ABC transporters do not contribute to extracellular translocation of hyaluronan in human breast cancer in vitro

Extracellular translocation of the polysaccharide, hyaluronan (HA) has been thought to be mediated via its transmembrane synthetic enzyme, hyaluronan synthase (HAS) but recent studies have indicated that the ATP-Binding-Cassette (ABC) transporter, MRP5 contributes to this process. Liberated and cell-associated HA contributes to breast cancer initiation and progression, and therefore the inhibition of ABC transporters and consequently HA transport could provide therapeutic benefit in the treatment of breast cancer. Quantitation of ABC transporter genes, MRP1-5, BCRP and MDR1 were determined in six breast cancer cell lines selected for their differential HA synthetic rates. Low endogenous expression of transporters was detected but no significant correlation existed between ABC transporter and HAS gene expression or HA production. A dose titration of up to ten times the IC₅₀ of ten small molecule ABC transporter inhibitors did not significantly inhibit HA export in four breast cancer cell lines. Unlike the changes observed after inhibition of HA synthesis by the characterised inhibitor 4-MU, inhibition of ABC transporters did not alter the cell morphology, HA glycocalyx or the intracellular quantity or localisation of HA. Collectively these data indicate that ABC transporters do not contribute to the extracellular transport of HA in breast cancer, supporting a role for the hyaluronan synthase in translocation.     

The N terminus of the HasA protein and the SecB chaperone cooperate in the efficient targeting and secretion of HasA via the ATP-binding cassette transporter.

Secretion of the HasA hemophore is mediated by a C-terminal secretion signal as part of an ATP-binding cassette (ABC) pathway in the Gram-negative bacterium Serratia marcescens. We reconstituted the HasA secretion pathway in Escherichia coli. In E. coli, this pathway required three specific secretion functions and SecB, the general chaperone of the Sec pathway that recognizes HasA. The secretion of the isolated C-terminal secretion signal was not SecB-dependent. We have previously shown that intracellular folded HasA can no longer be secreted, and we proposed a step in the secretion process before the recognition of the secretion signal. Here we show that the secretion of a fully functional HasA variant, lacking the first 10 N-terminal amino acids, was less efficient than that of HasA and was SecB-independent. The N terminus of HasA was required, along with SecB, for the efficient secretion of the whole protein. We have also previously shown that HasA inhibits the secretion of metalloproteases from Erwinia chrysanthemi by their specific ABC transporter. Here we show that this abortive interaction between HasA and the E. chrysanthemi metalloprotease ABC transporter required both SecB and the N terminus of HasA. N-terminal fragments of HasA displayed this abortive interaction in vivo and also interacted specifically in vitro with the ABC protein of the Prt system. SecB also interacted specifically in vitro with the ABC protein of the Prt system. Finally, the HasA variant, lacking the first 10 N-terminal amino acids did not display this abortive interaction with the Prt system. We suggest that the N-terminal domain of HasA specifically recognizes the ABC protein in a SecB-dependent fashion, facilitating functional interaction with the C-terminal secretion signal leading to efficient secretion.     

Identification, cloning, and characterization of rcsF, a new regulator gene for exopolysaccharide synthesis that suppresses the division mutation ftsZ84 in Escherichia coli K-12.

A new gene, designated rcsF, was located adjacent to drpA at the 5.2-min position of the genetic map of Escherichia coli. The deduced amino acid sequence encoded by the rcsF gene indicates a small protein of 133 amino acid residues with a calculated pI of 10.8 that is rich in proline, serine, alanine, and cysteine residues. When overexpressed as a result of its presence on a multicopy plasmid, rcsF confers a mucoid phenotype and restores colony formation to ftsZ84 mutant cells on L agar medium containing no added NaCl. These two phenotypes are not observed in rcsB mutant cells. Ion mutant cells harboring an rcsF mutation accumulate considerably lower levels of exopolysaccharides, whereas the presence of a multicopy rcsF plasmid not only increases capsule synthesis but also confers a mucoid phenotype at 37 degrees C, a temperature at which ion mutant cells are known not to form mucoid colonies. RcsF does not stimulate the expression of rcsB, indicating that it exerts its action through the RcsB protein, possibly by phosphorylation. It is also shown that RcsF stimulation of capsule synthesis is RcsA-dependent, whereas colony formation of ftsZ84 mutant cells can be restored by RcsF in the absence of RcsA.     

