Peptide translocation by the lysosomal ABC transporter TAPL is regulated by coupling efficiency and activation energy

The lysosomal polypeptide transporter TAPL belongs to the superfamily of ATP-binding cassette transporters. TAPL forms a homodimeric transport complex, which translocates oligo- and polypeptides into the lumen of lysosomes driven by ATP hydrolysis. Although the structure and the function of ABC transporters were intensively studied in the past, details about the single steps of the transport cycle are still elusive. Therefore, we analyzed the coupling of peptide binding, transport and ATP hydrolysis for different substrate sizes. Although longer and shorter peptides bind with the same affinity and are transported with identical Km values, they differ significantly in their transport rates. This difference can be attributed to a higher activation energy for the longer peptide. TAPL shows a basal ATPase activity, which is inhibited in the presence of longer peptides. Uncoupling between ATP hydrolysis and peptide transport increases with peptide length. Remarkably, also the type of nucleotide determines the uncoupling. While GTP is hydrolyzed as good as ATP, peptide transport is significantly reduced. In conclusion, TAPL does not differentiate between transport substrates in the binding process but during the following steps in the transport cycle, whereas, on the other hand, not only the coupling efficiency but also the activation energy varies depending on the size of peptide substrate

Single liposome analysis of peptide translocation by the ABC transporter TAPL

ATP-binding cassette (ABC) transporters use ATP to drive solute transport across biological membranes. Members of this superfamily have crucial roles in cell physiology, and some of the transporters are linked to severe diseases. However, understanding of the transport mechanism, especially of human ABC exporters, is scarce. We reconstituted the human lysosomal polypeptide ABC transporter TAPL, expressed in Pichia pastoris, into lipid vesicles (liposomes) and performed explicit transport measurements. We analyzed solute transport at the single liposome level by monitoring the coincident fluorescence of solutes and proteoliposomes in the focal volume of a confocal microscope. We determined a turnover number of eight peptides per minute, which is two orders of magnitude higher than previously estimated from macroscopic measurements. Moreover, we show that TAPL translocates peptides against a large concentration gradient. Maximal filling is not limited by an electrochemical gradient but by trans-inhibition. Countertransport and reversibility studies demonstrate that peptide translocation is a strictly unidirectional process. Altogether, these data are included in a refined model of solute transport by ABC exporters

The Propaganda Model and Intersectionality: Integrating Separate Paradigms

International audienceThe world is currently witnessing a revitalisation of the right and of authoritarian political tendencies. Right-wing forces across the globe have been able to push misogynist, homophobic and xenophobic discourses into the mainstream of politics and media. Whilst these developments have been fuelled by the neoliberal economic programmes unrolled since the 1970s, sexism and racism have always been anchored within the structures of real existing capitalism. This suggests, then, that many of the societal issues we are encountering today are rooted in structural disadvantage and oppression pertaining not only to economics and class but also to gender, race and ethnicity. Yet, approaches in Communication Studies and Cultural Studies have often engaged in separate interrogations of media misrepresentations in relation to either class and economics, or gender and/or race. On the other hand, intersectional scholarship has long highlighted how these societal spheres are interconnected and should thus be researched simultaneously. The Herman-Chomsky Propaganda Model constitutes the leading analytical tool to theorize and investigate media bias. The following contributions will conceptualize and illustrate how the PM relates to intersectional scholarship and societal structures. This will be done on the basis of theoretical elaborations and empirical case studies as well as broader discussions of the politics within the disciplines of Communications Studies and Cultural Studies. It will be demonstrated that the PM can be used to unveil interlocking media biases and misrepresentations deriving from parallel societal discriminations including classism, sexism and racism

Crystal structure and mechanistic basis of a functional homolog of the antigen transporter TAP

ABC transporters form one of the largest protein superfamilies in all domains of life, catalyzing the movement of diverse substrates across membranes. In this key position, ABC transporters can mediate multidrug resistance in cancer therapy and their dysfunction is linked to various diseases. Here, we describe the 2.7-Å X-ray structure of heterodimeric Thermus thermophilus multidrug resistance proteins A and B (TmrAB), which not only shares structural homology with the antigen translocation complex TAP, but is also able to restore antigen processing in human TAP-deficient cells. TmrAB exhibits a broad peptide specificity and can concentrate substrates several thousandfold, using only one single active ATP-binding site. In our structure, TmrAB adopts an asymmetric inward-facing state, and we show that the C-terminal helices, arranged in a zipper-like fashion, play a crucial role in guiding the conformational changes associated with substrate transport. In conclusion, TmrAB can be regarded as a model system for asymmetric ABC exporters in general, and for TAP in particular.publishe