Assembly Chaperone Research Articles

Biolayer interferometry of lipid nanodisc-reconstituted yeast vacuolar H+ -ATPase.

Vacuolar H+ -ATPase (V-ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V-ATPase activity is regulated by reversible disassembly, resulting in cytosolic V1 -ATPase and membrane-integral V0 proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein-protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein-protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc-reconstituted V-ATPase (V1 V0 ND). We show that V1 V0 ND can be immobilized on streptavidin-coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation.

Polymorphisms in the F Pocket of HLA-B27 Subtypes Strongly Affect Assembly, Chaperone Interactions, and Heavy-Chain Misfolding.

HLA-B27 is associated with the inflammatory spondyloarthritides (SpA), although subtypes HLA-B*27:06 and HLA-B*27:09 are not. These subtypes differ from the HLA-B*27:05 disease-associated allele primarily at residues 114 and 116 of the heavy chain, part of the F pocket of the antigen-binding groove. Dimerization of HLA-B27 during assembly has been implicated in disease onset. The purpose of this study was to investigate the factors that influence differences in dimerization between disease-associated and non-disease-associated HLA-B27 alleles. HLA-B*27:05 and mutants resembling the HLA-B*27:06 and 09 subtypes were expressed in the rat C58 T cell line, the human CEM T cell line and its calnexin-deficient variant CEM.NKR. Immunoprecipitation, pulse-chase experiments, flow cytometry, and immunoblotting were performed to study the assembly kinetics, heavy-chain dimerization, and chaperone associations. By expressing HLA-B*27:05, 06-like, and 09 alleles on a restrictive rat transporter associated with antigen processing background, we demonstrate that a tyrosine expressed at p116, either alone or together with an aspartic acid residue at p114, inhibited HLA-B27 dimerization and increased the assembly rate. F-pocket residues altered the associations with chaperones of the early major histocompatibility complex class I folding pathway. Calnexin was demonstrated to participate in endoplasmic reticulum (ER) stress-mediated degradation of dimers, whereas the oxidoreductase ERp57 does not appear to influence dimerization. Residues within the F pocket of the peptide-binding groove, which differ between disease-associated and non-disease-associated HLA-B27 subtypes, can influence the assembly process and heavy-chain dimerization, events which have been linked to the initiation of disease pathogenesis.

Molecular basis for protection of ribosomal protein L4 from cellular degradation

Eukaryotic ribosome biogenesis requires the nuclear import of ∼80 nascent ribosomal proteins and the elimination of excess amounts by the cellular degradation machinery. Assembly chaperones recognize nascent unassembled ribosomal proteins and transport them together with karyopherins to their nuclear destination. We report the crystal structure of ribosomal protein L4 (RpL4) bound to its dedicated assembly chaperone of L4 (Acl4), revealing extensive interactions sequestering 70 exposed residues of the extended RpL4 loop. The observed molecular recognition fundamentally differs from canonical promiscuous chaperone–substrate interactions. We demonstrate that the eukaryote-specific RpL4 extension harbours overlapping binding sites for Acl4 and the nuclear transport factor Kap104, facilitating its continuous protection from the cellular degradation machinery. Thus, Acl4 serves a dual function to facilitate nuclear import and simultaneously protect unassembled RpL4 from the cellular degradation machinery.

The Survival of Motor Neuron Protein Acts as a Molecular Chaperone for mRNP Assembly

Spinal muscular atrophy (SMA) is a motor neuron disease caused by reduced levels of the survival of motor neuron (SMN) protein. SMN is part of a multiprotein complex that facilitates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN has also been found to associate with mRNA-binding proteins, but the nature of this association was unknown. Here, we have employed a combination of biochemical and advanced imaging methods to demonstrate that SMN promotes the molecular interaction between IMP1 protein and the 3' UTR zipcode region of β-actin mRNA, leading to assembly of messenger ribonucleoprotein (mRNP) complexes that associate with the cytoskeleton to facilitate trafficking. We have identified defects in mRNP assembly in cells and tissues from SMA disease models and patients that depend on the SMN Tudor domain and explain the observed deficiency in mRNA localization and local translation, providing insight into SMA pathogenesis as a ribonucleoprotein (RNP)-assembly disorder.