Revising the role of the pneumococcal vex-vncRS locus in vancomycin tolerance

Vancomycin is used increasingly to treat invasive infections caused by multidrug-resistant Streptococcus pneumoniae. Although no vancomycin-resistant strains have been isolated to date, tolerant strains that fail to die rapidly and that cause relapsing disease have been described. The vex123-pep27-vncRS locus, consisting of an ABC transporter, a presumed signaling peptide, and a two-component system, respectively, has been implicated in vancomycin tolerance. Recent findings, however, challenged this model. The data presented here indicate that erythromycin in the growth medium induces a vancomycin-tolerant phenotype and that loss of function of Pep27 or VncRS does not alter autolysis. However, a role for the ABC transporter encoded by the vex123 genes in tolerance was confirmed. A vex3 mutant was considerably more tolerant to vancomycin treatment than the wild-type strain T4, and the strength of the phenotype depended on the orientation of the resistance cassette used to construct the mutant. Microarray results suggested a number of genes that might be involved in tolerance in the vex3 mutant. Although the exact function and regulation of the vex123-pep27-vncRS locus remains to be determined, several factors influence the autolysis behavior of S. pneumoniae, including the bacterial capsule, erythromycin, and the lytA and vex3 gene products.     

Secretion of the Serratia marcescens HasA protein by an ABC transporter.

We previously identified a Serratia marcescens extracellular protein, HasA, able to bind heme and required for iron acquisition from heme and hemoglobin by the bacterium. This novel type of extracellular protein does not have a signal peptide and does not show sequence similarities to other proteins. HasA secretion was reconstituted in Escherichia coli, and we show here that like many proteins lacking a signal peptide, HasA has a C-terminal targeting sequence and is secreted by a specific ATP binding cassette (ABC) transporter consisting of three proteins, one inner membrane protein with a conserved ATP binding domain, called the ABC; a second inner membrane protein; and a third, outer membrane component. Since the three S. marcescens components of the HasA transporter have not yet been identified, the reconstituted HasA secretion system is a hybrid. It consists of the two S. marcescens inner membrane-specific components, HasD and HasE, associated with an outer membrane component coming from another bacterial ABC transporter, such as the E. coli TolC protein, the outer membrane component of the hemolysin transporter, or the Erwinia chrysanthemi PrtF protein, the outer membrane component of the protease transporter. This hybrid transporter was first shown to allow the secretion of the S. marcescens metalloprotease and the E. chrysanthemi metalloproteases B and C. On account of that, the two S. marcescens components HasD and HasE were previously named PrtDSM and PrtESM, respectively. However, HasA is secreted neither by the PrtD-PrtE-PrtF transporter (the genuine E. chrysanthemi protease transporter) nor by the HlyB-HlhD-TolC transporter (the hemolysin transporter). Moreover, HasA, coexpressed in the same cell, strongly inhibits the secretion of proteases B and C by their own transporter, indicating that the E. chrysanthemi transporter recognizes HasA. Since PrtF could replace TolC in the constitution of the HasA transporter, this indicates that the secretion block does not take place at the level of the outer membrane component but rather at an earlier step of interaction between HasA and the inner membrane components.     

Homologous expression of the lipase and ABC transporter gene cluster, tliDEFA, enhances lipase secretion in Pseudomonas spp.

The ABC transporter TliDEF was found to be an efficient secretory apparatus for extracellular lipase TliA in Pseudomonas fluorescens. For the enhanced secretion of the lipase, we tried to coexpress tliA and tliDEF in various Pseudomonas species. Whereas the coexpression of tliA and tliDEF was required for the lipase secretion in P. fragi, the expression of tliA was sufficient for the lipase secretion in P. fluorescens, P. syringae, and P. putida, indicating the existence of compatible ABC transporter in these species. However, P. fluorescens harboring tliDEFA secreted much more lipase than P. fluorescens harboring only tliA, but the tliDEF was functional only at temperatures below 30 degrees C. The recombinant P. fluorescens overexpressing tliDEFA showed the highest secretion level, 217 U/ml. OD (optical density) (28 microg/ml. OD) of lipase in Luria-Bertani medium under microaerated conditions. With the increase of aeration, the lipase production was decreased and the lipase seemed to be degraded as the cells entered the cell death phase. These results demonstrate that P. fluorescens can be used as a host system for the secretory production of the lipase using the ABC transporter, thus producing lipase in over 14% of the total protein.     

