A MademoiseLLE domain binding platform links the key RNA transporter to endosomes.

Spatiotemporal expression can be achieved by transport and translation of mRNAs at defined subcellular sites. An emerging mechanism mediating mRNA trafficking is microtubule-dependent co-transport on shuttling endosomes. Although progress has been made in identifying various components of the endosomal mRNA transport machinery, a mechanistic understanding of how these RNA-binding proteins are connected to endosomes is still lacking. Here, we demonstrate that a flexible MademoiseLLE (MLLE) domain platform within RNA-binding protein Rrm4 of Ustilago maydis is crucial for endosomal attachment. Our structure/function analysis uncovered three MLLE domains at the C-terminus of Rrm4 with a functionally defined hierarchy. MLLE3 recognises two PAM2-like sequences of the adaptor protein Upa1 and is essential for endosomal shuttling of Rrm4. MLLE1 and MLLE2 are most likely accessory domains exhibiting a variable binding mode for interaction with currently unknown partners. Thus, endosomal attachment of the mRNA transporter is orchestrated by a sophisticated MLLE domain binding platform.

Flipping and other astonishing transporter dance moves in fungal drug resistance.

In all domains of life, transmembrane proteins from the ATP-binding cassette (ABC) transporter family drive the translocation of diverse substances across lipid bilayers. In pathogenic fungi, the ABC transporters of the pleiotropic drug resistance (PDR) subfamily confer antibiotic resistance and so are of interest as therapeutic targets. They also drive the quest for understanding how ABC transporters can generally accommodate such a wide range of substrates. The Pdr5 transporter from baker's yeast is representative of the PDR group and, ever since its discovery more than 30 years ago, has been the subject of extensive functional analyses. A new perspective of these studies has been recently provided in the framework of the first electron cryo-microscopy structures of Pdr5, as well as emergent applications of machine learning in the field. Taken together, the old and the new developments have been used to propose a mechanism for the transport process in PDR proteins. This mechanism involves a "flippase" step that moves the substrates from one leaflet of the bilayer to the other, as a central element of cellular efflux.

Pdr5: A master of asymmetry.

Pdr5 is a founding member of a large (pdr) subfamily of clinically and agriculturally significant fungal ABC transporters. The tremendous power of yeast genetics combined with biochemical and structural approaches revealed the astonishing asymmetry of this efflux pump. Asymmetry is manifested in Pdr5's ATP-binding sites, drug binding sites, signal transformation interface, and molecular exit gate. Even its mode of conformational switching is asymmetric with one half of the protein remaining nearly stationary. In the case of its ATP-binding sites, asymmetry is created by replacing a set of highly conserved residues with a characteristic set of deviant ones. This contrasts with the asymmetry of the molecular gate. There, a full complement of canonical residues is present, but structural features in the vicinity prevent some of these from forming a molecular plug during closure. Compared to their canonical-functioning counterparts, the deviant ATP site and these gating residues have different, essential functions. In addition to its remarkable asymmetry, the surprising observation that Pdr5 is a drug / proton co-transporter shines a new light on this remarkable protein.

Residues forming the gating regions of asymmetric multidrug transporter Pdr5 also play roles in conformational switching and protein folding.

ATP-binding cassette (ABC) multidrug transporters are large, polytopic membrane proteins that exhibit astonishing promiscuity for their transport substrates. These transporters unidirectionally efflux thousands of structurally and functionally distinct compounds. To preclude the reentry of xenobiotic molecules via the drug-binding pocket, these proteins contain a highly conserved molecular gate, essentially allowing the transporters to function as molecular diodes. However, the structure-function relationship of these conserved gates and gating regions are not well characterized. In this study, we combine recent single-molecule, cryo-EM data with genetic and biochemical analyses of residues in the gating region of the yeast multidrug transporter Pdr5, the founding member of a large group of clinically relevant asymmetric ABC efflux pumps. Unlike the symmetric ABCG2 efflux gate, the Pdr5 counterpart is highly asymmetric, with only four (instead of six) residues comprising the gate proper. However, other residues in the near vicinity are essential for the gating activity. Furthermore, we demonstrate that residues in the gate and in the gating regions have multiple functions. For example, we show that Ile-685 and Val-1372 are required not only for successful efflux but also for allosteric inhibition of Pdr5 ATPase activity. Our investigations reveal that the gating region residues of Pdr5, and possibly other ABCG transporters, play a role not only in molecular gating but also in allosteric regulation, conformational switching, and protein folding.

Interaction of RTX toxins with the host cell plasma membrane.

