Functional analysis of the ABC export systems TmrAB from Thermus thermophilus and MDL1 from Saccharomyces cerevisiae [Fuctional analysis of the ABC export systems TmrAB from Thermus thermophilus and MDL1 from Saccharomyces cerevisiae]

ATP‐binding cassette (ABC) transporters belong to one of the most abundant families of integral membrane proteins present in all three kingdoms of life. Members of this superfamily energize the transmembrane movement of a broad range of solutes and therefore play a crucial role in various cellular processes. In this study, the prokaryotic ABC complex TmrAB as well as the eukaryotic mitochondrial ABC transporter MDL1 were chosen as model system for functional analysis of ABC exporters. ...Die Translokation von gelösten Stoffen über zelluläre Membranen ist ein essentieller biologischer Prozess, der durch eine Vielfalt an integralen Membranproteinen vermittelt wird. Diese sind in den selektiven Austausch verschiedenster Stoffe bzw. Teilchen involviert und ermöglichen somit die Kommunikation zwischen den einzelnen Zellkompartimenten untereinander bzw. mit der extrazellulären Umgebung. Eine der größten Familien paraloger Proteine, die den vektoriellen Transport von Substanzen über Zellmembranen katalysieren, stellen die ATP‐binding cassette (ABC)‐Transporter dar. Mitglieder dieser Proteinfamilie sind in allen bisher untersuchten Organismen von Prokaryoten bis hin zu höheren Eukaryoten vertreten und übernehmen essentielle Funktionen in einer Vielzahl von zellulären Abläufen. ABC‐Transporter zeichnen sich durch eine breite Substratdiversität aus, d.h. sie energetisieren unter ATP‐Verbrauch die Translokation zahlreicher, strukturell und chemisch unterschiedlicher Substanzen wie Zucker, Lipide, Ionen, Aminosäuren, Proteine oder auch zelltoxische Stoffe. In Bakterien können sie sowohl als Importproteine fungieren, welche hauptsächlich die Aufnahme von Nährstoffen vermitteln, als auch als Exportproteine, deren Hauptaufgabe es ist, zelltoxische Substanzen aus der Zelle heraus zu schleusen. Eukaryotische ABC‐Transporter sind sowohl in der Plasmamembran als auch in den intrazellulären Membranen zu finden – beispielsweise in denen des Endoplasmatischen Retikulums, des Golgi Apparats, der Lysosomen, der Peroxisomen und der Mitochondrien. Sie fungieren als Exportproteine und sind z.B. an der Ionen‐Homöostase, der Antigenprozessierung, der Insulinfreisetzung oder am Cholesterol‐ und Lipidtransport beteiligt. ..

Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus

ATP-binding cassette (ABC) systems translocate a wide range of solutes across cellular membranes. The thermophilic Gram-negative eubacterium Thermus thermophilus, a model organism for structural genomics and systems biology, discloses ∼46 ABC proteins, which are largely uncharacterized. Here, we functionally analyzed the first two and only ABC half-transporters of the hyperthermophilic bacterium, TmrA and TmrB. The ABC system mediates uptake of the drug Hoechst 33342 in inside-out oriented vesicles that is inhibited by verapamil. TmrA and TmrB form a stable heterodimeric complex hydrolyzing ATP with a Km of 0.9 mm and kcat of 9 s−1 at 68 °C. Two nucleotides can be trapped in the heterodimeric ABC complex either by vanadate or by mutation inhibiting ATP hydrolysis. Nucleotide trapping requires permissive temperatures, at which a conformational ATP switch is possible. We further demonstrate that the canonic glutamate 523 of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding aspartate in TmrB (Asp-500) has only a regulatory role. Notably, exchange of this single noncanonic residue into a catalytic glutamate cannot rescue the function of the E523Q/D500E complex, implicating a built-in asymmetry of the complex. However, slow ATP hydrolysis in the newly generated canonic site (D500E) strictly depends on the formation of a posthydrolysis state in the consensus site, indicating an allosteric coupling of both active sites

Structural and functional fingerprint of the mitochondrial ATP-binding cassette transporter Mdl1 from Saccharomyces cerevisiae