MucR, a Novel Membrane-Associated Regulator of Alginate Biosynthesis in Pseudomonas aeruginosa

Alginate biosynthesis by Pseudomonas aeruginosa was shown to be regulated by the intracellular second messenger bis-(3′-5′)-cyclic-dimeric-GMP (c-di-GMP), and binding of c-di-GMP to the membrane protein Alg44 was required for alginate production. In this study, PA1727, a c-di-GMP-synthesizing enzyme was functionally analyzed and identified to be involved in regulation of alginate production. Deletion of the PA1727 gene in the mucoid alginate-overproducing P. aeruginosa strain PDO300 resulted in a nonmucoid phenotype and an about 38-fold decrease in alginate production; thus, this gene is designated mucR. The mucoid alginate-overproducing phenotype was restored by introducing the mucR gene into the isogenic ΔmucR mutant. Moreover, transfer of the MucR-encoding plasmid into strain PDO300 led to an about sevenfold increase in alginate production, wrinkly colony morphology, increased pellicle formation, auto-aggregation, and the formation of highly structured biofilms as well as the inhibition of swarming motility. Outer membrane protein profile analysis showed that overproduction of MucR mediates a strong reduction in the copy number of FliC (flagellin), required for flagellum-mediated motility. Translational reporter enzyme fusions with LacZ and PhoA suggested that MucR is located in the cytoplasmic membrane with a cytosolic C terminus. Deletion of the proposed C-terminal GGDEF domain abolished MucR function. MucR was purified and identified using tryptic peptide fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overall, experimental evidence was provided suggesting that MucR specifically regulates alginate biosynthesis by activation of alginate production through generation of a localized c-di-GMP pool in the vicinity of Alg44.     

The Arabidopsis Peroxisomal ABC Transporter,Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2Δ Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2Δ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2Δ mutant, and restored β-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.     

AtCCMA interacts with AtCcmB to form a novel mitochondrial ABC transporter involved in cytochrome c maturation in Arabidopsis

ABC transporters make a large and diverse family of proteins found in all phylae. AtCCMA is the nucleotide binding domain of a novel Arabidopsis mitochondrial ABC transporter. It is encoded in the nucleus and imported into mitochondria. Sub-organellar and topology studies find AtCCMA bound to the mitochondrial inner membrane, facing the matrix. AtCCMA exhibits an ATPase activity, and ATP/Mg(2+) can facilitate its dissociation from membranes. Blue Native PAGE shows that it is part of a 480-kDa complex. Yeast two-hybrid assays reveal interactions between AtCCMA and domains of CcmB, the mitochondria-encoded transmembrane protein of a conserved ABC transporter. All these properties designate the protein as the ortholog in plant mitochondria of the bacterial CcmA required for cytochrome c maturation. The transporter that involves AtCCMA defines a new category of eukaryotic ABC proteins because its transmembrane and nucleotide binding domains are encoded by separate genomes.     

An ABC Transporter Plays a Developmental Aggregation Role in Myxococcus xanthus

Myxococcus xanthus is a gram-negative bacterium which has a complex life cycle. Autochemotaxis, a process whereby cells release a self-generated signaling molecule, may be the principal mechanism facilitating directed motility in both the vegetative swarming and developmental aggregation stages of this life cycle. The process requires the Frz signal transduction system, including FrzZ, a protein which is composed of two domains, both showing homology to the enteric chemotaxis response regulator CheY. The first domain of FrzZ (FrzZ1), when expressed as bait in the yeast two-hybrid system and screened against a library, was shown to potentially interact with the C-terminal portion of a protein encoding an ATP-binding cassette (AbcA). The activation domain-AbcA fusion protein did not interact with the second domain of FrzZ (FrzZ2) or with two other M. xanthus response regulator-containing proteins presented as bait, suggesting that the FrzZ1-AbcA interaction may be specific. Cloning and sequencing of the upstream region of the abcA gene showed the ATP-binding cassette to be linked to a large hydrophobic, potentially membrane-spanning domain. This domain organization is characteristic of a subgroup of ABC transporters which perform export functions. Cloning and sequencing downstream of abcA indicated that the ABC transporter is at the start of an operon containing three open reading frames. An insertion mutation in the abcA gene resulted in cells displaying the frizzy aggregation phenotype, providing additional evidence that FrzZ and AbcA may be part of the same signal transduction pathway. Cells with mutations in genes downstream of abcA showed no developmental defects. Analysis of the proposed exporter role of AbcA in cell mixing experiments showed that the ABC transporter mutant could be rescued by extracellular complementation. We speculate that the AbcA protein may be involved in the export of a molecule required for the autochemotactic process.     