Repeats in ToXins (RTX) protein family is a group of exoproteins secreted by Type 1 secretion system (T1SS) of several Gram-negative bacteria. The term RTX is derived from the characteristic nonapeptide sequence (GGxGxDxUx) present at the C-terminus of the protein. This RTX domain binds to calcium ions in the extracellular medium after being secreted out of the bacterial cells, and this facilitates folding of the entire protein. The secreted protein then binds to the host cell membrane and forms pores via a complex pathway, which eventually leads to the cell lysis. In this review, we summarize two different pathways in which RTX toxins interact with host cell membrane and discuss the possible reasons for specific and unspecific activity of RTX toxins to different types of host cells.

Structure and Function of Hepatobiliary ATP Binding Cassette Transporters.

The liver is beyond any doubt the most important metabolic organ of the human body. This function requires an intensive crosstalk within liver cellular structures, but also with other organs. Membrane transport proteins are therefore of upmost importance as they represent the sensors and mediators that shuttle signals from outside to the inside of liver cells and/or vice versa. In this review, we summarize the known literature of liver transport proteins with a clear emphasis on functional and structural information on ATP binding cassette (ABC) transporters, which are expressed in the human liver. These primary active membrane transporters form one of the largest families of membrane proteins. In the liver, they play an essential role in for example bile formation or xenobiotic export. Our review provides a state of the art and comprehensive summary of the current knowledge of hepatobiliary ABC transporters. Clearly, our knowledge has improved with a breath-taking speed over the last few years and will expand further. Thus, this review will provide the status quo and will lay the foundation for new and exciting avenues in liver membrane transporter research.

A step forward to the optimized HlyA type 1 secretion system through directed evolution.

Secretion of proteins into the extracellular space has great advantages for the production of recombinant proteins. Type 1 secretion systems (T1SS) are attractive candidates to be optimized for biotechnological applications, as they have a relatively simple architecture compared to other classes of secretion systems. A paradigm of T1SS is the hemolysin A type 1 secretion system (HlyA T1SS) from Escherichia coli harboring only three membrane proteins, which makes the plasmid-based expression of the system easy. Although for decades the HlyA T1SS has been successfully applied for secretion of a long list of heterologous proteins from different origins as well as peptides, but its utility at commercial scales is still limited mainly due to low secretion titers of the system. To address this drawback, we engineered the inner membrane complex of the system, consisting of HlyB and HlyD proteins, following KnowVolution strategy. The applied KnowVolution campaign in this study provided a novel HlyB variant containing four substitutions (T36L/F216W/S290C/V421I) with up to 2.5-fold improved secretion for two hydrolases, a lipase and a cutinase. KEY POINTS: • An improvement in protein secretion via the use of T1SS • Reaching almost 400 mg/L of soluble lipase into the supernatant • A step forward to making E. coli cells more competitive for applying as a secretion host.

ABC Transporters in Bacterial Nanomachineries.

Members of the superfamily of ABC transporters are found in all domains of life. Most of these primary active transporters act as isolated entities and export or import their substrates in an ATP-dependent manner across biological membranes. However, some ABC transporters are also part of larger protein complexes, so-called nanomachineries that catalyze the vectorial transport of their substrates. Here, we will focus on four bacterial examples of such nanomachineries: the Mac system providing drug resistance, the Lpt system catalyzing vectorial LPS transport, the Mla system responsible for phospholipid transport, and the Lol system, which is required for lipoprotein transport to the outer membrane of Gram-negative bacteria. For all four systems, we tried to summarize the existing data and provide a structure-function analysis highlighting the mechanistical aspect of the coupling of ATP hydrolysis to substrate translocation.

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region.

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.

Quantification and Surface Localization of the Hemolysin A Type I Secretion System at the Endogenous Level and under Conditions of Overexpression.

