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Nature Communications Oct 2023The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for autophagosome-lysosome fusion in mammals, yet...
The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for autophagosome-lysosome fusion in mammals, yet reconstituting the mammalian HOPS complex remains a challenge. Here we propose a "hook-up" model for mammalian HOPS complex assembly, which requires two HOPS sub-complexes docking on membranes via membrane-associated Rabs. We identify Rab39A as a key small GTPase that recruits HOPS onto autophagic vesicles. Proper pairing with Rab2 and Rab39A enables HOPS complex assembly between proteoliposomes for its tethering function, facilitating efficient membrane fusion. GTP loading of Rab39A is important for the recruitment of HOPS to autophagic membranes. Activation of Rab39A is catalyzed by C9orf72, a guanine exchange factor associated with amyotrophic lateral sclerosis and familial frontotemporal dementia. Constitutive activation of Rab39A can rescue autophagy defects caused by C9orf72 depletion. These results therefore reveal a crucial role for the C9orf72-Rab39A-HOPS axis in autophagosome-lysosome fusion.
Topics: Animals; Autophagy; C9orf72 Protein; Catalysis; Guanosine Triphosphate; Mammals; Membrane Fusion; Vacuoles
PubMed: 37821429
DOI: 10.1038/s41467-023-42003-0 -
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 -
The Plant Cell Nov 2019
Topics: Carotenoids; Crocus; Membrane Transport Proteins; Vacuoles
PubMed: 31548255
DOI: 10.1105/tpc.19.00720 -
Nature Metabolism Oct 2023Amino acid homeostasis is critical for many cellular processes. It is well established that amino acids are compartmentalized using pH gradients generated between...
Amino acid homeostasis is critical for many cellular processes. It is well established that amino acids are compartmentalized using pH gradients generated between organelles and the cytoplasm; however, the dynamics of this partitioning has not been explored. Here we develop a highly sensitive pH reporter and find that the major amino acid storage compartment in Saccharomyces cerevisiae, the lysosome-like vacuole, alkalinizes before cell division and re-acidifies as cells divide. The vacuolar pH dynamics require the uptake of extracellular amino acids and activity of TORC1, the v-ATPase and the cycling of the vacuolar specific lipid phosphatidylinositol 3,5-bisphosphate, which is regulated by the cyclin-dependent kinase Pho85 (CDK5 in mammals). Vacuolar pH regulation enables amino acid sequestration and mobilization from the organelle, which is important for mitochondrial function, ribosome homeostasis and cell size control. Collectively, our data provide a new paradigm for the use of dynamic pH-dependent amino acid compartmentalization during cell growth/division.
Topics: Animals; Vacuoles; Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae; Homeostasis; Amino Acids; Cell Division; Cell Cycle; Hydrogen-Ion Concentration; Mammals
PubMed: 37640943
DOI: 10.1038/s42255-023-00872-1 -
The New Phytologist Jan 2021The coordinated control of ion transport across the two major membranes of differentiated plant cells, the plasma and the vacuolar membranes, is fundamental in cell... (Review)
Review
The coordinated control of ion transport across the two major membranes of differentiated plant cells, the plasma and the vacuolar membranes, is fundamental in cell physiology. The stomata responses to the fluctuating environmental conditions are an illustrative example. Indeed, they rely on the coordination of ion fluxes between the different cell compartments. The cytosolic environment, which is an interface between intracellular compartments, and the activity of the ion transporters localised in the different membranes influence one each other. Here we analyse the molecular mechanisms connecting and modulating the transport processes at both the plasma and the vacuolar membranes of guard cells.
Topics: Arabidopsis; Biological Transport; Cell Membrane; Ion Transport; Vacuoles
PubMed: 33007120
DOI: 10.1111/nph.16983 -
Autophagy Oct 2022Small 30-nm vesicles containing the integral membrane protein Atg9 provide the initial membrane source for autophagy in yeast. Atg23 is an Atg9 binding protein that is...
Small 30-nm vesicles containing the integral membrane protein Atg9 provide the initial membrane source for autophagy in yeast. Atg23 is an Atg9 binding protein that is required for Atg9 vesicle trafficking but whose exact function is unknown. In our recent paper, we explored the function of Atg23 using an approach combining cellular biology and biochemistry on purified protein. We determined that Atg23 is an elongated dimer spanning 320 Å in length. We also demonstrated that Atg23 is a membrane-binding and -tethering protein. Furthermore, we identified a series of amino acids residing in a putative coiled-coil region that when mutated prevent Atg23 dimer formation resulting in a stable Atg23 monomer. Last, we demonstrated that when monomeric Atg23 is expressed in yeast lacking Atg23, this leads to a loss of Atg23 puncta, a reduction in Atg9 puncta, a reduction in nonselective autophagy and a complete block in the cytoplasm-to-vacuole targeting (Cvt) pathway.
