yggS Encoding Pyridoxal 5′-Phosphate Binding Protein Is Required for Acidovorax citrulli Virulence

Bacterial fruit blotch, caused by seed-borne pathogen Acidovorax citrulli, poses a serious threat to the production of cucurbits globally. Although the disease can cause substantial economic losses, limited information is available about the molecular mechanisms of virulence. This study identified that, a random transposon insertion mutant impaired in the ability to elicit a hypersensitive response on tobacco. The disrupted gene in this mutant was determined to be Aave_0638, which is predicted to encode a YggS family pyridoxal phosphate-dependent enzyme. YggS is a highly conserved protein among multiple organisms, and is responsible for maintaining the homeostasis of pyridoxal 5′-phosphate and amino acids in cells. yggS deletion mutant of A. citrulli strain XjL12 displayed attenuated virulence, delayed hypersensitive response, less tolerance to H2O2 and pyridoxine, increased sensitivity to antibiotic β-chloro-D-alanine, and reduced swimming. In addition, RNA-Seq analysis demonstrated that yggS was involved in regulating the expression of certain pathogenicity-associated genes related to secretion, motility, quorum sensing and oxidative stress response. Importantly, YggS significantly affected type III secretion system and its effectors in vitro. Collectively, our results suggest that YggS is indispensable for A.citrulli virulence and expands the role of YggS in the biological processes.

Pib2 as an Emerging Master Regulator of Yeast TORC1

Cell growth is dynamically regulated in response to external cues such as nutrient availability, growth factor signals, and stresses. Central to this adaptation process is the Target of Rapamycin Complex 1 (TORC1), an evolutionarily conserved kinase complex that fine-tunes an enormous number of cellular events. How upstream signals are sensed and transmitted to TORC1 has been intensively studied in major model organisms including the budding yeast Saccharomyces cerevisiae. This field recently saw a breakthrough: the identification of yeast phosphatidylInositol(3)-phosphate binding protein 2 (Pib2) protein as a critical regulator of TORC1. Although the study of Pib2 is still in its early days, multiple groups have provided important mechanistic insights on how Pib2 relays nutrient signals to TORC1. There remain, on the other hand, significant gaps in our knowledge and mysteries that warrant further investigations. This is the first dedicated review on Pib2 that summarizes major findings and outstanding questions around this emerging key player in cell growth regulation.

First person – Anthony Ravussin

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Anthony Ravussin is first author on ‘The phosphatidylinositol 3-phosphate-binding protein SNX4 controls ATG9A recycling and autophagy’, published in JCS. Anthony is a postdoc in the lab of Professor Harald Stenmark at the Centre for Cancer Cell Reprogramming, University of Oslo, Norway, investigating the role of PI3P-binding proteins in the control of membrane dynamics.

The phosphatidylinositol 3-phosphate-binding protein SNX4 controls ATG9A recycling and autophagy

ABSTRACTLate endosomes and lysosomes (endolysosomes) receive proteins and cargo from the secretory, endocytic and autophagic pathways. Although these pathways and the degradative processes of endolysosomes are well characterized, less is understood about protein traffic from these organelles. In this study, we demonstrate the direct involvement of the phosphatidylinositol 3-phosphate (PI3P)-binding SNX4 protein in membrane protein recycling from endolysosomes, and show that SNX4 is required for proper autophagic flux. We show that SNX4 mediates recycling of the lipid scramblase ATG9A, which drives expansion of nascent autophagosome membranes, from endolysosomes to early endosomes, from where ATG9A is recycled to the trans-Golgi network in a retromer-dependent manner. Upon siRNA-mediated depletion of SNX4 or the retromer component VPS35, we observed accumulation of ATG9A on endolysosomes and early endosomes, respectively. Moreover, starvation-induced autophagosome biogenesis and autophagic flux were inhibited when SNX4 was downregulated. We propose that proper ATG9A recycling by SNX4 sustains autophagy by preventing exhaustion of the available ATG9A pool.This article has an associated First Person interview with the first author of the paper.

The phosphatidylinositol 3-phosphate binding protein SNX4 controls ATG9A recycling and autophagy

Late endosomes and lysosomes (endolysosomes) receive proteins and cargo from the secretory, endocytic and autophagic pathways. Whereas these pathways and the degradative processes of endolysosomes are well characterized, less is understood about protein traffic from these organelles. In this study, we demonstrate the direct involvement of the phosphatidylinositol 3-phosphate (PI3P) binding SNX4 protein in membrane protein recycling from endolysosomes, and show that SNX4 is required for proper autophagic flux. We show that SNX4 mediates recycling of the transmembrane autophagy machinery protein ATG9A from endolysosomes to early endosomes, from where ATG9A is recycled to the trans-Golgi network in a retromer-dependent manner. Upon siRNA-mediated depletion of SNX4 or the retromer component VPS35, we observed accumulation of ATG9A on endolysosomes and early endosomes, respectively. Moreover, starvation-induced autophagosome biogenesis and autophagic flux were inhibited when SNX4 was downregulated. Altogether, we propose that proper ATG9A recycling by SNX4 sustains autophagy by preventing exhaustion of the available ATG9A pool.

Short and simple sequences favored the emergence of N-helix phospho-ligand binding sites in the first enzymes

The ubiquity of phospho-ligands suggests that phosphate binding emerged at the earliest stage of protein evolution. To evaluate this hypothesis and unravel its details, we identified all phosphate-binding protein lineages in the Evolutionary Classification of Protein Domains database. We found at least 250 independent evolutionary lineages that bind small molecule cofactors and metabolites with phosphate moieties. For many lineages, phosphate binding emerged later as a niche functionality, but for the oldest protein lineages, phosphate binding was the founding function. Across some 4 billion y of protein evolution, side-chain binding, in which the phosphate moiety does not interact with the backbone at all, emerged most frequently. However, in the oldest lineages, and most characteristically in αβα sandwich enzyme domains, N-helix binding sites dominate, where the phosphate moiety sits atop the N terminus of an α-helix. This discrepancy is explained by the observation that N-helix binding is uniquely realized by short, contiguous sequences with reduced amino acid diversity, foremost Gly, Ser, and Thr. The latter two amino acids preferentially interact with both the backbone amide and the side-chain hydroxyl (bidentate interaction) to promote binding by short sequences. We conclude that the first αβα sandwich domains emerged from shorter and simpler polypeptides that bound phospho-ligands via N-helix sites.