Traffic ATPases Research Articles

Mutational analysis of the Escherichia coli phosphate-specific transport system, a member of the traffic ATPase (or ABC) family of membrane transporters. A role for proline residues in transmembrane helices.

The Escherichia coli Pst system is a periplasmic phosphate permease. A mutational analysis of the requirement for function of specific charged residues or proline residues in the two hydrophobic subunits (PstC and PstA) has been carried out. No residues, among 19 charged residues altered, were found to be essential for phosphate uptake, although some alterations resulted in partial effects. Evidence was obtained that the 3 residues, R220 in the PstA protein and R237 and E241 in the PstC protein, previously shown to be required for phosphate transport (Cox, G. B., Webb, D., Godovac-Zimmermann, J., and Rosenberg, H. (1988) J. Bacteriol. 170, 2283-2286; Cox, G. B., Webb, D., and Rosenberg, H. (1989) J. Bacteriol. 171, 1531-1534), interact with each other. A feature of the proposed structures of the PstA and PstC proteins was 2 pairs of proline residues in putative transmembrane helices 3 and 4. While individual substitutions of these proline residues by leucine resulted in loss of phosphate transport activity substitution by alanine only had partial effects. However, if the proline to alanine changes were paired then, depending on the particular subunit, markedly different effects were obtained. The double mutation in the PstA protein resulted in a permanently "closed" system, whereas the double mutation in the PstC protein resulted in a permanently "open" transport system.

Secretion of peptides and proteins lacking hydrophobic signal sequences: the role of adenosine triphosphate-driven membrane translocators.

In this review, we will emphasize the role of ATP-dependent membrane transporters in protein export and intracellular protein trafficking in prokaryotic and eukaryotic cells. ATP-binding-cassette (ABC)-transport proteins, also termed "traffic ATPases," belong to a superfamily of ubiquitous ATP-driven membrane transporters that share extensive sequence similarity and highly conserved domain organization. They are implicated in a remarkable variety of transmembrane transport processes, including the transport of ions, heavy metals, sugars, anticancer drugs, amino acids, oligopeptides, and proteins. Bacterial ABC-proteins include the well-characterized periplasmic permeases involved in nutrient uptake, but also include protein secretion systems, such as the exporter for the Escherichia coli enterotoxin hemolysin A. Prominent eukaryotic members of this superfamily include the human P-glycoprotein (which is associated with the phenomenon of multiple drug resistance in tumor cells), the product of the cystic fibrosis gene (CFTR), the gene (pfmdr) implicated in chloroquine resistance of the malarial parasite, putative peptide transporters encoded at the locus for the class II major histocompatibility complex (MHC), and the yeast Ste6 transporter which mediates export of a peptide hormone that lacks a classical hydrophobic signal peptide. The well-established function of prokaryotic ABC-transporters in the secretion of proteins without typical signal sequences, and the example set by the Ste6 transporter, have led to the reasonable hypothesis that certain ABC-proteins in animal cells may be operating by a similar mechanism to mediate the export of a new class of secretory proteins, those lacking a classical hydrophobic signal peptide.

The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis.

Many bacteria, both gram positive and gram negative, extrude in an energy-dependent manner the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5[and -6]-carboxyfluorescein (BCECF) (D. Molenaar, T. Abee, and W. N. Konings, Biochim. Biophys. Acta 1115:75-83, 1991). This efflux was studied in detail in Lactococcus lactis, and several indications that a transport system is involved were found. This transport system is most likely driven by ATP or a related compound. The evidence is that BCECF extrusion (i) occurs against a BCECF gradient, (ii) is strictly correlated with ATP concentration and not with the proton motive force, and (iii) is inhibited by vanadate and to a lesser extent by N,N'-dicyclohexylcarbodiimide. Most convincingly, a UV mutant with a strongly reduced efflux rate was isolated. Such a mutant was isolated from a BCECF-loaded and lactose-energized population by selection of highly fluorescent cells in a flow cytometer-cell sorter. The physiological function of this extrusion system is unknown, but its characteristics classify it among the traffic ATPases.

Traffic ATPases: a superfamily of transport proteins operating from Escherichia coli to humans.

This chapter contains sections titled: Introduction Architecture of Permeases Structure-Function Studies Relevance to Eukaryotic Carriers Future Prospects

Bacterial Periplasmic Permeases as Model Systems for the Superfamily of Traffic ATPases, Including the Multidrug Resistance Protein and the Cystic Fibrosis Transmembrane Conductance Regulator

This chapter discusses the composition and basic features of periplasmic permeases of gram-negative bacteria emphasizing those aspects of periplasmic transport that relate to their energy-coupling mechanism and structural organization. The mechanism of action of eukaryotic transporters is also presented in the chapter. Periplasmic permeases are composed of a receptor (a periplasmic substrate binding protein) and two to three membrane-bound components. All periplasmic permeases have the same overall composition. The similarity in the composition of these permeases indicates a conserved structural organization and mechanism of action despite the vast variety of substrates transported and a common evolutionary origin. Periplasmic permeases typically concentrate substrates against a very large concentration gradient. ATP binding and hydrolysis result in the conformational changes that are transmitted to the rest of the permease, thereby causing the opening of a pore or the creation of a substrate binding site in the membrane-bound complex. Conformational changes may be necessary as a signal for the receptor to release the substrate, which is deeply buried between its two lobes.