The ATP binding cassette transporter ABC1, is required for the engulfment of corpses generated by apoptotic cell death.

ATP binding cassette (ABC) transporters define a family of proteins with strong structural similarities conserved across evolution and devoted to the translocation of a variety of substrates across cell membranes. A few members of the family are known in mammals, but although all of them are medically relevant proteins, knowledge of their molecular function remains scanty. We report here a morphological and functional study of the recently identified mammalian ABC transporter, ABC1. Its expression during embryonic development correlates spatially and temporally with the areas of programmed cell death. More specifically, ABC1 is expressed in macrophages engaged in the engulfment and clearance of dead cells. Moreover, ABC1 transporter is required for engulfment since the ability of macrophages to ingest apoptotic bodies is severely impaired after antibody-mediated steric blockade of ABC1. A structural homologue of ABC1 has been identified in the Caenorhabditis elegans genome and maps close to the ced-7 locus. Since ced-7 phenotype is precisely defined by an imparied engulfment of cell corpses, it is tempting to surmise that ABC1 might be a mammalian homologue of ced-7.     

X-Linked Adrenoleukodystrophy: Genes,Mutations, and Phenotypes

X-linked adrenoleukodystrophy (X-ALD) is a complex and perplexing neurodegenerative disorder. The metabolic abnormality, elevated levels of very long-chain fatty acids in tissues and plasma, and the biochemical defect, reduced peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity, are ubiquitous features of the disease. However, clinical manifestations are highly variable with regard to time of onset, site of initial pathology and rate of progression. In addition, the abnormal gene in X-ALD is not the gene for VLCS. Rather, it encodes a peroxisomal membrane protein with homology to the ATP-binding cassette (ABC) transmembrane transporter superfamily of proteins. The X-ALD protein (ALDP) is closely related to three other peroxisomal membrane ABC proteins. In this report we summarize all known X-ALD mutations and establish the lack of an X-ALD genotype/phenotype correlation. We compare the evolutionary relationships among peroxisomal ABC proteins, demonstrate that ALDP forms homodimers with itself and heterodimers with other peroxisomal ABC proteins and present cDNA complementation studies suggesting that the peroxisomal ABC proteins have overlapping functions. We also establish that there are at least two peroxisomal VLCS activities, one that is ALDP dependent and one that is ALDP independent. Finally, we discuss variable expression of the peroxisomal ABC proteins and ALDP independent VLCS in relation to the variable clinical presentations of X-ALD.     

The FtsEX ABC transporter directs cellular differentiation in Bacillus subtilis

A fundamental challenge in developmental biology is to elucidate the regulatory events that trigger cellular differentiation. Sporulation in the Gram-positive bacterium Bacillus subtilis serves as a simple experimental model system to address this challenge. The hallmark of sporulation is the formation of an asymmetrically positioned septum that divides the cell into unequally sized progeny. Here we describe the role of an ABC transporter, comprising the FtsE and FtsX proteins, in the initiation of spore formation. We discovered that in the absence of this transporter, entry into sporulation is delayed and an atypical symmetric septum is formed instead of a polar one. We show that this phenotype can be suppressed by artificially activating the master regulator of sporulation, Spo0A, or by activating the histidine kinases that function upstream of Spo0A. Our data indicate that the FtsEX transporter is one of the top components in the hierarchy of factors required to initiate sporulation, and thus it is essential for establishing proper temporal activation of the process.