Substrate complexes and domain organization of the Salmonella flagellar export chaperones FlgN and FliT

The flagellar proteins FlgN and FliT have been proposed to act as substrate-specific export chaperones, facilitating incorporation of the enterobacterial hook-associated axial proteins (HAPs) FlgK/FlgL and FliD into the growing flagellum. In Salmonella typhimurium flgN and fliT mutants, the export of target HAPs was reduced, concomitant with loss of unincorporated flagellin into the surrounding medium. Gel filtration chromatography of wild-type S. typhimurium cell extracts identified stable pools of FlgN and FliT homodimers in the cytosol, but no chaperone-substrate complexes were evident. Nevertheless, stable unique complexes were assembled efficiently in vitro by co-incubation of FlgN and FliT with target HAPs purified from recombinant Escherichia coli. The sizes of the chaperone-substrate complexes indicated that, in each case, a chaperone homodimer binds to a substrate monomer. FlgN prevented in vitro aggregation of FlgK monomers, generating a soluble form of the HAP. Recombinant polypeptides spanning the potentially amphipathic C-terminal regions of FlgN or FliT could not complement in trans the chaperone deficiency of the respective flgN and fliT mutants, but efficient flagellar assembly was restored by homodimeric translational fusions of these domains to glutathione S-transferase, which bound FlgK and FlgL like the wild-type FlgN. These data provide further evidence for the substrate-specific chaperone function of FlgN and FliT and indicate that these chaperones comprise common N- and C-terminal domains mediating homodimerization and HAP substrate binding respectively. In support of this view, the flgN mutation was specifically complemented by a hybrid chaperone comprising the N-terminal half of FliT and the C-terminal half of FlgN.     

Flagellar hook and hook-associated proteins of Salmonella typhimurium and their relationship to other axial components of the flagellum

Within the bacterial flagellum the basal-body rod, the hook, the hook-associated proteins (HAPs), and the helical filament constitute an axial substructure whose elements share structural features and a common export pathway. We present here the amino acid sequences of the hook protein and the three HAPs of Salmonella typhimurium, as deduced from the DNA sequences of their structural genes (flgE, flgK, flgL and fliD, respectively). We compared these sequences with each other and with those for the filament protein (flagellin) and four rod proteins, which have been described previously (Joys, 1985; Homma et al., 1990; Smith & Selander, 1990). Hook protein most strongly resembled the distal rod protein (FlgG) and the proximal HAP (HAP1), which are thought to be attached to the proximal and distal ends of the hook, respectively; the similarities were most pronounced near the N and C termini. Hook protein and flagellin, which occupy virtually identical helical lattices, did not resemble each other strongly but showed some limited similarities near their termini. HAP3 and HAP2, which form the proximal and distal boundaries of the filament, showed few similarities to flagellin, each other, or the other axial proteins. With the exceptions of the N-terminal region of HAP2, and the C-terminal region of flagellin, proline residues were absent from the terminal regions of the axial proteins. Moreover, with the exception of the N-terminal region of HAP2, the terminal regions contained hydrophobic residues at intervals of seven residues. Together, these observations suggest that the axial proteins may have amphipathic alpha-helical structure at their N and C termini. In the case of the filament and the hook, the terminal regions are believed to be responsible for the quaternary interactions between subunits. We suggest that this is likely to be true of the other axial structures as well, and specifically that interaction between N-terminal and C-terminal alpha-helices may be important in the formation of the axial structures of the flagellum. Although consensus sequences were noted among some of the proteins, such as the rod, hook and HAP1, no consensus extended to the entire set of axial proteins. Thus the basis for recognition of a protein for export by the flagellum-specific pathway remains to be identified.     