Secretion systems are essential for Gram-negative bacteria, as these nanomachineries allow communication with the outside world by exporting proteins into the extracellular space or directly into the cytosol of a host cell. For example, type I secretion systems (T1SS) secrete a broad range of substrates across both membranes into the extracellular space. One well-known example is the hemolysin A (HlyA) T1SS from Escherichia coli, which consists of an ABC transporter (HlyB), a membrane fusion protein (HlyD), the outer membrane protein TolC, and the substrate HlyA, a member of the family of repeats in toxins (RTX) toxins. Here, we determined the amount of TolC at the endogenous level (parental strain, UTI89) and under conditions of overexpression [T7 expression system, BL21(DE3)-BD]. The overall amount of TolC was not influenced by the overexpression of the HlyBD complex. Moving one step further, we determined the localization of the HlyA T1SS by superresolution microscopy. In contrast to other bacterial secretion systems, no polarization was observed with respect to endogenous or overexpression levels. Additionally, the cell growth and division cycle did not influence polarization. Most importantly, the size of the observed T1SS clusters did not correlate with the recently proposed outer membrane islands. These data indicate that T1SS clusters at the outer membrane, generating domains of so-far-undescribed identity. IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains cause about 110 million urinary tract infections each year worldwide, representing a global burden to the health care system. UPEC strains secrete many virulence factors, among these, the TX toxin hemolysin A via a cognate T1SS into the extracellular space. In this study, we determined the endogenous copy number of the HlyA T1SS in UTI89 and analyzed the surface localization in BL21(DE3)-BD and UTI89, respectively. With approximately 800 copies of the T1SS in UTI89, this is one of the highest expressed bacterial secretion systems. Furthermore, and in clear contrast to other secretion systems, no polarized surface localization was detected. Finally, quantitative analysis of the superresolution data revealed that clusters of the HlyA T1SS are not related to the recently identified outer membrane protein islands. These data provide insights into the quantitative molecular architecture of the HlyA T1SS.

Monomeric bile acids modulate the ATPase activity of detergent-solubilized ABCB4/MDR3.

ABCB4, also called multidrug-resistant protein 3 (MDR3), is an ATP binding cassette transporter located in the canalicular membrane of hepatocytes that specifically translocates phosphatidylcholine (PC) lipids from the cytoplasmic to the extracellular leaflet. Due to the harsh detergent effect of bile acids, PC lipids provided by ABCB4 are extracted into the bile. While it is well known that bile acids are the major extractor of PC lipids from the membrane into bile, it is unknown whether only PC lipid extraction is improved or whether bile acids also have a direct effect on ABCB4. Using in vitro experiments, we investigated the modulation of ATP hydrolysis of ABC by different bile acids commonly present in humans. We demonstrated that all tested bile acids stimulated ATPase activity except for taurolithocholic acid, which inhibited ATPase activity due to its hydrophobic nature. Additionally, we observed a nearly linear correlation between the critical micelle concentration and maximal stimulation by each bile acid, and that this modulation was maintained in the presence of PC lipids. This study revealed a large effect of 24-nor-ursodeoxycholic acid, suggesting a distinct mode of regulation of ATPase activity compared with other bile acids. In addition, it sheds light on the molecular cross talk of canalicular ABC transporters of the human liver.

New developments in RiPP discovery, enzymology and engineering.

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.

The many facets of bile acids in the physiology and pathophysiology of the human liver.

Bile acids perform vital functions in the human liver and are the essential component of bile. It is therefore not surprising that the biology of bile acids is extremely complex, regulated on different levels, and involves soluble and membrane receptors as well as transporters. Hereditary disorders of these proteins manifest in different pathophysiological processes that result in liver diseases of varying severity. In this review, we summarize our current knowledge of the physiology and pathophysiology of bile acids with an emphasis on recently established analytical approaches as well as the molecular mechanisms that underlie signaling and transport of bile acids. In this review, we will focus on ABC transporters of the canalicular membrane and their associated diseases. As the G protein-coupled receptor, TGR5, receives increasing attention, we have included aspects of this receptor and its interaction with bile acids.

The ABC transporter G subfamily in Arabidopsis thaliana.

ABC transporters are ubiquitously present in all kingdoms and mediate the transport of a large spectrum of structurally different compounds. Plants possess high numbers of ABC transporters in relation to other eukaryotes; the ABCG subfamily in particular is extensive. Earlier studies demonstrated that ABCG transporters are involved in important processes influencing plant fitness. This review summarizes the functions of ABCG transporters present in the model plant Arabidopsis thaliana. These transporters take part in diverse processes such as pathogen response, diffusion barrier formation, or phytohormone transport. Studies involving knockout mutations reported pleiotropic phenotypes of the mutants. In some cases, different physiological roles were assigned to the same protein. The actual transported substrate(s), however, still remain to be determined for the majority of ABCG transporters. Additionally, the proposed substrate spectrum of different ABCG proteins is not always reflected by sequence identities between ABCG members. Applying only reverse genetics is thereby insufficient to clearly identify the substrate(s). We therefore stress the importance of in vitro studies in addition to in vivo studies in order to (i) clarify the substrate identity; (ii) determine the transport characteristics including directionality; and (iii) identify dimerization partners of the half-size proteins, which might in turn affect substrate specificity.