Is RAF1 protein from Synechocystis sp. PCC 6803 really needed in the cyanobacterial Rubisco assembly process?

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is responsible for carbon dioxide conversion during photosynthesis and, therefore, is the most important protein in biomass generation. Modifications of this biocatalyst toward improvements in its properties are hindered by the complicated and not yet fully understood assembly process required for the formation of active holoenzymes. An entire set of auxiliary factors, including chaperonin GroEL/GroES and assembly chaperones RbcX or Rubisco accumulation factor 1 (RAF1), is involved in the folding and subsequent assembly of Rubisco subunits. Recently, it has been shown that cyanobacterial RAF1 acts during the formation of the large Rubisco subunit (RbcL) dimer. However, both its physiological function and its necessity in the prokaryotic Rubisco formation process remain elusive. Here, we demonstrate that the Synechocystis sp. PCC 6803 strain with raf1 gene disruption shows the same growth rate as wild-type cells under standard conditions. Moreover, the Rubisco biosynthesis process seems to be unperturbed in mutant cells despite the absence of RbcL-RAF1 complexes. However, in the tested environmental conditions, sulfur starvation triggers the degradation of RbcL and subsequent proteolysis of other polypeptides in wild-type but not Δraf1 strains. Pull-down experiments also indicate that, apart from Rubisco, RAF1 co-purifies with phycocyanins. We postulate that RAF1 is not an obligatory factor in cyanobacterial Rubisco assembly, but rather participates in environmentally regulated Rubisco homeostasis.

Biogenesis and Metabolic Maintenance of Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO2 in photosynthesis by catalyzing the carboxylation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Rubisco is a remarkably inefficient enzyme, fixing only 2-10 CO2 molecules per second. Efforts to increase crop yields by bioengineering Rubisco remain unsuccessful, owing in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. The large subunit of Rubisco requires the chaperonin system for folding, and recent studies have shown that assembly of hexadecameric Rubisco is mediated by specific assembly chaperones. Moreover, Rubisco function can be inhibited by a range of sugar-phosphate ligands, including RuBP. Metabolic repair depends on remodeling of Rubisco by the ATP-dependent Rubisco activase and hydrolysis of inhibitory sugar phosphates by specific phosphatases. Here, we review our present understanding of the structure and function of these auxiliary factors and their utilization in efforts to engineer more catalytically efficient Rubisco enzymes.

Screening of potential biomarkers for prenatal diagnosis of trisomy 21

We aimed to identify key genes located on chromosome 21 as potential biomarkers for prenatal diagnosis of trisomy 21 (Ts21). The microarray data of GSE48051, including 10 cultivated amniocyte samples with Ts21 and 9 controls with normal euploid constitution, was obtained from Gene Expression Omnibus database. The differentially expressed genes (DEGs) in cultivated amniocyte samples with Ts21 compared to normal controls were screened using limma package. Then, we performed GO enrichment analysis using DAVID and chromosomal location of DEGs based on the information of the University of California Santa Cruz (UCSC) Genome Browser Database. Finally, protein–protein interaction (PPI) network analysis was performed using STRING. Total 155 DEGs in cultivated amniocyte samples with Ts21 were identified, including 89 up- and 66 down-regulated DEGs. The over-represented GO terms of DEGs were mainly related with apoptosis, programmed cell death and cell death. In total, 13 DEGs were located on chromosome 21, thereinto, only 6 DEGs were included into the PPI network, including superoxide dismutase 1 (SOD1), phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase (GART), downstream neighbour of SON (DONSON), ATP synthase, H + transporting, mitochondrial F1 complex, O subunit (ATP5O), chromatin assembly factor 1, subunit B (p60) (CHAF1B) and proteasome (prosome, macropain) assembly chaperone 1 (PSMG1). Our results suggest that SOD1, GART, DONSON, ATP5O, CHAF1B and PSMG1 may play important roles in the pathogenesis of Down syndrome and may serve as potential biomarkers for prenatal diagnosis of Ts21.

Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry.

Chromosomal structure of nuclear DNA is usually maintained by insertion of nucleosomes into preexisting chromatin, both on newly synthesized DNA atreplication forks and at sites of DNA damage. But during retrovirus infection, a histone-free DNA copy of the viral genome is synthesized that must be loaded with nucleosomes de novo. Here, we show that core histones are rapidly loaded onto unintegrated Moloney murine leukemia virus DNAs. Loading of nucleosomes requires nuclear entry, but does not require viral DNA integration. The histones associated with unintegrated DNAs become marked by covalent modifications, with a delay relative to thetime of core histone loading. Expression from unintegrated DNA can be enhanced by modulation of the histone-modifying machinery. The data show that histone loading onto unintegrated DNAs occurs very rapidly after nuclear entry and does not require prior establishment of an integrated provirus.

Tetanus vaccination is associated with differential DNA-methylation: Reduces the risk of asthma in adolescence

BackgroundVaccinations have been suggested to be associated with increased risk of allergic diseases. Tetanus vaccination is one of the most frequently administered vaccines as a part of wound management and was also found to be associated with increased serum IgE levels. We hypothesized that the vaccination modifies the risk of allergic diseases through epigenetic changes such as DNA methylation. MethodData on tetanus vaccination between 10 and 18years of age was collected from a birth cohort established on the Isle of Wight UK in 1989. DNA methylation data were collected from individuals at different ages (at birth [n=30], age 10 [n=34], age 18 [n=245] and during pregnancy [n=121]) using the Illumina Infinium HumanMethylation450K array. Firstly, we performed an epigenome-wide screening to identify cytosine-phosphate-guanine sites (CpGs) associated with tetanus vaccination in 18-year-olds. Secondly, we tested their association with asthma, allergic sensitization, eczema, serum IgE and pulmonary lung function (FVC, FEV1, FEV1/FVC, and FEF25-75%). We then described changes in the methylation of the selected CpG sites over age, and by vaccination status. ResultsTetanus vaccination was found to be associated with decreased methylation of cg14472551 (p value 0.5×10−5, FDR-adjusted p value 2.1×10−4) and increased methylation of cg01669161 (p value 0.0007, FDR-adjusted p value 0.014). Both CpGs, in turn, were associated with decreased risk of asthma at 18years of age. Cg14472551 is located in an intron of KIAA1549L, whose protein binds to a B-cell commitment transcription factor; cg01669161 is located between an antisense regulator of the proteasome assembly chaperone PSMG3, and TFAMP1, a pseudogene. Increased methylation of cg01669161 was also associated with decreased serum IgE levels. ConclusionDNA methylation changes following tetanus vaccination may offer a novel prospect to explain a differential occurrence of asthma in adolescence.

Pangenome and immuno-proteomics analysis of Acinetobacter baumannii strains revealed the core peptide vaccine targets.