Bacterial birth scar proteins mark future flagellum assembly site

Many prokaryotic protein complexes underlie polar asymmetry. In Caulobacter crescentus, a flagellum is built exclusively at the pole that arose from the previous cell division. The basis for this pole specificity is unclear but could involve a cytokinetic birth scar that marks the newborn pole as the flagellum assembly site. We identified two developmental proteins, TipN and TipF, which localize to the division septum and the newborn pole after division. We show that septal localization of TipN/F depends on cytokinesis. Moreover, TipF, a c-di-GMP phosphodiesterase homolog, is a flagellum assembly factor that relies on TipN for proper positioning. In the absence of TipN, flagella are assembled at ectopic locations, and TipF is mislocalized to such sites. Thus TipN and TipF establish a link between bacterial cytokinesis and polar asymmetry, demonstrating that division does indeed leave a positional mark in its wake to direct the biogenesis of a polar organelle.     

Excretion of unassembled hook-associated proteins by Salmonella typhimurium.

Hook-associated proteins (HAPs) were excreted into the culture medium of the Fla+ strain as well as into the growth medium of the filamentless mutants of Salmonella typhimurium. This indicates that the bacteria synthesize HAPs excessively, beyond the amount required for construction of flagella. The extra HAPs are shed into the culture medium after a definite amount of each HAP has been assembled into the flagellar structure.     

Heterologous expression of naturally evolved putative de novo proteins with chaperones

Over the past decade, evidence has accumulated that new protein‐coding genes can emerge de novo from previously non‐coding DNA. Most studies have focused on large scale computational predictions of de novo protein‐coding genes across a wide range of organisms. In contrast, experimental data concerning the folding and function of de novo proteins are scarce. This might be due to difficulties in handling de novo proteins in vitro, as most are short and predicted to be disordered. Here, we propose a guideline for the effective expression of eukaryotic de novo proteins in Escherichia coli. We used 11 sequences from Drosophila melanogaster and 10 from Homo sapiens, that are predicted de novo proteins from former studies, for heterologous expression. The candidate de novo proteins have varying secondary structure and disorder content. Using multiple combinations of purification tags, E. coli expression strains, and chaperone systems, we were able to increase the number of solubly expressed putative de novo proteins from 30% to 62%. Our findings indicate that the best combination for expressing putative de novo proteins in E. coli is a GST‐tag with T7 Express cells and co‐expressed chaperones. We found that, overall, proteins with higher predicted disorder were easier to express.StatementToday, we know that proteins do not only evolve by duplication and divergence of existing proteins but also arise from previously non‐coding DNA. These proteins are called de novo proteins. Their properties are still poorly understood and their experimental analysis faces major obstacles. Here, we aim to present a starting point for soluble expression of de novo proteins with the help of chaperones and thereby enable further characterization.     

Swimming with protists: perception, motility and flagellum assembly

In unicellular and multicellular eukaryotes, fast cell motility and rapid movement of material over cell surfaces are often mediated by ciliary or flagellar beating. The conserved defining structure in most motile cilia and flagella is the '9+2' microtubule axoneme. Our general understanding of flagellum assembly and the regulation of flagellar motility has been led by results from seminal studies of flagellate protozoa and algae. Here we review recent work relating to various aspects of protist physiology and cell biology. In particular, we discuss energy metabolism in eukaryotic flagella, modifications to the canonical assembly pathway and flagellum function in parasite virulence.     

The FliO, FliP, FliQ, and FliR proteins of Salmonella typhimurium: putative components for flagellar assembly.

The flagellar genes fliO, fliP, fliQ, and fliR of Salmonella typhimurium are contiguous within the fliLMNOPQR operon. They are needed for flagellation but do not encode any known structural or regulatory components. They may be involved in flagellar protein export, which proceeds by a type III export pathway. The genes have been cloned and sequenced. The sequences predict proteins with molecular masses of 13,068, 26,755, 9,592, and 28,933 Da, respectively. All four gene products were identified experimentally; consistent with their high hydrophobic residue content, they segregated with the membrane fraction. From N-terminal amino acid sequence analysis, we conclude that fliO starts immediately after fliN rather than at a previously proposed site downstream. FliP existed in two forms, a 25-kDa form and a 23-kDa form. N-terminal amino acid analysis of the 23-kDa form demonstrated that it had undergone cleavage of a signal peptide--a rare process for prokaryotic cytoplasmic membrane proteins. Site-directed mutation at the cleavage site resulted in impaired processing, which reduced, but did not eliminate, complementation of a fliP mutant in swarm plate assays. A cloned fragment encoding the mature form of the protein could also complement the fliP mutant but did so even more poorly. Finally, when the first transmembrane span of MotA (a cytoplasmic membrane protein that does not undergo signal peptide cleavage) was fused to the mature form of FliP, the fusion protein complemented very weakly. Higher levels of synthesis of the mutant proteins greatly improved function. We conclude that, for insertion of FliP into the membrane, cleavage is important kinetically but not absolutely required.     

