The role of ABC transporters in cuticular lipid secretion

The aerial surfaces of plants are enveloped by a waxy cuticle, which among other functions serves as a barrier to limit non-stomatal water loss and defend against pathogens. The cuticle is a complex three-dimensional structure composed of cutin (a lipid polyester matrix) and waxes (very long chain fatty acid derivatives), which are embedded within and layered on top of the cutin matrix. Biosynthesis of cuticular lipids is believed to take place solely within aerial epidermal cells. Once synthesized, both the waxes and the cutin precursors must leave the cytoplasm, pass through the hydrophilic apoplastic space, and finally assemble to form the cuticle. These processes of secretion and assembly are essentially unknown. Initial steps toward our understanding of these processes were the characterization of CER5/ABCG12/WBC12 and more recently ABCG11/WBC11, a pair of ABC transporters required for cuticular lipid secretion. ABCG12 is involved in wax secretion, as mutations in this gene result in a lower surface-load of wax and a concomitant accumulation of lipidic inclusions within the epidermal cell cytoplasm. Mutations in ABCG11 result in a similar wax phenotype as cer5 and similar cytoplasmic inclusions. In contrast to cer5, however, abcg11 mutants also show significantly reduced cutin, post-genital organ fusions, and reduced growth and fertility. Thus, for the first time, a transporter is implicated in cutin accumulation. This review will discuss the secretion of cuticular lipids, focusing on ABCG12, ABCG11 and the potential involvement of other ABC transporters in the ABCG subfamily.     

The Type 1 secretion pathway — The hemolysin system and beyond

Type 1 secretion systems (T1SS) are wide-spread among Gram-negative bacteria. An important example is the secretion of the hemolytic toxin HlyA from uropathogenic strains. Secretion is achieved in a single step directly from the cytosol to the extracellular space. The translocation machinery is composed of three indispensable membrane proteins, two in the inner membrane, and the third in the outer membrane. The inner membrane proteins belong to the ABC transporter and membrane fusion protein families (MFPs), respectively, while the outer membrane component is a porin-like protein. Assembly of the three proteins is triggered by accumulation of the transport substrate (HlyA) in the cytoplasm, to form a continuous channel from the inner membrane, bridging the periplasm and finally to the exterior. Interestingly, the majority of substrates of T1SS contain all the information necessary for targeting the polypeptide to the translocation channel — a specific sequence at the extreme C-terminus. Here, we summarize our current knowledge of regulation, channel assembly, translocation of substrates, and in the case of the HlyA toxin, its interaction with host membranes. We try to provide a complete picture of structure function of the components of the translocation channel and their interaction with the substrate. Although we will place the emphasis on the paradigm of Type 1 secretion systems, the hemolysin A secretion machinery from E. coli, we also cover as completely as possible current knowledge of other examples of these fascinating translocation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

High-level production of Serratia proteamaculans metalloprotease using a recombinant ABC protein exporter-mediated secretion system in Pseudomonas fluorescens

Serratia proteamaculans metalloprotease (SPP) was successfully secreted by a heterologous ABC protein exporter, the Pseudomonas fluorescens TliDEF, in recombinant host strains. Escherichia coli and P. fluorescens cells containing the SPP-encoding gene showed the extracellular protease activity only when the TliDEF-encoding gene cluster was coexpressed. Recombinant P. fluorescens produced an approximately 34.8-fold higher amount of extracellular SPP than did E. coli. The use of a more nutrient-rich medium and controlled dissolved oxygen conditions was effective in increasing SPP secretion in P. fluorescens batch fermentation (an 8.7-fold increase from 41.8 U/mL to 365.2 U/mL). Therefore, SPP, which could not be secreted without an ABC protein exporter, was produced in large quantities by applying the heterologous TliDEF exporter in P. fluorescens. The results also suggest that the use of the ABC protein exporter in P. fluorescens could be an efficient production platform for an industrially promising type I secretion pathway-dependent enzyme.     