BackgroundAcinetobacter baumannii has emerged as a significant nosocomial pathogen during the last few years, exhibiting resistance to almost all major classes of antibiotics. Alternative treatment options such as vaccines tend to be most promising and cost effective approaches against this resistant pathogen. In the current study, we have explored the pan-genome of A. baumannii followed by immune-proteomics and reverse vaccinology approaches to identify potential core vaccine targets.ResultsThe pan-genome of all available A. baumannii strains (30 complete genomes) is estimated to contain 7,606 gene families and the core genome consists of 2,445 gene families (~32 % of the pan-genome). Phylogenetic tree, comparative genomic and proteomic analysis revealed both intra- and inter genomic similarities and evolutionary relationships. Among the conserved core genome, thirteen proteins, including P pilus assembly protein, pili assembly chaperone, AdeK, PonA, OmpA, general secretion pathway protein D, FhuE receptor, Type VI secretion system OmpA/MotB, TonB dependent siderophore receptor, general secretion pathway protein D, outer membrane protein, peptidoglycan associated lipoprotein and peptidyl-prolyl cis-trans isomerase are identified as highly antigenic. Epitope mapping of the target proteins revealed the presence of antigenic surface exposed 9-mer T-cell epitopes. Protein-protein interaction and functional annotation have shown their involvement in significant biological and molecular processes. The pipeline is validated by predicting already known immunogenic targets against Gram negative pathogen Helicobacter pylori as a positive control.ConclusionThe study, based upon combinatorial approach of pan-genomics, core genomics, proteomics and reverse vaccinology led us to find out potential vaccine candidates against A. baumannii. The comprehensive analysis of all the completely sequenced genomes revealed thirteen putative antigens which could elicit substantial immune response. The integration of computational vaccinology strategies would facilitate in tackling the rapid dissemination of resistant A.baumannii strains. The scarcity of effective antibiotics and the global expansion of sequencing data making this approach desirable in the development of effective vaccines against A. baumannii and other bacterial pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2951-4) contains supplementary material, which is available to authorized users.

The Ribosome Cooperates with the Assembly Chaperone pICln to Initiate Formation of snRNPs

The formation of macromolecular complexes within the crowded environment of cells often requires aid from assembly chaperones. PRMT5 and SMN complexes mediate this task for the assembly of the common core of pre-mRNA processing small nuclear ribonucleoprotein particles (snRNPs). Core formation is initiated by the PRMT5-complex subunit pICln, which pre-arranges the core proteins into spatial positions occupied in the assembled snRNP. The SMN complex then accepts these pICln-bound proteins and unites them with small nuclear RNA (snRNA). Here, we have analyzed how newly synthesized snRNP proteins are channeled into the assembly pathway to evade mis-assembly. We show that they initially remain bound to the ribosome near the polypeptide exit tunnel and dissociate upon association with pICln. Coincident with its release activity, pICln ensures the formation of cognate heterooligomers and their chaperoned guidance into the assembly pathway. Our study identifies the ribosomal quality control hub as a site where chaperone-mediated assembly of macromolecular complexes can be initiated.

Adhesive Dimerization of Human P-Cadherin Catalyzed by a Chaperone-like Mechanism

Orderly assembly of classical cadherins governs cell adhesion and tissue maintenance. A key event is the strand-swap dimerization of the extracellular ectodomains of two cadherin molecules from apposing cells. Here we have determined crystal structures of P-cadherin in six different conformational states to elaborate a motion picture of its adhesive dimerization at the atomic level. The snapshots revealed that cell-adhesive dimerization is facilitated by several intermediate states collectively termed X-dimer in analogy to other classical cadherins. Based on previous studies and on the combined structural, kinetic, thermodynamic, biochemical, and cellular data reported herein, we propose that the adhesive dimerization of human P-cadherin is achieved by a stepwise mechanism analogous to that of assembly chaperones. This mechanism, applicable to type I classical cadherins, confers high specificity and fast association rates. We expect these findings to guide innovative therapeutic approaches targeting P-cadherin in cancer.

Recognition and targeting mechanisms by chaperones in flagellum assembly and operation

The flagellum is a complex bacterial nanomachine that requires the proper assembly of several different proteins for its function. Dedicated chaperones are central in preventing aggregation or undesired interactions of flagellar proteins, including their targeting to the export gate. FliT is a key flagellar chaperone that binds to several flagellar proteins in the cytoplasm, including its cognate filament-capping protein FliD. We have determined the solution structure of the FliT chaperone in the free state and in complex with FliD and the flagellar ATPase FliI. FliT adopts a four-helix bundle and uses a hydrophobic surface formed by the first three helices to recognize its substrate proteins. We show that the fourth helix constitutes the binding site for FlhA, a membrane protein at the export gate. In the absence of a substrate protein FliT adopts an autoinhibited structure wherein both the binding sites for substrates and FlhA are occluded. Substrate binding to FliT activates the complex for FlhA binding and thus targeting of the chaperone-substrate complex to the export gate. The activation and targeting mechanisms reported for FliT appear to be shared among the other flagellar chaperones.

