29,30]. Another interesting attribute of protein complexes is their modularity. Different molecular machines often use the same protein or the same protein module (where a module consist of two or more proteins) to exert their different functions. For example, the scaffolding protein Yotiao stably associates with both the N-methyl-D-aspartate (NMDA) receptor and the potassium channel hKCNQ1/ hKCNE1, where it functions as a docking platform for both protein kinase A and protein phosphatase 1 [38]. Similarly, the ATM/PtdIns-3 kinase-related protein TRRAP targets different histone acetylases at the sites of transcriptional initiation [39]. The heat shock proteins Hsp90 and Hsp70 form stable complexes involved in the folding of proteins, but Hsp70 has been found stably associated with class I histone deacetylases, where it may be required for nucleosome remodeling and ef®cient histone deacetylation [40]. Hsp90 and p23 were found stably associated with the human telomerase protein, where they probably contribute to the conformational tweaking likely to occur during the reverse transcription process [41].

Networks of protein complexes: an ideal platform for systems biology analysis

In a similar manner to the domains from which they are built, proteins are used combinatorially to build higherlevel assemblies in an intricate spatial and temporal hierarchy that ultimately ends at the level of organelle or cell (Figure 3). Genome-wide analysis of multi-protein complexes in Saccharomyces cerevisiae revealed that more than 80% of the yeast proteins are assembled in protein complexes that are highly interconnected through the sharing of certain components [20]. The yeast proteome thus appears to be organized in a large network of protein complexes whose attributes appear ubiquitous (i.e. modularity and tethering to each other). The networks of protein complexes depicted in [20,22] represent a higher level of proteome organization that Current Opinion in Chemical Biology 2003, 7:21±27

24 Proteomics and genomics

Table 1 Protein complexes are more than the sum of their parts: some prominent examples. Complex/biological activity

Description of the complex

Subunits

References

Polymerase II complex mRNA synthesis

Selection of start site Building of the `core module', active sites Binding of the complex to DNA, maintenance of the RNA±DNA duplex, access of nucleotide to the catalytic site and translocation the polymerase along the template DNA Complex assembly, structural role Orchestration of activity, coupling to RNA-processing machineries and TFIIB complex Targeting of the complex at the origin of replication: Primer synthesis ± Initiation, binding to ssDNA, binding to RNA±DNA duplex, translocation of the complex along the DNA template ± Translocation of the primer from the primase to the polymerase a catalytic site DNA duplication Coupling of polymerase a-primase complex to the remaining replication machinery Binding/anchoring of the complex to actin filament Orientation of Arp2 and Arp3 at a 708 angle to the parent actin filament Actin nucleation Binding of the regulatory component WASp/Scar Regulation of the activity of the complex, transduction of extra-cellular signals to the complex Activation of protein synthesis through phosphorylation Sensing of the nutrient availability, inhibition of mTOR mTOR stability, localization or substrate specificity Recruitment of the complex to E2F- and Myc-responsive genes (specific Methylation of Lys9 on histone H3

Rpb9 Rpb1, 2 Rpb1, 2, 5, 6, 9

[29,30]

DNApola-primase complex DNA duplication

Arp2/3 complex Branched growth of actin filaments

mTOR complex Nutrient-sensitive complex E2F-6 complex Silencing of ETF- and Myc-responsive genes in quiescent cells (G0)

AP2 complex; clathrin adaptor

Vesicle trafficking hKCNQ1 complex Slow outward potassium ion current channel

SCAP/SREBP complex

Cholesterol homeostasis

Binding to Lys9-methylated histone H3. Stabilization of the complex. Docking of the polycomb proteins Propagation of chromatin inactivation (Polycomb group proteins (PcG)) Binding to phosphatidyl inositols, targeting AP2 complex to the membrane Phosphorylation-induced conformational changes required for binding to canonical motifs on transmembrane cargos Binding of canonical motifs on transmembrane cargos Interaction with clathrin Recruitment of accessory/regulatory proteins Slow outward potassium ion current channel b adrenergic-dependent stimulation of hKCNQ1 activity by phosphorylation Inhibition of hKCNQ1 activity by dephosphorylation Targeting of PKA and PP1 to hKCNQ1 Expression of genes involved in lipid/cholesterol synthesis. Requires the translocation of SREBP to the Golgi where it is cleaved by membrane proteases; its NH2-terminal fragment activates transcription Escorts SREBP from the ER to the Golgi. Cholesterol binding/sensing Sterol-dependent retention of SCAP-SREBP in the ER. Inhibition

goes beyond representations of protein networks [42,43]. It represents a ®rst draft on the molecular integration/ regulation of the activity of cellular machineries. It can be envisaged, that the network of complexes, because of its functional content, can be an ideal platform to integrate Current Opinion in Chemical Biology 2003, 7:21±27