Molecular chaperones,stress proteins and redox homeostasis

Protection against oxidative stress is highly interrelated with the function of the most ancient cellular defense system, the network of molecular chaperones, heat shock, or stress-proteins. These ubiquitous, conserved proteins help other proteins and macromolecules to fold or re-fold and reach their final, native conformation. Redox regulation of protein folding becomes especially important during the preparation of extracellular proteins to the outside oxidative milieu, which should take place in a gradual and step-by-step controlled manner in the endoplasmic reticulum or in the periplasm. Several chaperones, such as members of the Hsp33 family in yeast and the plethora of small heat shock proteins as well as one of the major chaperones, Hsp70 are able to act against cytoplasmic oxidative damage. Abrupt changes of cellular redox status lead to chaperone induction. The function of several chaperones is tightly regulated by the surrounding redox conditions. Moreover, our recent data suggest that chaperones may act as a central switchboard for the transmission of redox changes in the life of the cell.     

Excretion of unassembled flagellin by Salmonella typhimurium mutants deficient in hook-associated proteins.

Of the flagellar filamentless mutants of Salmonella typhimurium, the flaV, flaU, and flaW mutants, which are defective in hook-associated proteins, synthesized flagellin molecules, but flagella did not polymerize at the tips of the mutant hooks and were excreted into the culture medium as intact monomers.     

Localization and stoichiometry of hook-associated proteins within Salmonella typhimurium flagella.

The localization of hook-associated proteins (HAP1, HAP2, and HAP3) in Salmonella typhimurium flagella was studied by using specific antibodies together with a second antibody conjugated with colloidal gold. HAP1 and HAP3 were localized at the hook-filament junction, as has been suggested previously. HAP2, however, was localized at the filament tip. This finding supports the idea that HAP2 acts to induce polymerization of endogenous flagellin at the filament tip, and HAP1 and HAP3 are junction proteins to connect hook with filament. Analysis of the protein composition of short flagella from a mutant indicated that a single flagellum contains about 10 to 20 HAP1, 10 to 20 HAP2, and 10 to 40 HAP3 molecules.     

A motile but non-swarming mutant of Proteus mirabilis lacks FlgN, a facilitator of flagella filament assembly

A Tn phoA mutant of Proteus mirabilis was isolated, which had lost the ability to swarm, yet was still motile. The transposon had inserted into flgN , a flagella gene encoding a 147-amino-acid protein of undefined function. Proteus flgN is arranged in an operon with the class III anti-σ²⁸ gene, flgM , flanked by the class II genes, flgA , flgBCD and flhBA , and a novel putative virulence-related gene. The flgN mutation caused a substantial reduction in cell surface-associated flagellin, particularly during differentiation to the normally hyperflagellated swarm cell. This was not due to an effect on flagella gene expression or a typical defect in the flagella export apparatus as there was no class III gene downregulation by FlgM feedback, or intracellular flagellin accumulation. Loss of FlgN nevertheless caused a severe reduction in the incorporation of pulse-labelled flagellin into the membrane/flagellum fraction of differentiating cells. Substantial amounts of both non-oligomeric flagellin and flagellin degradation products appeared in the extracellular medium, although the few mature filaments made by the mutant were no more sensitive to proteolysis than those of the wild type. FlgN appeared soluble and active in the cytosol. The data suggest that the function of FlgN is to facilitate the initiation of flagella filament assembly, a role that may be especially critical in attaining the much higher concentration of surface flagellin required for swarming. Proteus FlgN has leucine zipper-like motifs arranged on potential amphipathic helices, a feature conserved in cytosolic chaperones for the exported substrates of flagella-related type III virulence systems. While gel filtration of FlgN from the soluble cell fraction did not establish an interaction with flagellin, it indicated that FlgN may associate with an unknown component and/or form an oligomer.     

