Costa Georgopoulos

Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli

The E. coli chaperonin GroEL and its cofactor GroES promote protein folding by sequestering nonnative polypeptides in a cage-like structure. Here we define the contribution of this system to protein folding across the entire E. coli proteome. Approximately 250 different proteins interact with GroEL, but most of these can utilize either GroEL or the upstream chaperones trigger factor (TF) and DnaK for folding. Obligate GroEL-dependence is limited to only approximately 85 substrates, including 13 essential proteins, and occupying more than 75% of GroEL capacity. These proteins appear to populate kinetically trapped intermediates during folding; they are stabilized by TF/DnaK against aggregation but reach native state only upon transfer to GroEL/GroES. Interestingly, substantially enriched among the GroEL substrates are proteins with (betaalpha)8 TIM-barrel domains. We suggest that the chaperonin system may have facilitated the evolution of this fold into a versatile platform for the implementation of numerous enzymatic functions.

Polypeptide flux through bacterial Hsp70: DnaK cooperates with trigger factor in chaperoning nascent chains.

A role for DnaK, the major E. coli Hsp70, in chaperoning de novo protein folding has remained elusive. Here we show that under nonstress conditions DnaK transiently associates with a wide variety of nascent and newly synthesized polypeptides, with a preference for chains larger than 30 kDa. Deletion of the nonessential gene encoding trigger factor, a ribosome-associated chaperone, results in a doubling of the fraction of nascent polypeptides interacting with DnaK. Combined deletion of the trigger factor and DnaK genes is lethal under normal growth conditions. These findings indicate important, partially overlapping functions of DnaK and trigger factor in de novo protein folding and explain why the loss of either chaperone can be tolerated by E. coli.

The E. coli dnaK gene product, the hsp70 homolog, can reactivate heat-inactivated RNA polymerase in an ATP hydrolysis-dependent manner

Pelham previously proposed that the hsp70 family of heat shock proteins could prevent the formation and/or allow the dissolution of protein aggregates created during stress conditions. We confirmed this hypothesis by showing that the E. coli hsp70 homolog, the dnaK gene product, protects the host RNA polymerase enzyme from heat inactivation in an ATP-independent reaction. In addition, we show that heat-inactivated and aggregated RNA polymerase is both disaggregated and reactivated following simultaneous incubation with DnaK protein and hydrolyzable ATP. The DnaK756 mutant protein has lost the ability to disaggregate the inactivated RNA polymerase enzyme. Our results demonstrate that the DnaK protein contributes to E. colis growth not only by protecting some enzymes from denaturation but also by reactivating some once they are misfolded or aggregated.

Modulation of the Escherichia coli sigmaE (RpoE) heat-shock transcription-factor activity by the RseA, RseB and RseC proteins.

The σE (RpoE) transcription factor of Escherichia coli regulates the expression of genes whose products are devoted to extracytoplasmic activities. The σE regulon is induced upon misfolding of proteins in the periplasm or the outer membrane. Similar to other alternative sigma factors, the activity of σE is tightly regulated in E. coli. We have previously shown that σE is positively autoregulated at the transcriptional level. DNA sequencing, coupled with transcriptional analyses, have shown that σE is encoded by the first gene of a four‐gene operon. The second gene of this operon, rseA, encodes an anti‐σE activity. This was demonstrated at both the genetic and biochemical levels. For example, mutations in rseA constitutively increase σE activity. Consistent with this, overproduction of RseA leads to an inhibitory effect on σE activity. Topological analysis of RseA suggests the existence of one transmembrane domain, with the N‐terminal part localized in the cytoplasm. Overproduction of this N‐terminal domain alone was shown to inhibit σE activity. These observations were confirmed in vitro, because either purified RseA or only its purified N‐terminal domain inhibited transcription from EσE‐dependent promoters. Furthermore, RseA and σE co‐purify, and can be co‐immunoprecipitated, and chemically cross‐linked. The σE activity is further modulated by the products of the remaining genes in this operon, rseB and rseC. RseB is a periplasmic protein, which negatively regulates σE activity and specifically interacts with the C‐terminal periplasmic domain of RseA. In contrast, RseC is an inner membrane protein that positively modulates σE activity. Most of these protein–protein interactions were verified in vivo using the yeast two‐hybrid system.

The dnaK protein modulates the heat-shock response of Escherichia coli

E. coli bacteria respond to a sudden upward shift in temperature by transiently overproducing a small subset of their proteins, one of which is the product of the dnaK gene. Mutations in dnaK have been previously shown to affect both DNA and RNA synthesis in E. coli. Bacteria carrying the dnaK756 mutation fail to turn off the heat-shock response at 43 degrees C. Instead, they continue to synthesize the heat-shock proteins in large amounts and underproduce other proteins. Both reversion and P1 transduction analyses have shown that the failure to turn off the heat-shock response is the result of the dnaK756 mutation. In addition, bacteria that overproduce the dnaK protein at all temperatures undergo a drastically reduced heat-shock response at high temperature. We conclude that the dnaK protein is an inhibitor of the heat-shock response in E. coli.

Function of Trigger Factor and DnaK in Multidomain Protein Folding: Increase in Yield at the Expense of Folding Speed

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.