Subunit interactions in ABC transporters: towards a functional architecture

The ABC superfamily is a diverse group of integral membrane proteins involved in the ATP-dependent transport of solutes across biological membranes in both prokaryotes and eukaryotes. Although ABC transporters have been studied for over 30 years, very little is known about the mechanism by which the energy of ATP hydrolysis is used to transport substrate across the membrane. The recent report of the high resolution crystal structure of HisP, the nucleotide-binding subunit of the histidine permease complex of Salmonella typhimurium, represents a significant breakthrough toward the elucidation of the mechanism of solute translocation by ABC transporters. In this review, we use data from the crystallographic structures of HisP and other nucleotide-binding proteins, combined with sequence analysis of a subset of atypical ABC transporters, to argue a new model for the dimerisation of the nucleotide-binding domains that embraces the notion that the C motif from one subunit forms part of the ATP-binding site in the opposite subunit. We incorporate this dimerisation of the ATP-binding domains into our recently reported beta-barrel model for P-glycoprotein and present a general model for the cooperative interaction of the two nucleotide-binding domains and the translocation of mechanical energy to the transmembrane domains in ABC transporters.     

The potential roles of retinoids in combating drug resistance in cancer: implications of ATP-binding cassette (ABC) transporters

Multidrug resistance (MDR) means that tumour cells become unresponsive during or after the course of treatment to one or more of chemotherapeutic drugs. Chemotherapeutic resistance critically limits the treatment outcomes and remains a key challenge for clinicians. The alternation in intracellular drug concentration through the modulation of its transport across the plasma membrane is the major cause for MDR and is adopted by various mediators, including ATP-requiring enzymes (ATPases). Among these ATPases, ABC transporters have been extensively studied, and found to be highly implicated in tumorigenesis and MDR. The present review sheds light on the documented effects of retinoids on ABC enzymes to understand their mechanism in combating cancer cell resistance. This would open the gate to test the mechanism and applicability of different new synthetic retinoids in literature and market as modulators of ATP-dependent efflux pumping activity, and promote their applicability in diminishing anti-cancer drug resistance.     

Nitensidine A,a guanidine alkaloid from Pterogyne nitens,is a novel substrate for human ABC transporter ABCB1

The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity.     

The YheI/YheH heterodimer from Bacillus subtilis is a multidrug ABC transporter

ABC (ATP-binding cassette) transporters form the largest family of membrane proteins in micro-organisms where they are able to transport a wide variety of substrates against a concentration gradient, in an ATP-dependent process. Two genes from the same putative Bacillus subtilis operon, yheI and yheH, encoding possibly two different ABC transporters, were overexpressed in Escherichia coli in high yield, either separately or jointly. Using membrane vesicles, it is shown here that both subunits were required to detect, (i) the transport of four structurally unrelated drugs, and (ii) a vanadate-sensitive ATPase activity. Mutation of the invariant Walker-A lysine to an alanine residue in both subunits led to an inactive transporter. Moreover, after membrane solubilization by detergent, both wild-type subunits co-purified on a Ni-Agarose affinity column while only the YheH subunit contained a hexa-histidine tag. This shows that YheI and YheH are indeed able to interact together to form a heterodimer. Importantly, expression of both yheI and yheH genes in B. subtilis could be strongly stimulated by addition of sub-inhibitory concentrations of various unrelated antibiotics. Therefore, B. subtilis YheI/YheH forms a new heterodimeric multidrug ABC transporter possibly involved in multiple antibiotic resistance in vivo.     

空肠弯曲菌Ⅵ型分泌系统研究进展

Ⅵ型分泌系统(Type Ⅵ Secretion System,T6SS)是一种倒置于细胞膜上的类噬菌体样结构,能够输送效应蛋白并在定殖和生态位建立中发挥作用.近年来,在空肠弯曲菌(Campylobacter jejuni)中发现了T6SS同源基因且能够表达组装成结构完整的T6SS,但T6SS对空肠弯曲菌的毒力影响尚不清...     

The rate of folding dictates substrate secretion by the Escherichia coli hemolysin type 1 secretion system

Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.     

Structure and dynamic association of an assembly platform subcomplex of the bacterial type II secretion system

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ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis

Aluminum (Al) toxicity in acid soils is a worldwide agricultural problem that severely limits crop productivity through inhibition of root growth. Previously, Arabidopsis mutants with increased Al sensitivity were isolated in order to identify genes important for Al tolerance in plants. One mutant, als3, exhibited extreme root growth inhibition in the presence of Al, suggesting that this mutation negatively impacts a gene required for Al tolerance. Map-based cloning of the als3-1 mutation resulted in the isolation of a novel gene that encodes a previously undescribed ABC transporter-like protein, which is highly homologous to a putative bacterial metal resistance protein, ybbM. Northern analysis for ALS3 expression revealed that it is found in all organs examined, which is consistent with the global nature of Al sensitivity displayed by als3, and that expression increases in roots following Al treatment. Based on GUS fusion and in situ hybridization analyses, ALS3 is primarily expressed in leaf hydathodes and the phloem throughout the plant, along with the root cortex following Al treatment. Immunolocalization indicates that ALS3 predominantly accumulates in the plasma membrane of cells that express ALS3. From our results, it appears that ALS3 encodes an ABC transporter-like protein that is required for Al resistance/tolerance and may function to redistribute accumulated Al away from sensitive tissues in order to protect the growing root from the toxic effects of Al.     

细菌Ⅵ型分泌系统效应蛋白的装配及功能的研究进展

蛋白分泌作为细胞之间传递信号的途径之一,在微生物生存竞争中也扮演着重要的角色。革兰氏阴性菌可以通过Ⅵ型分泌系统(type Ⅵ secretion system, T6SS)将效应蛋白传递至胞外或原核和真核微生物中,从而介导微生物间的竞争或宿主-细菌的相互作用,最终建立竞争优势。本文主要总结了T6SS的结构与组成,并重点对效应蛋白的装配以及其与免疫蛋白的作用机制的研究进展进行阐述,为以后靶向T6SS抗菌药物的研制提供新思路。     

Domestication of a housekeeping transglycosylase for assembly of a Type VI secretion system

The type VI secretion system (T6SS) is an anti‐bacterial weapon comprising a contractile tail anchored to the cell envelope by a membrane complex. The TssJ, TssL, and TssM proteins assemble a 1.7‐MDa channel complex that spans the cell envelope, including the peptidoglycan layer. The electron microscopy structure of the TssJLM complex revealed that it has a diameter of ~18 nm in the periplasm, which is larger than the size of peptidoglycan pores (~2 nm), hence questioning how the T6SS membrane complex crosses the peptidoglycan layer. Here, we report that the MltE housekeeping lytic transglycosylase (LTG) is required for T6SS assembly in enteroaggregative Escherichia coli. Protein–protein interaction studies further demonstrated that MltE is recruited to the periplasmic domain of TssM. In addition, we show that TssM significantly stimulates MltE activity in vitro and that MltE is required for the late stages of T6SS membrane complex assembly. Collectively, our data provide the first example of domestication and activation of a LTG encoded within the core genome for the assembly of a secretion system.     

The overexpression of a new ABC transporter in Leishmania is related to phospholipid trafficking and reduced infectivity

This paper reports the characterization of a new ABC transporter (LtrABC1.1), related to the human ABCA subfamily, in the protozoan parasite Leishmania tropica. LtrABC1.1 is a tandem duplicated gene flanked by inverted repeats. LtrABC1.1 is expressed mainly in the flagellar pocket of the parasite. Drug resistance studies in Leishmania overexpressing LtrABC1.1 showed the transporter not to confer resistance to a range of unrelated drugs. LtrABC1.1 appears to be involved in lipid movements across the plasma membrane of the parasite since overexpression reduces the accumulation of fluorescent phospholipid analogues. The activity of this protein may also affect membrane movement processes since secreted acid phosphatase (SAP) activity was significantly lower in promastigotes overexpressing LtrABC1.1. In vitro infection experiments with macrophages indicated LtrABC1.1-transfected parasites to be significantly less infective. Together, these results suggest that this new ABC transporter could play a role in lipid movements across the plasma membrane, and that its activity might influence vesicle trafficking. This is the first ABCA-like transporter described in unicellular eukaryotes.     

The putative ABC transporter YheH/YheI is involved in the signalling pathway that activates KinA during sporulation initiation

The primary kinases that control the supply of phosphate to the phosphorelay are KinA and KinB, although it is not yet known what type of signal(s) activates these kinases. Our systematic study of protein-protein interactions using yeast two-hybrid analysis revealed an interaction between KinA and YheH. YheH with the preceding gene product YheI is categorized as an ABC transporter. Overexpression of yheH/yheI in the kinB mutant resulted in a reduced sporulation efficiency. Moreover, reporter assays using Spo0A approximately P dependent promoters revealed that the deficiency in sporulation is probably due to a failure in the activation of Spo0A. Our results further suggest that the N-terminal region of YheH may play an important role in sensing the signal to be delivered to the C-terminally bound KinA.