Topics: Amino Acids; Autophagy; Autophagy-Related Proteins; Membrane Proteins; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vacuoles
PubMed: 35867625
DOI: 10.1080/15548627.2022.2105107 -
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 -
International Journal of Molecular... Sep 2022Autophagy is a highly conserved self-degradation mechanism in eukaryotes. Excess or harmful intracellular content can be encapsulated by double-membrane autophagic... (Review)
Review
Autophagy is a highly conserved self-degradation mechanism in eukaryotes. Excess or harmful intracellular content can be encapsulated by double-membrane autophagic vacuoles and transferred to vacuoles for degradation in plants. Current research shows three types of autophagy in plants, with macroautophagy being the most important autophagic degradation pathway. Until now, more than 40 autophagy-related (ATG) proteins have been identified in plants that are involved in macroautophagy, and these proteins play an important role in plant growth regulation and stress responses. In this review, we mainly introduce the research progress of autophagy in plant vegetative growth (roots and leaves), reproductive growth (pollen), and resistance to biotic (viruses, bacteria, and fungi) and abiotic stresses (nutrients, drought, salt, cold, and heat stress), and we discuss the application direction of plant autophagy in the future.
Topics: Autophagy; Gene Expression Regulation, Plant; Plants; Seeds; Stress, Physiological; Vacuoles
PubMed: 36232711
DOI: 10.3390/ijms231911410 -
Cell Reports Aug 2023The nucleolus is the most prominent membraneless organelle within the nucleus. How the nucleolar structure is regulated is poorly understood. Here, we identified two...
The nucleolus is the most prominent membraneless organelle within the nucleus. How the nucleolar structure is regulated is poorly understood. Here, we identified two types of nucleoli in C. elegans. Type I nucleoli are spherical and do not have visible nucleolar vacuoles (NoVs), and rRNA transcription and processing factors are evenly distributed throughout the nucleolus. Type II nucleoli contain vacuoles, and rRNA transcription and processing factors exclusively accumulate in the periphery rim. The NoV contains nucleoplasmic proteins and is capable of exchanging contents with the nucleoplasm. The high-order structure of the nucleolus is dynamically regulated in C. elegans. Faithful rRNA processing is important to prohibit NoVs. The depletion of 27SA rRNA processing factors resulted in NoV formation. The inhibition of RNA polymerase I (RNAPI) transcription and depletion of two conserved nucleolar factors, nucleolin and fibrillarin, prohibits the formation of NoVs. This finding provides a mechanism to coordinate structure maintenance and gene expression.
Topics: Animals; Caenorhabditis elegans; Nuclear Proteins; Vacuoles; Cell Nucleolus; Cell Nucleus; RNA, Ribosomal
PubMed: 37537842
DOI: 10.1016/j.celrep.2023.112915 -
PloS One 2022Vacuoles in plants and fungi play critical roles in cell metabolism and osmoregulation. To support these functions, vacuoles change their morphology, e.g. they fragment...
Vacuoles in plants and fungi play critical roles in cell metabolism and osmoregulation. To support these functions, vacuoles change their morphology, e.g. they fragment when these organisms are challenged with draught, high salinity or metabolic stress (e.g. acetate accumulation). In turn, morphology reflects an equilibrium between membrane fusion and fission that determines size, shape and copy number. By studying Saccharomyces cerevisiae and its vacuole as models, conserved molecular mechanisms responsible for fusion have been revealed. However, a detailed understanding of vacuole fission and how these opposing processes respond to metabolism or osmoregulation remain elusive. Herein we describe a new fluorometric assay to measure yeast vacuole fission in vitro. For proof-of-concept, we use this assay to confirm that acetate, a metabolic stressor, triggers vacuole fission and show it blocks homotypic vacuole fusion in vitro. Similarly, hypertonic stress induced by sorbitol or glucose caused robust vacuole fission in vitro whilst inhibiting fusion. Using wortmannin to inhibit phosphatidylinositol (PI) -kinases or rGyp1-46 to inactivate Rab-GTPases, we show that acetate stress likely targets PI signaling, whereas osmotic stress affects Rab signaling on vacuole membranes to stimulate fission. This study sets the stage for further investigation into the mechanisms that change vacuole morphology to support cell metabolism and osmoregulation.
Topics: Acetates; Membrane Fusion; Osmotic Pressure; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vacuoles
PubMed: 35834522
DOI: 10.1371/journal.pone.0271199