The essential Escherichia coli msbA gene, a multicopy suppressor of null mutations in the htrB gene, is related to the universally conserved family of ATP‐dependent translocators

We report the characterization of the msbA gene, isolated as a multicopy suppressor of the HtrB temperature‐sensitive phenotype. The msbA gene maps to 20.5 min on the Escherichia coli genetic map and encodes a protein with an estimated molecular mass of 64460 Da, with the properties of an integral membrane protein. The amino acid sequence of MsbA is very similar to those of the family of ATP‐dependent translocators, which includes the haemolysin B protein of E. coli and the mammalian multidrug resistance (MDR) proteins. Mutational analysis of msbA indicates that it may form an operon with a downstream gene, orfE, and that both of these genes are essential for bacterial viability under all growth conditions tested.

Mitochondrial Hsp60 Chaperonopathy Causes an Autosomal-Recessive Neurodegenerative Disorder Linked to Brain Hypomyelination and Leukodystrophy

Hypomyelinating leukodystrophies (HMLs) are disorders involving aberrant myelin formation. The prototype of primary HMLs is the X-linked Pelizaeus-Merzbacher disease (PMD) caused by mutations in PLP1. Recently, homozygous mutations in GJA12 encoding connexin 47 were found in patients with autosomal-recessive Pelizaeus-Merzbacher-like disease (PMLD). However, many patients of both genders with PMLD carry neither PLP1 nor GJA12 mutations. We report a consanguineous Israeli Bedouin kindred with clinical and radiological findings compatible with PMLD, in which linkage to PLP1 and GJA12 was excluded. Using homozygosity mapping and mutation analysis, we have identified a homozygous missense mutation (D29G) not previously described in HSPD1, encoding the mitochondrial heat-shock protein 60 (Hsp60) in all affected individuals. The D29G mutation completely segregates with the disease-associated phenotype. The pathogenic effect of D29G on Hsp60-chaperonin activity was verified by an in vivo E. coli complementation assay, which demonstrated compromised ability of the D29G-Hsp60 mutant protein to support E. coli survival, especially at high temperatures. The disorder, which we have termed MitCHAP-60 disease, can be distinguished from spastic paraplegia 13 (SPG13), another Hsp60-associated autosomal-dominant neurodegenerative disorder, by its autosomal-recessive inheritance pattern, as well as by its early-onset, profound cerebral involvement and lethality. Our findings suggest that Hsp60 defects can cause neurodegenerative pathologies of varying severity, not previously suspected on the basis of the SPG13 phenotype. These findings should help to clarify the important role of Hsp60 in myelinogenesis and neurodegeneration.

Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes

SummaryA λ hybrid phage (λSda1), containing an 8.1 kb EcoRI DNA fragment from the Escherichia coli chromosome, was selected on the basis of its ability to suppress bacterial thermosensitivity caused by the dnaA46 mutation. We have shown that this suppression is due to a recA+-dependent amplification of the 8.1 kb fragment; consistent with this observation, cloning of the 8.1 kb fragment into a high copy number plasmid (pBR325) leads also to suppression of dnaA46. In the suppressed strains growing at high temperature, bidirectional replication starts in or near the oriC region and requires the presence of the DnaA polypeptide. These findings suggest that the overproduction of a gene product(s), encoded by the cloned 8.1 kb fragment, can restore dnaA-dependent initiation of replication at high temperature in the oriC region. Genetic mapping shows that the groES (mopB) and groEL (mopA) genes are located on the 8.1 kb suppressor fragment. Further analysis, including in vitro mutagenesis and subcloning, demonstrates that the amplification of the groES and groEL genes is both necessary and sufficient to suppress the temperature sensitive phenotype of the dnaA46 mutation.

MUTATIONAL ANALYSIS AND PROPERTIES OF THE MSBA GENE OF ESCHERICHIA COLI, CODING FOR AN ESSENTIAL ABC FAMILY TRANSPORTER

The htrB gene was discovered because its insertional inactivation interfered with Escherichia coli growth and viability at temperatures above 32.5°C, as a result of accumulation of phospholipids. The msbA gene was originally discovered because when cloned on a low‐copy‐number plasmid vector it was able to suppress the temperature‐sensitive growth phenotype of an htrB null mutant as well as the accumulation of phospholipids. The msbA gene product belongs to the superfamily of ABC transporters, a universally conserved family of proteins characterized by a highly conserved ATP‐binding domain. The msbA gene is essential for bacterial viability at all temperatures. In order to understand the physiological role of the MsbA protein, we mutated the ATP‐binding domain using random PCR mutagenesis. Six independent mutants were isolated and characterized. Four of these mutations resulted in single‐amino‐acid substitutions in non‐conserved residues and were able to support cell growth at 30°C but not at 43°C. The remaining two mutations behaved as recessive lethals, and resulted in single‐amino‐acid substitutions in Walker motif B, one of the two highly conserved regions of the ATP‐binding domain. Despite the fact that neither of these two mutant proteins can support E. coli growth, they both retained the ability to bind ATP in vitro. In addition, we present evidence to show that W‐acetyl [3H]‐glucosamine, a precursor of lipopolysaccharides, accumulates at the non‐permissive temperature in the inner membrane of either htrB null or msbA conditional lethal strains. Translocation of the precursor to the outer membrane is restored by transformation with a plasmid containing the wild‐type msbA gene. A possible role for MsbA