Clathrin-lndependent endocytosis and signalling of interleukin 2 receptors IL-2R endocytosis and signalling.

Interleukin 2 receptors (IL-2R) belong to the cytokine receptor family and share subunits with other members of the family. They are essential in T cell activation and in maintaining homeostatic immune responses. These receptors do not have an intrinsic kinase activity and use multiple signalling pathways. Their endocytic pathway is different from that of classic growth factor receptors in that it does not follow the classic clathrin-coated pit and vesicle route. After uptake, one of the IL-2R chains, alpha, recycles to the plasma membrane, whereas the two other chains, beta and gamma, are targeted to late endosomes/lysosomes and degraded. This involves ubiquitination of the receptor as a sorting signal. Links between the signalling events, internalisation and intracellular sorting of these receptors are reviewed.

Disulfide bond formation in secreton component PulK provides a possible explanation for the role of DsbA in pullulanase secretion.

When expressed in Escherichia coli, the 15 Klebsiella oxytoca pul genes that encode the so-called Pul secreton or type II secretion machinery promote pullulanase secretion and the assembly of one of the secreton components, PulG, into pili. Besides these pul genes, efficient pullulanase secretion also requires the host dsbA gene, encoding a periplasmic disulfide oxidoreductase, independently of disulfide bond formation in pullulanase itself. Two secreton components, the secretin pilot protein PulS and the minor pseudopilin PulK, were each shown to posses an intramolecular disulfide bond whose formation was catalyzed by DsbA. PulS was apparently destabilized by the absence of its disulfide bond, whereas PulK stability was not dramatically affected either by a dsbA mutation or by the removal of one of its cysteines. The pullulanase secretion defect in a dsbA mutant was rectified by overproduction of PulK, indicating reduced disulfide bond formation in PulK as the major cause of the secretion defect under the conditions tested (in which PulS is probably present in considerable excess of requirements). PulG pilus formation was independent of DsbA, probably because PulK is not needed for piliation.

Pilus formation and protein secretion by the same machinery in Escherichia coli.

The secreton (type II secretion) and type IV pilus biogenesis branches of the general secretory pathway in Gram-negative bacteria share many features that suggest a common evolutionary origin. Five components of the secreton, the pseudopilins, are similar to subunits of type IV pili. Here, we report that when the 15 genes encoding the pullulanase secreton of Klebsiella oxytoca were expressed on a high copy number plasmid in Escherichia coli, one pseudopilin, PulG, was assembled into pilus-like bundles. Assembly of the 'secreton pilus' required most but not all of the secreton components that are essential for pullulanase secretion, including some with no known homologues in type IV piliation machineries. Two other pseudopilins, pullulanase and two outer membrane-associated secreton components were not associated with pili. Thus, PulG is probably the major component of the pilus. Expression of a type IV pilin gene, the E.coli K-12 gene ppdD, led to secreton-dependent incorporation of PpdD pilin into pili without diminishing pullulanase secretion. This is the first demonstration that pseudopilins can be assembled into pilus-like structures.

PpdD type IV pilin of Escherichia coli K-12 can Be assembled into pili in Pseudomonas aeruginosa.

Escherichia coli K-12 possesses at least 16 chromosomal genes related to genes involved in the formation of type IV pili in other gram-negative bacteria. However, E. coli K-12 does not produce type IV pili when grown under standard laboratory conditions. The results of reverse transcription-PCR, operon fusion analysis, and immunoblotting demonstrated that several of the putative E. coli piliation genes are expressed at very low levels. Increasing the level of expression of the major pilin gene (ppdD) and the linked assembly genes hofB and hofC (homologues of the Pseudomonas aeruginosa type IV pilus assembly genes pilB and pilC) did not lead to pilus production. However, expression of the ppdD gene in P. aeruginosa led to assembly of PpdD into pili that were recognized by antibodies directed against the PpdD protein. Assembly of PpdD into pili in P. aeruginosa was dependent on the expression of the pilB and pilC genes and independent of expression of the P. aeruginosa pilin structural gene pilA.

