The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; ...however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood. To learn how certain ESCRT-IIIs shape positively curved membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B + IST1, and IST1-only filaments by cryo-EM. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand on CHMP1B, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction thins the underlying bilayer, lowering the barrier to membrane fission. Our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, constriction and bilayer thinning.
Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the ...endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a β-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the 'Endosomal SNX-BAR sorting complex for promoting exit 1' (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.
Alanine-, serine-, cysteine-preferring transporter 2 (ASCT2, SLC1A5) is responsible for the uptake of glutamine into cells, a major source of cellular energy and a key regulator of mammalian target ...of rapamycin (mTOR) activation. Furthermore, ASCT2 expression has been reported in several human cancers, making it a potential target for both diagnostic and therapeutic purposes. Here we identify ASCT2 as a membrane-trafficked cargo molecule, sorted through a direct interaction with the PDZ domain of sorting nexin 27 (SNX27). Using both membrane fractionation and subcellular localization approaches, we demonstrate that the majority of ASCT2 resides at the plasma membrane. This is significantly reduced within CrispR-mediated SNX27 knockout (KO) cell lines, as it is missorted into the lysosomal degradation pathway. The reduction of ASCT2 levels in SNX27 KO cells leads to decreased glutamine uptake, which, in turn, inhibits cellular proliferation. SNX27 KO cells also present impaired activation of the mTOR complex 1 (mTORC1) pathway and enhanced autophagy. Taken together, our data reveal a role for SNX27 in glutamine uptake and amino acid–stimulated mTORC1 activation via modulation of ASCT2 intracellular trafficking.
Macroautophagy is a physiological cellular response to nutrient stress, which leads to the engulfment of cytosolic contents by a double-walled membrane structure, the phagophore. Phagophores seal to ...become autophagosomes, which then fuse with lysosomes to deliver their contents for degradation. Macroautophagy is regulated by numerous cellular factors, including the Class III PI3K (phosphoinositide 3-kinase) Vps34 (vacuolar protein sorting 34). The autophagic functions of Vps34 require its recruitment to a complex that includes Vps15, Beclin-1 and Atg14L (autophagy-related 14-like protein) and is known as Vps34 Complex I. We have now identified NRBF2 (nuclear receptor-binding factor 2) as a new member of Vps34 Complex I. NRBF2 binds to complexes that include Vps34, Vps15, Beclin-1 and ATG-14L, but not the Vps34 Complex II component UVRAG (UV radiation resistance-associated gene). NRBF2 directly interacts with Vps15 via the Vps15 WD40 domain as well as other regions of Vps15. The formation of GFP-LC3 (light chain 3) punctae and PE (phosphatidylethanolamine)-conjugated LC3 (LC3-II) in serum-starved cells was inhibited by NRBF2 knockdown in the absence and presence of lysosomal inhibitors, and p62 levels were increased. Thus NRBF2 plays a critical role in the induction of starvation-induced autophagy as a specific member of Vps34 Complex I.
Waste threatens human health and the environment. Public participation, as an essential link in the whole waste management chain, is important for countries to achieve carbon neutrality and promote ...sustainable development. However, some countries are still in the initial stage of policy practice, for example, China's waste sorting policy is still in the pilot stage and has not yet covered the whole country. Therefore, it is necessary to understand whether waste sorting policy has a catalytic effect on public waste sorting behavior, and based on the Theory of Planned Behavior, knowledge and intention were proposed to have chain-mediating effect. With a large-sample national survey, we compared the differences in public waste sorting behavior between areas with and without waste sorting policies and examined the mechanism. Results showed that the public in areas with waste sorting policies participated more in actual waste sorting behavior than the public in areas without waste sorting policies, and the proposed effects of knowledge and intention were verified. The research results provide valuable insights for policymakers and stakeholders from multiple perspectives of policy implementation, policy instruments, and population differences.
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•Waste sorting policy implementation increases public waste sorting behaviors in China.•Knowledge and intention serially mediate the positive effect of policy implementation.•A large-scale national survey was conducted with cross-regional comparison.
The eukaryotic TORC1 kinase is a homeostatic controller of growth that integrates nutritional cues and mediates signals primarily from the surface of lysosomes or vacuoles. Amino acids activate TORC1 ...via the Rag GTPases that combine into structurally conserved multi-protein complexes such as the EGO complex (EGOC) in yeast. Here we show that Ego1, which mediates membrane-anchoring of EGOC via lipid modifications that it acquires while traveling through the trans-Golgi network, is separately sorted to vacuoles and perivacuolar endosomes. At both surfaces, it assembles EGOCs, which regulate spatially distinct pools of TORC1 that impinge on functionally divergent effectors: vacuolar TORC1 predominantly targets Sch9 to promote protein synthesis, whereas endosomal TORC1 phosphorylates Atg13 and Vps27 to inhibit macroautophagy and ESCRT-driven microautophagy, respectively. Thus, the coordination of three key regulatory nodes in protein synthesis and degradation critically relies on a division of labor between spatially sequestered populations of TORC1.