Rpb3, 10, 11, 12 Rbp1 PolA2 Prim1 Prim2

[28,27]

Prim2 PolA PolA2 [45,46]

p34, p40 p16, p20 ARP2, ARP3 p21, ARP2, 3 WASp/Scar mTOR Raptor Raptor E2F-6, DP 1, Mga, Max, Hp1g Eu-HMTase1, NG36/G9a

[34] [47]

HP1g

RING1, RING2, MBLR, h-l(3)mbt- like protein, YAF2 a, m2 adaptin

[48]

m2 adaptin m2, b2 adaptins b2 adaptin a, b2 adaptin hKCNQ1, hKCNE1 PKA, RII PP1 Yotiao SREBP

[38]

[49]

SCAP INSIG-1

further data. For example, protein complexes sharing the same subcellular location could be pooled in larger superstructures forming the building blocks of organelles (Figure 3). At the gene product level, genetic and single nucleotide polymorphism (SNP) data might become www.current-opinion.com

Protein complexes and proteome organization Gavin and Superti-Furga 25

Figure 3

Domains, ~20 Å

Proteins, 50–100 Å

Proteins complexes, 500–1000 Å

Organelles, 5000–10000 Å (5–10mm)

Current Opinion in Chemical Biology

The different levels of cellular functional organization. A wide repertoire of protein domains has been characterized that contribute to the biochemical/ biophysical properties of proteins. Domains are used combinatorially to form proteins with various biochemical functions. Similar to the domains they are built of, protein and protein modules are used combinatorially to build higher-level assemblies or protein complexes that fulfil various biological roles. As the size of these complexes is, on average, an order of magnitude larger than the individual components, the level of organization of complexes `bridges' the molecular and cellular world of organelles and large subcellular assemblies.

interpretable when envisaged combinatorially because of the functional cohesion of the corresponding gene products. Ultimately, the desire is to have several maps ordered hierarchically that would allow us to track and navigate the molecular organization of the cell by cellular structures or by function. We may only be a few years from such integrated cellular `atlases' [44].

Organization of the human proteome

The major rules and the general architecture observed in the yeast proteome are likely to be conserved in higher eukaryotes. Preliminary evidence comes from our analysis of several protein complexes in human, where we have not detected signi®cant differences in the number of components from yeast (unpublished observations). The difference in terms of biological complexity between yeast and human may not rely entirely on a higher number of www.current-opinion.com

proteins, differential splicing and post-translational modi®cations. An important aspect is likely to be the employment of these different forms combinatorially. So far, we have observed a degree of `promiscuity' in human proteins that compares well with that seen in yeast [20]. A comprehensive analysis of protein complexes in human may still appear a daunting task. It has been observed, however, that conservation among species extends from single proteins (orthologues) to their molecular environment. The analysis of the yeast proteome revealed a set of protein complexes that are enriched in orthologous proteins [20]. This represents a `core' conserved proteome likely to ful®l the essential functions of eukaryotic cells. These data are being used to derive a `humanized' proteome map that serves as a blueprint for exploring the human proteome. Current Opinion in Chemical Biology 2003, 7:21±27

26 Proteomics and genomics

Understanding the molecular and functional circuitry among protein complexes in human cells, as well as the changes associated with time, disease states or cell type will be an invaluable tool for medicine. For example, the majority of drug targets are proteins. Understanding of protein complex connectivity and modularity allows us to track the pathways and cellular functions affected by a given drug. It provides a molecular basis for the appreciation of drug secondary effects. In turn, this streamlines toxicology studies but may also provide suggestions for new medical uses of existing drugs.

Conclusions

The elucidation of the full repertoire of genes in several organisms and signi®cant breakthroughs in the ®elds of functional genomics and proteomics have fundamentally revolutionized the manner in which we explore the molecular basis of life and disease. After years of reductionism (one gene one function) it becomes obvious that it is time for integration and for more holistic approaches. Because they represent more relevant units of biological activity and because of their modular and dynamic nature, protein complexes have taken the centre stage in a broad panel of research areas including biology, pharmacology, structural biology, mathematics and computational biology. The understanding of these machines may be key to decipher the often indirect and convoluted relationship between genotype and phenotype.

Acknowledgements

We thank David B Jackson, Rob Russell and Tewis Bouwmeester for critical reading of this review. We are also grateful to Frank Weisbrodt for editing the ®gures.

References and recommended reading

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