Genetic dissection of the outer membrane secretin PulD: are there distinct domains for multimerization and secretion specificity?

Linker and deletion mutagenesis and gene fusions were used to probe the possible domain structure of the dodecameric outer membrane secretin PulD from the pullulanase secretion pathway of Klebsiella oxytoca. Insertions of 24 amino acids close to or within strongly predicted and highly conserved amphipathic beta strands in the C-terminal half of the polypeptide (the beta domain) abolished sodium dodecyl sulfate (SDS)-resistant multimer formation that is characteristic of this protein, whereas insertions elsewhere generally had less dramatic effects on multimer formation. However, the beta domain alone did not form SDS-resistant multimers unless part of the N-terminal region of the protein (the N domain) was produced in trans. All of the insertions except one, close to the C terminus of the protein, abolished function. The N domain alone was highly unstable and did not form SDS-resistant multimers even when the beta domain was present in trans. We conclude that the beta domain is a major determinant of multimer stability and that the N domain contributes to multimer formation. The entire or part of the N domain of PulD could be replaced by the corresponding region of the OutD secretin from the pectate lyase secretion pathway of Erwinia chrysanthemi without abolishing pullulanase secretion. This suggests that the N domain of PulD is not involved in substrate recognition, contrary to the role proposed for the N domain of OutD, which binds specifically to pectate lyase secreted by E. chrysanthemi (V. E. Shevchik, J. Robert-Badouy, and G. Condemine, EMBO J. 16:3007-3016, 1997).

The requirement for DsbA in pullulanase secretion is independent of disulphide bond formation in the enzyme.

Results from previous studies have suggested that an intramolecular disulphide bond in the exoprotein pullulanase is needed for its recognition and transport across the outer membrane. This interpretation of the data is shown here to be incorrect: pullulanase devoid of all potential disulphide bonds is secreted with apparently the same efficiency as the wild-type protein. Furthermore, the periplasmic disulphide bond, oxidoreductase DsbA, previously shown to catalyse the formation of a disulphide bond in pullulanase and to decrease its transit time in the periplasm, is shown here to be required for the rapid secretion of pullulanase devoid of disulphide bonds. Several possible explanations for the role of DsbA in pullulanase secretion are discussed.

Recent progress and future directions in studies of the main terminal branch of the general secretory pathway in Gram-negative bacteria--a review.

The main terminal branch (MTB) of the general secretory pathway is used by a wide variety of Gram- bacteria to transport exoproteins from the periplasm to the outside milieu. Recent work has led to the identification of the function of two of its 14 (or more) components: an enzyme with type-IV prepilin peptidase activity and a chaperone-like protein required for the insertion of another of the MTB components into the outer membrane. Despite these important discoveries, little tangible progress has been made towards identifying MTB components that determine secretion specificity (presumably by binding to cognate exoproteins) or which form the putative channel through which exoproteins are transported across the outer membrane. However, the idea that the single integral outer membrane component of the MTB could line the wall of this channel, and the intriguing possibility that other components of the MTB form a rudimentary type-IV pilus-like structure that might span the periplasm both deserve more careful examination. Although Escherichia coli K-12 does not normally secrete exoproteins, its chromosome contains an apparently complete set of genes coding for MTB components. At least two of these genes code for functional proteins, but the operon in which twelve of the genes are located does not appear to be expressed. We are currently searching for conditions which allow these genes to be expressed with the eventual aim of identifying the protein(s) that E. coli K-12 can secrete.

Pullulanase: model protein substrate for the general secretory pathway of gram-negative bacteria.

Pullulanase of Klebsiella oxytoca is one of a wide variety of extracellular proteins that are secreted by Gram-negative bacteria by the complex main terminal branch (MTB) of the general secretory pathway. The roles of some of the 14 components of the MTB are now becoming clear. In this review it is proposed that most of these proteins form a complex, the secretion, that spans the cell envelope to control the opening and closing of channel in the outer membrane. Progress toward the goal of testing this model is reviewed.