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•Rag GTPases and TORC1 assemble at the surfaces of both endosomes and vacuoles•These spatially and functionally distinct pools of TORC1 target specific effectors•Vacuolar TORC1 promotes protein synthesis through its proximal effector Sch9•Endosomal TORC1 curbs macro- and microautophagy via Atg13 and Vps27, respectively
The Rag GTPase-TORC1 signaling module integrates amino acid signals to control eukaryotic growth in a homeostatic manner. Here, Hatakeyama et al. show that functionally autonomous pools of these modules assemble at vacuolar and endosomal membranes to balance spatially distinct key regulatory nodes in protein synthesis and degradation, respectively.
•Personal moral norms, waste sorting knowledge and incentive measures have been considered.•Personal moral norms and waste sorting knowledge positively affect waste sorting intention.•Incentive ...measures narrow the gap between waste sorting intention and behavior.
Waste sorting is essential to address the current predicament of waste management. Though it is important, insufficient attention has been paid to explore residents’ waste sorting intention and behavior and understand its formation process. To narrow the research gap, this research built a theoretical research model by adding personal moral norms and waste sorting knowledge into the theory of planned behavior to explicate residents’ waste sorting intention and behavior formation process. Meanwhile, given the discrepancy between waste sorting intention and actual behavior, this research also explored the effect of external conditions, such as incentive measures, on this discrepancy. Based on survey data from 397 Chinese residents, this research found that attitudes, subjective norms, perceived behavioral control, personal moral norms and waste sorting knowledge were directly and significantly related to residents’ waste sorting intention. Waste sorting knowledge also had an indirect influence on residents’ waste sorting intention through attitudes and perceived behavioral control. Additionally, this research corroborated the discrepancy between waste sorting intention and behavior, and suggested that the link between intention and behavior was contingent on incentive measures. Incentive measures strengthened the effect of intention on behavior. This research is useful for understanding residents’ waste sorting intention and behavior and valuable for encouraging residents to sort waste in their daily lives.
SignalP is the currently most widely used program for prediction of signal peptides from amino acid sequences. Proteins with signal peptides are targeted to the secretory pathway, but are not ...necessarily secreted. After a brief introduction to the biology of signal peptides and the history of signal peptide prediction, this chapter will describe all the options of the current version of SignalP and the details of the output from the program. The chapter includes a case study where the scores of SignalP were used in a novel way to predict the functional effects of amino acid substitutions in signal peptides.
The PDZ-domain-containing sorting nexin 27 (SNX27) promotes recycling of internalized transmembrane proteins from endosomes to the plasma membrane by linking PDZ-dependent cargo recognition to ...retromer-mediated transport. Here, we employed quantitative proteomics of the SNX27 interactome and quantification of the surface proteome of SNX27- and retromer-suppressed cells to dissect the assembly of the SNX27 complex and provide an unbiased global view of SNX27-mediated sorting. Over 100 cell surface proteins, many of which interact with SNX27, including the glucose transporter GLUT1, the Menkes disease copper transporter ATP7A, various zinc and amino acid transporters, and numerous signalling receptors, require SNX27-retromer to prevent lysosomal degradation and maintain surface levels. Furthermore, we establish that direct interaction of the SNX27 PDZ domain with the retromer subunit VPS26 is necessary and sufficient to prevent lysosomal entry of SNX27 cargo. Our data identify the SNX27-retromer as a major endosomal recycling hub required to maintain cellular nutrient homeostasis.
The mammalian genome encodes 49 proteins that possess a PX (phox-homology) domain, responsible for membrane attachment to organelles of the secretory and endocytic system via binding of ...phosphoinositide lipids. The PX domain proteins, most of which are classified as SNXs (sorting nexins), constitute an extremely diverse family of molecules that play varied roles in membrane trafficking, cell signalling, membrane remodelling and organelle motility. In the present review, we present an overview of the family, incorporating recent functional and structural insights, and propose an updated classification of the proteins into distinct subfamilies on the basis of these insights. Almost all PX domain proteins bind PtdIns3P and are recruited to early endosomal membranes. Although other specificities and localizations have been reported for a select few family members, the molecular basis for binding to other lipids is still not clear. The PX domain is also emerging as an important protein-protein interaction domain, binding endocytic and exocytic machinery, transmembrane proteins and many other molecules. A comprehensive survey of the molecular interactions governed by PX proteins highlights the functional diversity of the family as trafficking cargo adaptors and membrane-associated scaffolds regulating cell signalling. Finally, we examine the mounting evidence linking PX proteins to different disorders, in particular focusing on their emerging importance in both pathogen invasion and amyloid production in Alzheimer's disease.
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