Impaired Chaperone Activity of Human Heat Shock Protein Hsp27 Site‐Specifically Modified with Argpyrimidine

AbstractNon‐enzymatic posttranslational modifications (nPTMs) affect at least ∼30 % of human proteins, but our understanding of their impact on protein structure and function is limited. Studies of nPTMs are difficult because many modifications are not included in common chemical libraries or protein expression systems and should be introduced site‐specifically. Herein, we probed the effect of the nPTM argpyrimidine on the structure and function of human protein Hsp27, which acquires argpyrimidine at residue 188 in vivo. We developed a synthetic approach to an argpyrimidine building block, which we then incorporated at position 188 of Hsp27 through protein semisynthesis. This modification did not affect the protein secondary structure, but perturbed the oligomeric assembly and impaired chaperone activity. Our work demonstrates that protein function can be altered by a single nPTM and opens up a new area of investigation only accessible by methods that allow site‐selective protein modification.

Impaired Chaperone Activity of Human Heat Shock Protein Hsp27 Site-Specifically Modified with Argpyrimidine.

Non-enzymatic posttranslational modifications (nPTMs) affect at least ∼30 % of human proteins, but our understanding of their impact on protein structure and function is limited. Studies of nPTMs are difficult because many modifications are not included in common chemical libraries or protein expression systems and should be introduced site-specifically. Herein, we probed the effect of the nPTM argpyrimidine on the structure and function of human protein Hsp27, which acquires argpyrimidine at residue 188 in vivo. We developed a synthetic approach to an argpyrimidine building block, which we then incorporated at position 188 of Hsp27 through protein semisynthesis. This modification did not affect the protein secondary structure, but perturbed the oligomeric assembly and impaired chaperone activity. Our work demonstrates that protein function can be altered by a single nPTM and opens up a new area of investigation only accessible by methods that allow site-selective protein modification.

PSMD10/gankyrin induces autophagy to promote tumor progression through cytoplasmic interaction with ATG7 and nuclear transactivation of ATG7 expression

ABSTRACTAlthough autophagy is most critical for survival of cancer cells, especially in fast-growing tumors, the mechanism remains to be fully characterized. Herein we report that PSMD10/gankyrin promotes autophagy in hepatocellular carcinoma (HCC) in response to starvation or stress through 2 complementary routes. PSMD10 was physically associated with ATG7 in the cytoplasm, and this association was enhanced by initial nutrient deprivation. Subsequently, PSMD10 translocated into the nucleus and bound cooperatively with nuclear HSF1 (heat shock transcription factor 1) onto the ATG7 promoter, upregulated ATG7 expression in the advanced stage of starvation. Intriguingly, the type of PSMD10-mediated autophagy was independent of the proteasome system, although PSMD10 has been believed to be an indispensable chaperone for assembly of the 26S proteasome. A significant correlation between PSMD10 expression and ATG7 levels was detected in human HCC biopsies, and the combination of these 2 parameters is a powerful predictor of poor prognosis. The median survival of sorafenib-treated HCC patients with high expression of PSMD10 was much shorter than those with low expression of PSMD10. Furthermore, PSMD10 augmented autophagic flux to resist sorafenib or conventional chemotherapy, and inhibition of autophagy suppressed PSMD10-mediated resistance. We conclude that these results present a novel mechanism involving modulation of ATG7 by PSMD10 in sustaining autophagy, promoting HCC cell survival against starvation or chemotherapy. Targeting of PSMD10 might therefore be an attractive strategy in HCC treatment by suppressing autophagy and inducing HCC cell sensitivity to drugs.

The Escherichia coli P and Type 1 Pilus Assembly Chaperones PapD and FimC Are Monomeric in Solution.