Protein binding and disruption by Clp/Hsp100 chaperones

Clp/Hsp100 chaperones work with other cellular chaperones and proteases to control the quality and amounts of many intracellular proteins. They employ an ATP-dependent protein unfoldase activity to solubilize protein aggregates or to target specific classes of proteins for degradation. The structural complexity of Clp/Hsp100 proteins combined with the complexity of the interactions with their macromolecular substrates presents a considerable challenge to understanding the mechanisms by which they recognize and unfold substrates and deliver them to downstream enzymes. Fortunately, high-resolution structural data is now available for several of the chaperones and their functional partners, which together with mutational data on the chaperones and their substrates has provided a glimmer of light at the end of the Clp/Hsp100 tunnel.     

Methylation of Sm proteins by a complex containing PRMT5 and the putative U snRNP assembly factor pICln

Seven Sm proteins, termed B/B', D1, D2, D3, E, F, and G, assemble in an ordered manner onto U snRNAs to form the Sm core of the spliceosomal snRNPs U1, U2, U4/U6, and U5. The survival of motor neuron (SMN) protein binds to Sm proteins and mediates in the context of a macromolecular (SMN-) complex the assembly of the Sm core. Binding of SMN to Sm proteins is enhanced by modification of specific arginine residues in the Sm proteins D1 and D3 to symmetrical dimethylarginines (sDMAs), suggesting that assembly might be regulated at the posttranslational level. Here we provide evidence that the previously described pICln-complex, consisting of Sm proteins, the methyltransferase PRMT5, pICln, and two novel factors, catalyzes the sDMA modification of Sm proteins. In vitro studies further revealed that the pICln complex inhibits the spontaneous assembly of Sm proteins onto a U snRNA. This effect is mediated by pICln via its binding to the Sm fold of Sm proteins, thereby preventing specific interactions between Sm proteins required for the formation of the Sm core. Our data suggest that the pICln complex regulates an early step in the assembly of U snRNPs, possibly the transfer of Sm proteins to the SMN-complex.     

Microtubules, mitochondria, and molecular chaperones: a new hypothesis for in vivo assembly of microtubules

In Chinese hamster ovary cells, a number of independent mutants selected for resistance to antimitotic drugs have been found to be specifically altered in two major cellular proteins, designated P1 (relative mass (Mr) approximately 60-63 kilodaltons (kDa] and P2 (Mr approximately 69-70 kDa), which appeared microtubule related by a number of genetic and biochemical criteria. Antibodies to P1 have been found to bind specifically to mitochondria that showed specific association with microtubules in interphase cells. Biochemical and cDNA sequence studies on P1 showed that this protein, which is localized in the matrix compartment, is the mammalian homolog of the highly conserved chaperonin family of proteins (other members include the GroEL protein of Escherichia coli, the 60-kDa heat-shock protein of yeast, and the rubisco subunit binding protein of plant chloroplasts). The chaperonin proteins in various systems play a transient but essential molecular chaperone role in the proper folding of polypeptide chains and their assembly into oligomeric protein complexes. Our studies on P2 protein established that it corresponds to the constitutive form of the major 70-kDa heat-shock protein of mammalian cells (i.e., hsc70), which also acts as a molecular chaperone in the intracellular transport of nascent proteins to organelles such as mitochondria and endoplasmic reticulum. To account for the above, as well as a number of other observations (e.g., binding of fluorescent-labeled antimitotic drugs to mitochondria, association of tubulin with mitochondria as well as other membranes, and high affinity binding of antimitotic drugs to free tubulin but not to assembled microtubules), a new model for the in vivo assembly of interphase microtubules is proposed. The model ascribes a central role to the mitochondrially localized chaperonin (i.e., P1) protein in the intracellular formation of tubulin dimers and in their addition to the growth sites in microtubules. The proposed model also explains a number of other observations related to microtubule assembly in the literature.