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The Journal of Cell Biology Dec 2023Autophagy is a lysosomal/vacuolar delivery system that degrades cytoplasmic material. During autophagy, autophagosomes deliver cellular components to the vacuole,...
Autophagy is a lysosomal/vacuolar delivery system that degrades cytoplasmic material. During autophagy, autophagosomes deliver cellular components to the vacuole, resulting in the release of a cargo-containing autophagic body (AB) into the vacuole. AB membranes must be disrupted for degradation of cargo to occur. The lipase Atg15 and vacuolar proteases Pep4 and Prb1 are known to be necessary for this disruption and cargo degradation, but the mechanistic underpinnings remain unclear. In this study, we establish a system to detect lipase activity in the vacuole and show that Atg15 is the sole vacuolar phospholipase. Pep4 and Prb1 are required for the activation of Atg15 lipase function, which occurs following delivery of Atg15 to the vacuole by the MVB pathway. In vitro experiments reveal that Atg15 is a phospholipase B of broad substrate specificity that is likely implicated in the disruption of a range of membranes. Further, we use isolated ABs to demonstrate that Atg15 alone is able to disrupt AB membranes.
Topics: Autophagosomes; Autophagy; Lipase; Vacuoles; Autophagy-Related Proteins; Cell Membrane; Phospholipases
PubMed: 37917025
DOI: 10.1083/jcb.202306120 -
Journal of Molecular Biology Feb 2017Macroautophagy is a degradation process in which autophagosomes are generated to isolate and transport various materials, including damaged organelles and protein... (Review)
Review
Macroautophagy is a degradation process in which autophagosomes are generated to isolate and transport various materials, including damaged organelles and protein aggregates, as cargos to the lysosomes or vacuoles. Bulk autophagy is one of the two types of macroautophagy, which is triggered by starvation and targets non-specific cargos. The second type, that is, selective autophagy, identifies and preferentially degrades specific cargos via receptor recognition. Cytoplasm-to-vacuole targeting (Cvt) is a selective autophagy pathway that specifically transports vacuolar hydrolases into the vacuole in budding yeast cells and has been extensively studied as a model of selective autophagy. In the present review, we focused on the Cvt pathway, especially on the recent structural insights into cargo assembly, receptor recognition, and recruitment mechanisms of the Cvt machinery. Elucidating the Cvt pathway would help in understanding the basic molecular mechanisms of various types of selective autophagy.
Topics: Amino Acid Sequence; Animals; Autophagy; Autophagy-Related Proteins; Biological Transport; Carrier Proteins; Cytoplasm; DNA-(Apurinic or Apyrimidinic Site) Lyase; Humans; Lysosomes; Protein Conformation; Vacuoles
PubMed: 28077284
DOI: 10.1016/j.jmb.2017.01.003 -
Frontiers in Cellular and Infection... 2021While most bacterial species taken up by macrophages are degraded through processing of the bacteria-containing vacuole through the endosomal-lysosomal degradation... (Review)
Review
While most bacterial species taken up by macrophages are degraded through processing of the bacteria-containing vacuole through the endosomal-lysosomal degradation pathway, intravacuolar pathogens have evolved to evade degradation through the endosomal-lysosomal pathway. All intra-vacuolar pathogens possess specialized secretion systems (T3SS-T7SS) that inject effector proteins into the host cell cytosol to modulate myriad of host cell processes and remodel their vacuoles into proliferative niches. Although intravacuolar pathogens utilize similar secretion systems to interfere with their vacuole biogenesis, each pathogen has evolved a unique toolbox of protein effectors injected into the host cell to interact with, and modulate, distinct host cell targets. Thus, intravacuolar pathogens have evolved clear idiosyncrasies in their interference with their vacuole biogenesis to generate a unique intravacuolar niche suitable for their own proliferation. While there has been a quantum leap in our knowledge of modulation of phagosome biogenesis by intravacuolar pathogens, the detailed biochemical and cellular processes affected remain to be deciphered. Here we discuss how the intravacuolar bacterial pathogens , and utilize their unique set of effectors injected into the host cell to interfere with endocytic, exocytic, and ER-to-Golgi vesicle traffic. However, is the main exception for a bacterial pathogen that proliferates within the hydrolytic lysosomal compartment, but its T4SS is essential for adaptation and proliferation within the lysosomal-like vacuole.
Topics: Bacterial Proteins; Golgi Apparatus; Host-Pathogen Interactions; Legionella; Lysosomes; Vacuoles
PubMed: 34858868
DOI: 10.3389/fcimb.2021.722433 -
MBio Oct 2023Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. , the causative agent, has perfected many ways to replicate...
Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. , the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the / system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as .
Topics: Humans; Vacuoles; Dictyostelium; Endoplasmic Reticulum; Mycobacterium marinum; Mycobacterium tuberculosis; Tuberculosis
PubMed: 37676004
DOI: 10.1128/mbio.00943-23 -
Proceedings of the National Academy of... Sep 2022Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane...
Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.
Topics: Autophagosomes; Cell Membrane; Endoplasmic Reticulum; Macroautophagy; Saccharomyces cerevisiae; Vacuoles
PubMed: 36122245
DOI: 10.1073/pnas.2209823119 -
Scientific Reports Oct 2023Vesicular transport driven by membrane trafficking systems conserved in eukaryotes is critical to cellular functionality and homeostasis. It is known that homotypic...
