Jensen, C. | bioRxiv Science

bioRxiv2018

N-terminal β-strand underpins biochemical specialization of an ATG8 isoform

ATG8 is a highly-conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and numerous proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein-protein interaction resource, obtained using in planta immunoprecipitation followed by mass spectrometry, to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal {beta}-strand--and, in particular, a single amino acid polymorphism--underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this proteins ATG8 interacting motif. Additional proteomics experiments indicated that the N-terminal {beta}-strand shapes the ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants.

plant biology↗

bioRxiv2018

The ClpX chaperone controls the Staphylococcus aureus cell cycle but can be bypassed by β-lactam antibiotics

The worldwide spread of Staphylococcus aureus strains resistant to almost all {beta}-lactam antibiotics is of major clinical concern. {beta}-lactams interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Here we show that sub-lethal doses of {beta}-lactams stimulate the growth of S. aureus mutants lacking the widely conserved chaperone ClpX. S. aureus clpX mutants have a severe growth defect at temperatures below 37{degrees}C, and we reasoned that a better understanding of this growth defect could provide novel insights into how {beta}-lactam antibiotics interfere with growth of S. aureus. We demonstrate that ClpX is important for coordinating the S. aureus cell cycle, and that S. aureus cells devoid of ClpX fail to divide, or lyze spontaneously, at high frequency unless {beta}-lactams are added to the growth medium. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30{degrees}C, but not at 37{degrees}C. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, {beta}-lactams restored septum synthesis and prevented premature autolytic splitting of clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with {beta}-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, underscoring a functional link between the PBP activity and WTA biosynthesis. The finding that {beta} -lactams can prevent lysis and restore septum synthesis of a mutant with dysregulated cell division lends support to the idea that PBPs function as coordinators of cell division and that {beta} -lactams do not kill S. aureus simply by weakening the cell wall.\n\nAuthor SummaryThe bacterium Staphylococcus aureus is a major cause of human disease, and the rapid spread of S. aureus strains that are resistant to almost all {beta}-lactam antibiotics has made treatment increasingly difficult. {beta}-lactams interfere with cross-linking of the bacterial cell wall but the killing mechanism of this important class of antibiotics is still not fully understood. Here we provide novel insight into this topic by examining a defined S. aureus mutant that has the unusual property of growing markedly better in the presence of {beta}-lactams. Without {beta}-lactams this mutant dies spontaneously at a high frequency due to premature separation of daughter cells during cell division. Cell death of the mutant can, however, be prevented either by exposure to {beta}-lactam antibiotics or by inhibiting synthesis of wall teichoic acid, a major component of the cell wall in Gram-positive bacteria with a conserved role in activation of autolytic splitting of daughter cells. The finding that the detrimental effect of {beta}-lactam antibiotics can be reversed by a mutation that affect the coordination of cell division emphasizes the idea that {beta}-lactams do not kill S. aureus simply by weakening the cell wall but rather by interference with the coordination of cell division.

microbiology↗