Structure-function analysis of the histidine permease and comparison with cystic fibrosis mutations.

Traffic ATPases constitute a superfamily of transporters that include prokaryotic permeases and medically important eukaryotic proteins, such as the multidrug resistance P-glycoprotein and the cystic fibrosis gene product. We present a structure-function analysis of a member of this superfamily, the prokaryotic histidine permease, using mutations generated both in vitro and in vivo, and assaying several biochemical functions. The analysis supports a previously predicted structural model and allows the assignment of specific functions to several predicted structural features. Mutations in the secondary structure features which form the nucleotide-binding pocket in general cause the loss of ATP binding activity. Mutations in the helical domain retain ATP binding activity. Several mutations have been identified which may affect the signaling mechanism between ATP hydrolysis and membrane translocation. We relate our findings to those emerging from the recent biochemical and genetic analyses of cystic fibrosis mutations.

Bacterial periplasmic permeases belong to a family of transport proteins operating fromEscherichia colito human: Traffic ATPases

Bacterial periplasmic transport systems are complex permeases composed of a soluble substrate-binding receptor and a membrane-bound complex containing 2-4 proteins. Recent developments have clearly demonstrated that these permeases are energized by the hydrolysis of ATP. Several in vitro systems have allowed a detailed study of the essential parameters functioning in these permeases. Several of the component proteins have been shown to interact with each other and the actual substrate for the transport process has been shown to be the liganded soluble receptor. The affinity of this substrate for the membrane complex is approximately 10 microM. The involvement of ATP in energy coupling is mediated by one of the proteins in the membrane complex. For each specific permease, this protein is a member of a family of conserved proteins which bind ATP. The similarity between the members of this family is high and extends itself beyond the consensus motifs for ATP binding. Interestingly, over the last few years, several eukaryotic membrane-bound proteins have been discovered which bear a high level of homology to the family of the conserved components of bacterial periplasmic permeases. Most of these proteins are known to, or can be inferred to participate in a transport process, such as in the case of the multidrug resistance protein (MDR), the STE6 gene product of yeast, and possibly the cystic fibrosis protein. This homology suggests a similarity in the mechanism of action and possibly a common evolutionary origin. This exciting development will stimulate progress in both the prokaryotic and eukaryotic areas of research by the use of overlapping procedures and model building. We propose that this universal class of permeases be called 'Traffic ATPases' to distinguish them from other types of transport systems, and to highlight their involvement in the transport of a vast variety of substrates in either direction relative to the cell interior and their use of ATP as energy source.

Bacterial periplasmic permeases belong to a family of transport proteins operating from Escherichia coli to human: Traffic ATPases

Bacterial periplasmic transport systems are complex permeases composed of a soluble substrate-binding receptor and a membrane-bound complex containing 2–4 proteins. Recent developments have clearly demonstrated that these permeases are energized by the hydrolysis of ATP. Several in vitro systems have allowed a detailed study of the essential parameters functioning in these permeases. Several of the compotent proteins have been shown to interact with each other and the actual substrate for the transport process has been shown to be the liganded soluble receptor. The affinity of this substrate for the membrane complex is approximately 10 μM. The involvement of ATP in energy coupling is mediated by one of the proteins in the membrane complex. For each specific permease, this protein is a member of a family of conserved proteins which bind ATP. The similarity between the members of this family is high and extends itself beyond the consensus motifs for ATP binding. Interestingly, over the last few years, several eukaryotic membrane-bound proteins have been discovered which bear a high level of homology to the family of the conserved components of bacterial periplasmic permeases. Most of these proteins are known to, or can be inferred to participate in a transport process, such as in the case of the multidrug resistance protein (MDR), the STE6 gene product of yeast, and possibly the cystic fibrosis protein. This homology suggests a similarity in the mechanism of action and possibly a common evolutionary origin. This exciting deveplopment will stimulate progress in both the prokaryotic and eukaryotic areas of research by the use of overlapping procedures and model building. We propose that this universal class of permeases be called ‘Traffic ATPases’ to distinguish them from other types of transport systems, and to highlight their involvement in the transport of a vast variety of substrate in either direction relative to the cell interior and their use of ATP as energy source.

Bacterial periplasmic permeases belong to a family of transport proteins operating from to human: Traffic ATPases

Bacterial periplasmic permeases belong to a family of transport proteins operating from to human: Traffic ATPases