Vesicular transport driven by membrane trafficking systems conserved in eukaryotes is critical to cellular functionality and homeostasis. It is known that homotypic fusion and vacuole protein sorting (HOPS) and class C core endosomal vacuole tethering (CORVET) interact with Rab-GTPases and SNARE proteins to regulate vesicle transport, fusion, and maturation in autophagy and endocytosis pathways. In this study, we identified two novel "Hybrid" tethering complexes in mammalian cells in which one of the subunits of HOPS or CORVET is replaced with the subunit from the other. Substrates taken up by receptor-mediated endocytosis or pinocytosis were transported by distinctive pathways, and the newly identified hybrid complexes contributed to pinocytosis in the presence of HOPS, whereas receptor-mediated endocytosis was exclusively dependent on HOPS. Our study provides new insights into the molecular mechanisms of the endocytic pathway and the function of the vacuolar protein sorting-associated (VPS) protein family.
Topics: Animals; Vacuoles; Vesicular Transport Proteins; Endosomes; Endocytosis; SNARE Proteins; Membrane Fusion; Saccharomyces cerevisiae Proteins; Mammals
PubMed: 37907479
DOI: 10.1038/s41598-023-45418-3 -
Frontiers in Cellular and Infection... 2017Bacteria of the genus cause diseases ranging from gastroenteritis to life-threatening typhoid fever and are among the most successful intracellular pathogens known.... (Review)
Review
Bacteria of the genus cause diseases ranging from gastroenteritis to life-threatening typhoid fever and are among the most successful intracellular pathogens known. After the invasion of the eukaryotic cell, exhibits contrasting lifestyles with different replication rates and subcellular locations. Although hyper-replicates in the cytosol of certain host cell types, most invading bacteria remain within vacuoles in which the pathogen proliferates at moderate rates or persists in a dormant-like state. Remarkably, these cytosolic and intra-vacuolar intracellular lifestyles are not mutually exclusive and can co-exist in the same infected host cell. The mechanisms that direct the invading bacterium to follow the cytosolic or intra-vacuolar "pathway" remain poorly understood. studies show predominance of either the cytosolic or the intra-vacuolar population depending on the host cell type invaded by the pathogen. The host and pathogen factors controlling phagosomal membrane integrity and, as consequence, the egress into the cytosol, are intensively investigated. Other aspects of major interest are the host defenses that may affect differentially the cytosolic and intra-vacuolar populations and the strategies used by the pathogen to circumvent these attacks. Here, we summarize current knowledge about these intracellular subpopulations and discuss how they emerge during the interaction of this pathogen with the eukaryotic cell.
Topics: Animals; Cells; Cytosol; Host-Pathogen Interactions; Humans; Mice; Salmonella; Salmonella Infections; Vacuoles
PubMed: 29046870
DOI: 10.3389/fcimb.2017.00432 -
Current Biology : CB Feb 2015
Topics: Plant Development; Plant Physiological Phenomena; Plant Proteins; Vacuoles
PubMed: 25689903
DOI: 10.1016/j.cub.2014.11.056 -
PLoS Pathogens Jul 2023A key element of Plasmodium biology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the...
A key element of Plasmodium biology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the parasite-encasing parasitophorous vacuole membrane, exported proteins utilise a channel-forming protein complex termed the Plasmodium translocon of exported proteins (PTEX). PTEX is obligatory for parasite survival, both in vitro and in vivo, suggesting that at least some exported proteins have essential metabolic functions. However, to date only one essential PTEX-dependent process, the new permeability pathways, has been described. To identify other essential PTEX-dependant proteins/processes, we conditionally knocked down the expression of one of its core components, PTEX150, and examined which pathways were affected. Surprisingly, the food vacuole mediated process of haemoglobin (Hb) digestion was substantially perturbed by PTEX150 knockdown. Using a range of transgenic parasite lines and approaches, we show that two major Hb proteases; falcipain 2a and plasmepsin II, interact with PTEX core components, implicating the translocon in the trafficking of Hb proteases. We propose a model where these proteases are translocated into the PV via PTEX in order to reach the cytostome, located at the parasite periphery, prior to food vacuole entry. This work offers a second mechanistic explanation for why PTEX function is essential for growth of the parasite within its host RBC.
Topics: Animals; Plasmodium falciparum; Vacuoles; Protein Transport; Protozoan Proteins; Erythrocytes; Parasites; Peptide Hydrolases
PubMed: 37523385
DOI: 10.1371/journal.ppat.1011006 -
Cells Dec 2019Ribosomes are essential for protein synthesis in all organisms and their biogenesis and number are tightly controlled to maintain homeostasis in changing environmental... (Review)
Review
Ribosomes are essential for protein synthesis in all organisms and their biogenesis and number are tightly controlled to maintain homeostasis in changing environmental conditions. While ribosome assembly and quality control mechanisms have been extensively studied, our understanding of ribosome degradation is limited. In yeast or animal cells, ribosomes are degraded after transfer into the vacuole or lysosome by ribophagy or nonselective autophagy, and ribosomal RNA can also be transferred directly across the lysosomal membrane by RNautophagy. In plants, ribosomal RNA is degraded by the vacuolar T2 ribonuclease RNS2 after transport by autophagy-related mechanisms, although it is unknown if a selective ribophagy pathway exists in plants. In this review, we describe mechanisms of turnover of ribosomal components in animals and yeast, and, then, discuss potential pathways for degradation of ribosomal RNA and protein within the vacuole in plants.
Topics: Animals; Autophagy; Humans; Lysosomes; RNA; Ribosomes; Vacuoles
PubMed: 31835634
DOI: 10.3390/cells8121603