Lethal (2) giant discs (Lgd)/CC2D1 is required for the full activity of the ESCRT machinery.

BACKGROUND: A major task of the endosomal sorting complex required for transport (ESCRT) machinery is the pinching off of cargo-loaded intraluminal vesicles (ILVs) into the lumen of maturing endosomes (MEs), which is essential for the complete degradation of transmembrane proteins in the lysosome. The ESCRT machinery is also required for the termination of signalling through activated signalling receptors, as it separates their intracellular domains from the cytosol. At the heart of the machinery lies the ESCRT-III complex, which is required for an increasing number of processes where membrane regions are abscised away from the cytosol. The core of ESCRT-III, comprising four members of the CHMP protein family, organises the assembly of a homopolymer of CHMP4, Shrub in Drosophila, that is essential for abscission. We and others identified the tumour-suppressor lethal (2) giant discs (Lgd)/CC2D1 as a physical interactor of Shrub/CHMP4 in Drosophila and mammals, respectively. RESULTS: Here, we show that the loss of function of lgd constitutes a state of reduced activity of Shrub/CHMP4/ESCRT-III. This hypomorphic shrub mutant situation causes a slight decrease in the rate of ILV formation that appears to result in incomplete incorporation of Notch into ILVs. We found that the forced incorporation in ILVs of lgd mutant MEs suppresses the uncontrolled and ligand-independent activation of Notch. Moreover, the analysis of Su(dx) lgd double mutants clarifies their relationship and suggests that they are not operating in a linear pathway. We could show that, despite prolonged lifetime, the MEs of lgd mutants have a similar ILV density as wild-type but less than rab7 mutant MEs, suggesting the rate in lgd mutants is slightly reduced. The analysis of the MEs of wild-type and mutant cells in the electron microscope revealed that the ESCRT-containing electron-dense microdomains of ILV formation at the limiting membrane are elongated, indicating a change in ESCRT activity. Since lgd mutants can be rescued to normal adult flies if extra copies of shrub (or its mammalian ortholog CHMP4B) are added into the genome, we conclude that the net activity of Shrub is reduced upon loss of lgd function. Finally, we show that, in solution, CHMP4B/Shrub exists in two conformations. LGD1/Lgd binding does not affect the conformational state of Shrub, suggesting that Lgd is not a chaperone for Shrub/CHMP4B. CONCLUSION: Our results suggest that Lgd is required for the full activity of Shrub/ESCRT-III. In its absence, the activity of the ESCRT machinery is reduced. This reduction causes the escape of a fraction of cargo, among it Notch, from incorporation into ILVs, which in turn leads to an activation of this fraction of Notch after fusion of the ME with the lysosome. Our results highlight the importance of the incorporation of Notch into ILV not only to assure complete degradation, but also to avoid uncontrolled activation of the pathway.

Stimulation of ABCB4/MDR3 ATPase activity requires an intact phosphatidylcholine lipid.

ABCB4/MDR3 is located in the canalicular membrane of hepatocytes and translocates PC-lipids from the cytoplasmic to the extracellular leaflet. ABCB4 is an ATP-dependent transporter that reduces the harsh detergent effect of the bile salts by counteracting self-digestion. To do so, ABCB4 provides PC lipids for extraction into bile. PC lipids account for 40% of the entire pool of lipids in the canalicular membrane with an unknown distribution over both leaflets. Extracted PC lipids end up in so-called mixed micelles. Mixed micelles are composed of phospholipids, bile salts, and cholesterol. Ninety to ninety-five percent of the phospholipids are members of the PC family, but only a subset of mainly 16.0-18:1 PC and 16:0-18:2 PC variants are present. To elucidate whether ABCB4 is the key discriminator in this enrichment of specific PC lipids, we used in vitro studies to identify crucial determinants in substrate selection. We demonstrate that PC-lipid moieties alone are insufficient for stimulating ABCB4 ATPase activity, and that at least two acyl chains and the backbone itself are required for a productive interaction. The nature of the fatty acids, like length or saturation has a quantitative impact on the ATPase activity. Our data demonstrate a two-step enrichment and protective function of ABCB4 to mitigate the harsh detergent effect of the bile salts, because ABCB4 can translocate more than just the PC-lipid variants found in bile.

An A666G mutation in transmembrane helix 5 of the yeast multidrug transporter Pdr5 increases drug efflux by enhancing cooperativity between transport sites.

Resistance to antimicrobial and chemotherapeutic agents is a significant clinical problem. Overexpression of multidrug efflux pumps often creates broad-spectrum resistance in cancers and pathogens. We describe a mutation, A666G, in the yeast ABC transporter Pdr5 that shows greater resistance to most of the tested compounds than does an isogenic wild-type strain. This mutant exhibited enhanced resistance without increasing either the amount of protein in the plasma membrane or the ATPase activity. In fluorescence quenching transport assays with rhodamine 6G in purified plasma membrane vesicles, the initial rates of rhodamine 6G fluorescence quenching of both the wild type and mutant showed a strong dependence on the ATP concentration, but were about twice as high in the latter. Plots of the initial rate of fluorescence quenching versus ATP concentration exhibited strong cooperativity that was further enhanced in the A666G mutant. Resistance to imazalil sulfate was about 3-4x as great in the A666G mutant strain as in the wild type. When this transport substrate was used to inhibit the rhodamine 6G transport, the A666G mutant inhibition curves also showed greater cooperativity than the wild-type strain. Our results suggest a novel and important mechanism: under selection, Pdr5 mutants can increase drug resistance by improving cooperative interactions between drug transport sites.

Biochemical and structural characterization of murine GBP7, a guanylate binding protein with an elongated C-terminal tail.

Guanylate-binding proteins (GBPs) constitute a family of interferon-inducible guanosine triphosphatases (GTPases) that are key players in host defense against intracellular pathogens ranging from protozoa to bacteria and viruses. So far, human GBP1 and GBP5 as well as murine GBP2 (mGBP2) have been biochemically characterized in detail. Here, with murine GBP7 (mGBP7), a GBP family member with an unconventional and elongated C-terminus is analyzed. The present study demonstrates that mGBP7 exhibits a concentration-dependent GTPase activity and an apparent GTP turnover number of 20 min-1. In addition, fluorescence spectroscopy analyses reveal that mGBP7 binds GTP with high affinity (KD = 0.22 µM) and GTPase activity assays indicate that mGBP7 hydrolyzes GTP to GDP and GMP. The mGBP7 GTPase activity is inhibited by incubation with γ-phosphate analogs and a K51A mutation interfering with GTP binding. SEC-MALS analyses give evidence that mGBP7 forms transient dimers and that this oligomerization pattern is not influenced by the presence of nucleotides. Moreover, a structural model for mGBP7 is provided by homology modeling, which shows that the GTPase possesses an elongated C-terminal (CT) tail compared with the CaaX motif-containing mGBP2 and human GBP1. Molecular dynamics simulations indicate that this tail has transmembrane characteristics and, interestingly, confocal microscopy analyses reveal that the CT tail is required for recruitment of mGBP7 to the parasitophorous vacuole of Toxoplasma gondii.

FK506 Resistance of Saccharomyces cerevisiae Pdr5 and Candida albicans Cdr1 Involves Mutations in the Transmembrane Domains and Extracellular Loops.

The 23-membered-ring macrolide tacrolimus, a commonly used immunosuppressant, also known as FK506, is a broad-spectrum inhibitor and an efflux pump substrate of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters. Little, however, is known about the molecular mechanism by which FK506 inhibits PDR transporter drug efflux. Thus, to obtain further insights we searched for FK506-resistant mutants of Saccharomyces cerevisiae cells overexpressing either the endogenous multidrug efflux pump Pdr5 or its Candida albicans orthologue, Cdr1. A simple but powerful screen gave 69 FK506-resistant mutants with, between them, 72 mutations in either Pdr5 or Cdr1. Twenty mutations were in just three Pdr5/Cdr1 equivalent amino acid positions, T550/T540 and T552/S542 of extracellular loop 1 (EL1) and A723/A713 of EL3. Sixty of the 72 mutations were either in the ELs or the extracellular halves of individual transmembrane spans (TMSs), while 11 mutations were found near the center of individual TMSs, mostly in predicted TMS-TMS contact points, and only two mutations were in the cytosolic nucleotide-binding domains of Pdr5. We propose that FK506 inhibits Pdr5 and Cdr1 drug efflux by slowing transporter opening and/or substrate release, and that FK506 resistance of Pdr5/Cdr1 drug efflux is achieved by modifying critical intramolecular contact points that, when mutated, enable the cotransport of FK506 with other pump substrates. This may also explain why the 35 Cdr1 mutations that caused FK506 insensitivity of fluconazole efflux differed from the 13 Cdr1 mutations that caused FK506 insensitivity of cycloheximide efflux.