The ATP-binding cassette half-transporter Mdl1 from Saccharomyces cerevisiae has been proposed to be involved in the quality control of misassembled respiratory chain complexes by exporting degradation products generated by the m-AAA proteases from the matrix. Direct functional or structural data of the transport complex are, however, not known so far. After screening expression in various hosts, Mdl1 was overexpressed 100-fold to 1% of total mitochondrial membrane protein in S. cerevisiae. Based on detergent screens, Mdl1 was solubilized and purified to homogeneity. Mdl1 showed a high binding affinity for MgATP (Kd = 0.26 μm) and an ATPase activity with a Km of 0.86 mm (Hill coefficient of 0.98) and a turnover rate of 2.6 ATP/s. Mutagenesis of the conserved glutamate downstream of the Walker B motif (E599Q) or the conserved histidine of the H-loop (H631A) abolished ATP hydrolysis, whereas ATP binding was not affected. Mdl1 reconstituted into liposomes showed an ATPase activity similar to the solubilized complex. By single particle electron microscopy, a first three-dimensional structure of the mitochondrial ATP-binding cassette transporter was derived at 2.3-nm resolution, revealing a homodimeric complex in an open conformation

A dual-reporter system for investigating and optimizing protein translation and folding in E. coli

Heterologous expression of recombinant proteins often results in misfolding, aggregation and degradation. Here, we show an in vivo dual-biosensor system that simultaneously assesses protein translation and protein folding, thereby enabling rapid screening of expression strains as well as mutant libraries

SERINC5 Is an Unconventional HIV Restriction Factor That Is Upregulated during Myeloid Cell Differentiation

<jats:p>Classical antiviral restriction factors promote cellular immunity by their ability to interfere with virus replication and induction of their expression by proinflammatory cytokines such as interferons. The serine incorporator proteins SERINC3 and SERINC5 potently reduce the infectivity of HIV-1 particles when overexpressed, and RNA interference or knockout approaches in T cells have indicated antiviral activity also of the endogenous proteins. Due to lack of reagents for detection of endogenous SERINC proteins, it is still unclear whether SERINC3/5 are expressed to functionally relevant levels in different primary target cells of HIV infection and how the expression levels of these innate immunity factors are regulated. In the current study, analysis of <i>SERINC3/5</i> mRNA steady-state levels in primary lymphoid and monocyte-derived cells revealed selective induction of their expression upon differentiation of myeloid cells. Contrary to classical antiviral restriction factors, various antiviral α-interferon subtypes and proinflammatory interleukins had no effect on <i>SERINC</i> levels, which were also not dysregulated in CD4+ T cells and monocytes isolated from patients with chronic HIV-1 infection. Notably, HIV-1 particles produced by terminally differentiated monocyte-derived macrophages with high <i>SERINC5</i> expression, but not by low-expressing monocytes, showed a Nef-dependent infectivity defect. Overall, these findings suggest endogenous expression of SERINC5 to antivirally active levels in macrophages. Our results classify SERINC5 as an unconventional HIV-1 restriction factor whose expression is specifically induced upon differentiation of cells towards the myeloid lineage.</jats:p&gt

Asymmetric ATP Hydrolysis Cycle of the Heterodimeric Multidrug ABC Transport Complex TmrAB from Thermus thermophilus*

ATP-binding cassette (ABC) systems translocate a wide range of solutes across cellular membranes. The thermophilic Gram-negative eubacterium Thermus thermophilus, a model organism for structural genomics and systems biology, discloses ∼46 ABC proteins, which are largely uncharacterized. Here, we functionally analyzed the first two and only ABC half-transporters of the hyperthermophilic bacterium, TmrA and TmrB. The ABC system mediates uptake of the drug Hoechst 33342 in inside-out oriented vesicles that is inhibited by verapamil. TmrA and TmrB form a stable heterodimeric complex hydrolyzing ATP with a Km of 0.9 mm and kcat of 9 s−1 at 68 °C. Two nucleotides can be trapped in the heterodimeric ABC complex either by vanadate or by mutation inhibiting ATP hydrolysis. Nucleotide trapping requires permissive temperatures, at which a conformational ATP switch is possible. We further demonstrate that the canonic glutamate 523 of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding aspartate in TmrB (Asp-500) has only a regulatory role. Notably, exchange of this single noncanonic residue into a catalytic glutamate cannot rescue the function of the E523Q/D500E complex, implicating a built-in asymmetry of the complex. However, slow ATP hydrolysis in the newly generated canonic site (D500E) strictly depends on the formation of a posthydrolysis state in the consensus site, indicating an allosteric coupling of both active sites