Identification of two regions of Klebsiella oxytoca pullulanase that together are capable of promoting beta-lactamase secretion by the general secretory pathway.

Pullulanase (PulA) is a 116 kDa amylolytic lipoprotein secreted by the Gram-negative bacterium Klebsiella oxytoca via the general secretory pathway. A deletion strategy was used in an attempt to determine the nature and the location of the secretion signal(s) in PulA presumed to be necessary for its specific secretion. The starting material was a gene fusion coding for an efficiently secreted PulA-beta-lactamase hybrid protein. Successive series of exonuclease III-generated deletions were used to remove internal segments of PulA from this hybrid. A simple plate test allowed the identification of truncated hybrids that retained beta-lactamase activity and that were secreted. Two non-adjacent regions, A and B (78 and 80 amino acids, respectively), were together necessary and sufficient to promote beta-lactamase translocation across the outer membrane. Secretion of PulA itself was markedly reduced when either of these regions was deleted, and was completely abolished when both regions were eliminated.

Secretion of active beta-lactamase to the medium mediated by the Escherichia coli haemolysin transport pathway.

An in frame gene fusion containing the coding region for mature beta-lactamase and the 3'-end of hylA encoding the haemolysin secretion signal, was constructed under the control of a lac promoter. The resulting 53 kDa hybrid protein was specifically secreted to the external medium in the presence of the haemolysin translocator proteins, HlyB and HlyD. The specific activity of the beta-lactamase portion of the secreted protein (measured by the hydrolysis of penicillin G), approximately 1 U/microgram protein, was close to that of authentic, purified TEM-beta-lactamase. This is an important example of a hybrid protein that is enzymatically active, and secreted via the haemolysin pathway. Previous studies have indicated that haemolysin is secreted directly into the medium, bypassing the periplasm, to which beta-lactamase is normally targeted. This study indicated, therefore, that normal folding of an active beta-lactamase, can occur, at least when fused to the HlyA C-terminus, without the necessity of entering the periplasm. Despite the secretion of approximately 5 micrograms/ml levels of the active beta-lactamase fusion into the medium, there was maximally only a 50% detectable increase in the LD50 for resistance to ampicillin at the individual cell level. This result suggests that, normally, resistance to ampicillin requires a high concentration of the enzyme close to killing targets, i.e. in the periplasm, in order to achieve significant levels of protection.

Extracellular secretion of pullulanase is unaffected by minor sequence changes but is usually prevented by adding reporter proteins to its N- or C-terminal end.

Linker insertions in the pullulanase structural gene (pulA) were examined for their effects on pullulanase activity and cell surface localization in Escherichia coli carrying the cognate secretion genes from Klebsiella oxytoca. Of the 23 insertions, 11 abolished pullulanase activity but none were found to prevent secretion. To see whether more drastic changes affected secretion, we fused up to five reporter proteins (E. coli periplasmic alkaline phosphatase, E. coli periplasmic maltose-binding protein, periplasmic TEM beta-lactamase, Erwinia chrysanthemi extracellular endoglucanase Z, and Bacillus subtilis extracellular levansucrase) to three different positions in the pullulanase polypeptide: close to the N terminus of the mature protein, at the C terminus of the protein, or at the C terminus of a truncated pullulanase variant lacking the last 256 amino acids. Only 3 of the 13 different hybrids were efficiently secreted: 2 in which beta-lactamase was fused to the C terminus of full-length or truncated pullulanase and 1 in which maltose-binding protein was fused close to the N terminus of pullulanase. Affinity-purified endoglucanase-pullulanase and pullulanase-endoglucanase hybrids exhibited apparently normal levels of pullulanase activity, indicating that the conformation of the pullulanase segment of the hybrid had not been dramatically altered by the presence of the reporter. However, pullulanase-endoglucanase hybrids were secreted efficiently if the endoglucanase component comprised only the 60-amino-acid, C-terminal cellulose-binding domain, suggesting that at least one factor limiting hybrid protein secretion might be the size of the reporter.