Thiol-switch control in A/B toxin cell entry and compartmental trafficking

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Mechanistic understanding of protein disulfide bond (SS) formation, rearrangement and redox control is a demanding challenge in eukaryotic cell biology as SS proteins are not only present in the endoplasmic reticulum (ER) and the mitochondrial intermembrane space, but also in the cytosol and almost all other compartments. On the other hand, SS bond cleavage also occurs in these compartments, not only for damage repair but also for intracellular signaling. However, the underlying mechanisms of thiol switch control, including electron donors and acceptors, substrate recognition and genetic/cellular factors, are still poorly understood, in particular mechanisms controlling protein thiols in low pH compartments such as the endocytic and the late secretory pathway. Our data on a viral A/B toxin (K28) and its strategy to kill eukaryotic target cells revealed that the most critical reductive cleavage of the single SS bond connecting both toxin subunits in the A/B dimer must only take place in the cytosol and has to be prevented during retrograde toxin traffic. This predicts a cellular control system, in particular in low pH compartments such as endosomes and Golgi, to sense and regulate inter-/intramolecular thiol switching in SS-bonded proteins. Likewise, K28 retrotranslocation from the ER into the cytosol involves redox-dependent chaperon activities of PDI and Hsp40 cochaperones to bring the A/B toxin into a translocation competent conformation without disrupting its SS-bonded structure but still allowing ER exit.Protecting toxin immunity likewise requires a cellular control system, in particular to regulate SS bond switching in the early endosome and during retrograde toxin transport through the secretory pathway. Here we intend to use K28 as model to study the regulation of intracellular SS bond formation/rearrangement within the endocytic and secretory pathway to understand the underlying principles of protein redox switch control in eukaryotic cells. By dissecting toxin immunity in a genetic screen, we intend to identify yet unknown cellular factors controlling spatiotemporal protein thiol switching in various compartments under conditions of intracellular pH changes. Since in vitro, A-toxin release from the dimer occurs spontaneously at pH >6.0, we would like to understand how this is prevented in low pH compartments such as endosomes and Golgi. Under in vitro conditions of neutral pH, K28 also forms SS-bonded oligomers that cause total loss of in vivo toxicity; we would like to understand how this is being prevented in the natural situation of killing, in particular in the ER lumen (at pH 7.2) where K28 should even have a stronger tendency to form inactive oligomers; at the same time, A-subunit release in the ER is cytotoxic and must be prevented to avoid suicide. By using K28 as tool, analysis of these mechanisms will foster our understanding of A/B toxin action and identify cellular factors controlling redox switching.