The chaperone/usher pathway is used by Gram-negative bacteria to assemble adhesive surface structures known as pili or fimbriae. Uropathogenic strains of Escherichia coli use this pathway to assemble P and type 1 pili, which facilitate colonization of the kidney and bladder, respectively. Pilus assembly requires a periplasmic chaperone and outer membrane protein termed the usher. The chaperone allows folding of pilus subunits and escorts the subunits to the usher for polymerization into pili and secretion to the cell surface. Based on previous structures of mutant versions of the P pilus chaperone PapD, it was suggested that the chaperone dimerizes in the periplasm as a self-capping mechanism. Such dimerization is counterintuitive because the chaperone G1 strand, important for chaperone-subunit interaction, is buried at the dimer interface. Here, we show that the wild-type PapD chaperone also forms a dimer in the crystal lattice; however, the dimer interface is different from the previously solved structures. In contrast to the crystal structures, we found that both PapD and the type 1 pilus chaperone, FimC, are monomeric in solution. Our findings indicate that pilus chaperones do not sequester their G1 β-strand by forming a dimer. Instead, the chaperones may expose their G1 strand for facile interaction with pilus subunits. We also found that the type 1 pilus adhesin, FimH, is flexible in solution while in complex with its chaperone, whereas the P pilus adhesin, PapGII, is rigid. Our study clarifies a crucial step in pilus biogenesis and reveals pilus-specific differences that may relate to biological function. Pili are critical virulence factors for many bacterial pathogens. Uropathogenic E. coli relies on P and type 1 pili assembled by the chaperone/usher pathway to adhere to the urinary tract and establish infection. Studying pilus assembly is important for understanding mechanisms of protein secretion, as well as for identifying points for therapeutic intervention. Pilus biogenesis is a multistep process. This work investigates the oligomeric state of the pilus chaperone in the periplasm, which is important for understanding early assembly events. Our work unambiguously demonstrates that both PapD and FimC chaperones are monomeric in solution. We further demonstrate that the solution behavior of the FimH and PapGII adhesins differ, which may be related to functional differences between the two pilus systems.

Assembly of an Evolutionarily Conserved Alternative Proteasome Isoform in Human Cells

Targeted intracellular protein degradation in eukaryotes is largely mediated by the proteasome. Here, we report the formation of an alternative proteasome isoform in human cells, previously found only in budding yeast, that bears an altered subunit arrangement in the outer ring of the proteasome core particle. These proteasomes result from incorporation of an additional α4 (PSMA7) subunit in the position normally occupied by α3 (PSMA4). Assembly of "α4-α4" proteasomes depends on the relative cellular levels of α4 and α3 and on the proteasome assembly chaperone PAC3. The oncogenic tyrosine kinases ABL and ARG and the tumor suppressor BRCA1 regulate cellular α4 levels and formation of α4-α4 proteasomes. Cells primed to assemble α4-α4 proteasomes exhibit enhanced resistance to toxic metal ions. Taken together, our results establish the existence of an alternative mammalian proteasome isoform and suggest a potential role in enabling cells to adapt to environmental stresses.

Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly.

Biogenesis of the 12-subunit RNA polymerase II (Pol II) transcription complex requires so-called GPN-loop GTPases, but the function of these enzymes is unknown. Here we report the first crystal structure of a eukaryotic GPN-loop GTPase, the Saccharomyces cerevisiae enzyme Npa3 (a homolog of human GPN1, also called RPAP4, XAB1, and MBDin), and analyze its catalytic mechanism. The enzyme was trapped in a GDP-bound closed conformation and in a novel GTP analog-bound open conformation displaying a conserved hydrophobic pocket distant from the active site. We show that Npa3 has chaperone activity and interacts with hydrophobic peptide regions of Pol II subunits that form interfaces in the assembled Pol II complex. Biochemical results are consistent with a model that the hydrophobic pocket binds peptides and that this can allosterically stimulate GTPase activity and subsequent peptide release. These results suggest that GPN-loop GTPases are assembly chaperones for Pol II and other protein complexes.

Operon Gene Order Is Optimized for Ordered Protein Complex Assembly.

SummaryThe assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization.