Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments

All microorganisms possess a positive turgor, and maintenance of this outward-directed pressure is essential since it is generally considered as the driving force for cell expansion. Exposure of microorganisms to high-osmolality environments triggers rapid fluxes of cell water along the osmotic gradient out of the cell, thus causing a reduction in turgor and dehydration of the cytoplasm. To counteract the outflow of water, microorganisms increase their intracellular solute pool by amassing large amounts of organic osmolytes, the so-called compatible solutes. These osmoprotectants are highly congruous with the physiology of the cell and comprise a limited number of substances including the disaccharide trehalose, the amino acid proline, and the trimethylammonium compound glycine betaine. The intracellular amassing of compatible solutes as an adaptive strategy to high-osmolality environments is evolutionarily well-conserved in Bacteria, Archaea, and Eukarya. Furthermore, the nature of the osmolytes that are accumulated during water stress is maintained across the kingdoms, reflecting fundamental constraints on the kind of solutes that are compatible with macromolecular and cellular functions. Generally, compatible solutes can be amassed by microorganisms through uptake and synthesis. Here we summarise the molecular mechanisms of compatible solute accumulation in Escherichia coli and Bacillus subtilis, model organisms for the gram-negative and gram-positive branches of bacteria. Received: 12 May 1998 / Accepted: 24 July 1998  

MRP Subfamily Transporters and Resistance to Anticancer Agents

The MRP subfamily of ABC transporters from mammals consists of at least seven members, six of which have been implicated in the transport of amphipathic anions. MRP1, MRP2, and MRP3 bear a close structural resemblance, confer resistance to a variety of natural products as well as methotrexate, and have the facility for transporting glutathione and glucuronate conjugates. MRP1 is a ubiquitously expressed efflux pump for the products of phase II of xenobiotic detoxification, while MRP2, whose hereditary deficiency results in Dubin–Johnson syndrome, functions to extrude organic anions into the bile. MRP3 is distinguished by its capacity to transport the monoanionic bile constituent glycocholate, and may function as a basolateral back-up system for the detoxification of hepatocytes when the usual canalicular route is impaired by cholestatic conditions. MRP4 and MRP5 resemble each other more closely than they resemble MRPs 1–3 and confer resistance to purine and nucleotide analogs which are either inherently anionic, as in the case of the anti-AIDS drug PMEA, or are phosphorylated and converted to anionic amphiphiles in the cell, as in the case of 6-MP. Given their capacity for transporting cyclic nucleotides, MRP4 and MRP5 have also been implicated in a broad range of cellular signaling processes. The drug resistance activity and physiological substrates of MRP6 are unknown. However, its hereditary deficiency results in pseudoxanthoma elasticum, a multisystem disorder affecting skin, eyes, and blood vessels. It is hoped that elucidation of the resistance profiles and physiological functions of the different members of the MRP subfamily will provide new insights into the molecular basis of clinical drug resistance and spawn new strategies for combating this phenomenon.  

Getting In or Out: Early Segregation Between Importers and Exporters in the Evolution of ATP-Binding Cassette (ABC) Transporters

ATP-binding cassette (ABC) systems, also called traffic ATPases, are found in eukaryotes and prokaryotes and almost all participate in the transport of a wide variety of molecules. ABC systems are characterized by a highly conserved ATPase module called here the ABC module, involved in coupling transport to ATP hydrolysis. We have used the sequence of one of the first representatives of bacterial ABC transporters, the MalK protein, to collect 250 closely related sequences from a nonredundant protein sequence database. The sequences collected by this objective method are all known or putative ABC transporters. After having eliminated short protein sequences and duplicates, the 197 remaining sequences were subjected to a phylogenetic analysis based on a mutational similarity matrix. An unrooted tree for these modules was found to display two major branches, one grouping all collected uptake systems and the other all collected export systems. This remarkable disposition strongly suggests that the divergence between these two functionally different types of ABC systems occurred once in the history of these systems and probably before the differentiation of prokaryotes and eukaryotes. We discuss the implications of this finding and we propose a model accounting for the generation and the diversification of ABC systems. Received: 23 February 1997 / Accepted: 7 April 1998  

Stable suppression of MDR1 gene expression and function by RNAi in Caco-2 cells

Vector-based RNAi was used to establish a stable Caco-2 cell line with a persistent knockdown of multidrug resistant gene 1 (MDR1) and P-glycoprotein (P-gp). Several positive clones were collected, many of which showed significantly reduced levels of MDR1 mRNA and P-gp compared to wt Caco-2 cells. Selected clones were sub-cultivated for six passages and real-time PCR showed that MDR1 expression remained significantly reduced (up to 96%) over this period of time. RNAi-MDR1 clones frozen long term also kept their low MDR1 expression levels when re-cultured. Permeability studies were performed across RNAi-MDR1 clone cell monolayers, and the efflux of cyclosporine A, digoxin, vinblastine, and vincristine showed 58%, 61%, 91%, and 78% decrease in active transport, respectively, compared to wt Caco-2 cells. This stably modified Caco-2 cell line provides a novel tool for studies on MDR1 and other ABC transporter protein gene cellular functions.  

Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1

Directional transport of the phytohormone auxin is required for the establishment and maintenance of plant polarity, but the underlying molecular mechanisms have not been fully elucidated. Plant homologs of human multiple drug resistance/P-glycoproteins (MDR/PGPs) have been implicated in auxin transport, as defects in MDR1 (AtPGP19) and AtPGP1 result in reductions of growth and auxin transport in Arabidopsis (atpgp1, atpgp19), maize (brachytic2) and sorghum (dwarf3). Here we examine the localization, activity, substrate specificity and inhibitor sensitivity of AtPGP1. AtPGP1 exhibits non-polar plasma membrane localization at the shoot and root apices, as well as polar localization above the root apex. Protoplasts from Arabidopsis pgp1 leaf mesophyll cells exhibit reduced efflux of natural and synthetic auxins with reduced sensitivity to auxin efflux inhibitors. Expression of AtPGP1 in yeast and in the standard mammalian expression system used to analyze human MDR-type proteins results in enhanced efflux of indole-3-acetic acid (IAA) and the synthetic auxin 1-naphthalene acetic acid (1-NAA), but not the inactive auxin 2-NAA. AtPGP1-mediated efflux is sensitive to auxin efflux and ABC transporter inhibitors. As is seen in planta, AtPGP1 also appears to mediate some efflux of IAA oxidative breakdown products associated with apical sites of high auxin accumulation. However, unlike what is seen in planta, some additional transport of the benzoic acid is observed in yeast and mammalian cells expressing AtPGP1, suggesting that other factors present in plant tissues confer enhanced auxin specificity to PGP-mediated transport.  

The Role of Half-Transporters in Multidrug Resistance

ATP-binding cassette proteins comprise a superfamily of transporter proteins, a subset of which have been implicated in multidrug resistance. Although P-glycoprotein was described over 15 years ago, the recent expansion in the number of transporters identified has prompted renewed interest in the role of drug transporters in clinical drug resistance. These newly identified transporters include additional members of the MRP family, ABC2, and a new half-transporter, MXR/BCRP/ABCP1. This half-transporter confers high levels of resistance to mitoxantrone, anthracyclines, and the camptothecins SN-38 and topotecan. At 72 kDa, MXR localizes to the plasma membrane in cells which highly overexpress the protein either through gene amplification or though gene rearrangement. Future studies will be aimed at identifying an inhibitor, and attempting to translate recognition of this new transporter into a target for anticancer treatment.  

Immunity to lantibiotics

Bacteria producing bacteriocins have to be protected from being killed by themselves. This mechanism of self-protection or immunity is especially important if the bacteriocin does not need a specific receptor for its action, as is the case for the type A lantibiotics forming pores in the cytoplasmic membrane. At least two different systems of immunity have evolved in this group of bacteriocins containing modified amino acids as a result of posttranslational modification. The immunity mechanism of Pep5 in Staphylococcus epidermidis is based on inhibition of pore formation by a small 69-amino acid protein weakly associated with the outer surface of the cytoplasmic membrane. In Lactococcus lactis and Bacillus subtilis the putative immunity lipoproteins NisI and SpaI, respectively, are also located at the outer surface of the cytoplasmic membrane, suggesting that a similar mechanism might be utilized by the producers of nisin and subtilin. In addition an ABC-transport system consisting of two membrane proteins, (NisEG, SpaG and the hydrophobic domain of SpaF, and EpiEG) and a cytoplasmic protein (NisF, the cytoplasmic domain of SpaF, and EpiF) play a role in immunity of nisin, subtilin and epidermin by import, export or inhibition of pore formation by the membrane components of the transport systems. Almost nothing is known of the immunity determinants of newly described and other type of lantibiotics.  

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.