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The Chinese Journal of Dental Research Mar 2024Coordination and information exchange among the various organelles ensure the precise and orderly functioning of eukaryotic cells. Interaction between the cytoplasm and... (Review)
Review
Coordination and information exchange among the various organelles ensure the precise and orderly functioning of eukaryotic cells. Interaction between the cytoplasm and nucleoplasm is crucial for many physiological processes. Macromolecular protein transport into the nucleus requires assistance from the nuclear transport system. These proteins typically contain a nuclear localisation sequence that guides them to enter the nucleus. Understanding the mechanism of nuclear import of macromolecular proteins is important for comprehending cellular processes. Investigation of disease-related alterations can facilitate the development of novel therapeutic strategies and provide additional evidence for clinical trials. This review provides an overview of the proteins involved in nuclear transport and the mechanisms underlying macromolecular protein transport.
Topics: Active Transport, Cell Nucleus; Protein Transport; Cell Nucleus; Cytoplasm; Eukaryotic Cells
PubMed: 38546518
DOI: 10.3290/j.cjdr.b5136729 -
Proceedings of the National Academy of... Aug 2023Certain transmembrane and membrane-tethered signaling proteins export from cilia as BBSome cargoes via the outward BBSome transition zone (TZ) diffusion pathway,...
Certain transmembrane and membrane-tethered signaling proteins export from cilia as BBSome cargoes via the outward BBSome transition zone (TZ) diffusion pathway, indispensable for maintaining their ciliary dynamics to enable cells to sense and transduce extracellular stimuli inside the cell. Murine Rab-like 2 (Rabl2) GTPase resembles Arf-like 3 (ARL3) GTPase in promoting outward TZ passage of the signaling protein cargo-laden BBSome. During this process, ARL3 binds to and recruits the retrograde IFT train-dissociated BBSome as its effector to diffuse through the TZ for ciliary retrieval, while how RABL2 and ARL3 cross talk in this event remains uncertain. Here, we report that RABL2 in a GTP-bound form (RABL2) cycles through cilia via IFT as an IFT-B1 cargo, dissociates from retrograde IFT trains at a ciliary region right above the TZ, and converts to RABL2 for activating ARL3 as an ARL3 guanine nucleotide exchange factor. This confers ARL3 to detach from the ciliary membrane and become available for binding and recruiting the phospholipase D (PLD)-laden BBSome, autonomous of retrograde IFT association, to diffuse through the TZ for ciliary retrieval. Afterward, RABL2 exits cilia by being bound to the ARL3/BBSome entity as a BBSome cargo. Our data identify ciliary signaling proteins exported from cilia via the RABL2-ARL3 cascade-mediated outward BBSome TZ diffusion pathway. According to this model, hedgehog signaling defect-induced Bardet-Biedl syndrome caused by mutations in humans could be well explained in a mutation-specific manner, providing us with a mechanistic understanding behind the outward BBSome TZ passage required for proper ciliary signaling.
Topics: Humans; ADP-Ribosylation Factors; Cilia; GTP Phosphohydrolases; Guanosine Triphosphate; Hedgehog Proteins; Membrane Proteins; Protein Transport; rab GTP-Binding Proteins; Chlamydomonas
PubMed: 37579161
DOI: 10.1073/pnas.2302603120 -
Journal of Chemical Information and... Aug 2023NarK nitrate/nitrite antiporter imports nitrate (a mineral form of the essential element nitrogen) into the cell and exports nitrite (a metabolite that can be toxic in...
NarK nitrate/nitrite antiporter imports nitrate (a mineral form of the essential element nitrogen) into the cell and exports nitrite (a metabolite that can be toxic in high concentrations) out of the cell. However, many details about its operational mechanism remain poorly understood. In this work, we performed steered molecular dynamics simulations of anion translocations and quantum-chemistry model calculations of the binding sites to study the wild-type NarK protein and its R89K mutant. Our results shed light on the importance of the two strictly conserved binding-site arginine residues (R89 and R305) and two glycine-rich signature motifs (G164-M176 and G408-F419) in anion movement through the pore. We also observe conformational changes of the protein during anion migration. For the R89K mutant, our quantum calculations reveal a competition for a proton between the anion (especially nitrite) and lysine, which can potentially slow down or even trap the anion in the pore. Our findings provide a possible explanation for the striking experimental finding that the arginine-to-lysine mutation, despite preserving the charge, impedes or abolishes anion transport in such mutants of NarK and other similar nitrate/nitrite exchangers.
Topics: Nitrites; Nitrates; Anion Transport Proteins; Models, Molecular; Protein Structure, Tertiary; Binding Sites; Cell Membrane; Mutation
PubMed: 37585651
DOI: 10.1021/acs.jcim.3c00295 -
Molecular Biology of the Cell Mar 2024DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and...
DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and experimental protein extracts to native chromatography and quantifying the contents of each fraction using mass spectrometry, it enables the quantitative detection of alterations to protein complexes and abundances. Here, we applied DIFFRAC to investigate the consequences of genetic loss of Ift122, a subunit of the intraflagellar transport-A (IFT-A) protein complex that plays a vital role in the formation and function of cilia and flagella, on the proteome of . A single DIFFRAC experiment was sufficient to detect changes in protein behavior that mirrored known effects of IFT-A loss and revealed new biology. We uncovered several novel IFT-A-regulated proteins, which we validated through live imaging in multiciliated cells, shedding new light on both the ciliary and non-ciliary functions of IFT-A. Our findings underscore the robustness of DIFFRAC for revealing proteomic changes in response to genetic or biochemical perturbation.
Topics: Protein Transport; Proteome; Proteomics; Biological Transport; Cilia; Flagella; Phenotype
PubMed: 38170584
DOI: 10.1091/mbc.E23-03-0084 -
Journal of the American Chemical Society Dec 2023The aberrant localization of proteins in cells is a key factor in the development of various diseases, including cancer and neurodegenerative disease. To better...
The aberrant localization of proteins in cells is a key factor in the development of various diseases, including cancer and neurodegenerative disease. To better understand and potentially manipulate protein localization for therapeutic purposes, we engineered bifunctional compounds that bind to proteins in separate cellular compartments. We show these compounds induce nuclear import of cytosolic cargoes, using nuclear-localized BRD4 as a "carrier" for co-import and nuclear trapping of cytosolic proteins. We use this system to calculate kinetic constants for passive diffusion across the nuclear pore and demonstrate single-cell heterogeneity in response to these bifunctional molecules with cells requiring high carrier to cargo expression for complete import. We also observe incorporation of cargo into BRD4-containing condensates. Proteins shown to be substrates for nuclear transport include oncogenic mutant nucleophosmin (NPM1c) and mutant PI3K catalytic subunit alpha (PIK3CA), suggesting potential applications to cancer treatment. In addition, we demonstrate that chemically induced localization of BRD4 to cytosolic-localized DNA-binding proteins, namely, IRF1 with a nuclear export signal, induces target gene expression. These results suggest that induced localization of proteins with bifunctional molecules enables the rewiring of cell circuitry, with significant implications for disease therapy.
Topics: Humans; Nuclear Proteins; Cell Nucleus; Neurodegenerative Diseases; Transcription Factors; Active Transport, Cell Nucleus; Cell Cycle Proteins
PubMed: 37992275
DOI: 10.1021/jacs.3c06179 -
The Journal of Cell Biology Jul 2023Trafficking of cell-surface proteins from endosomes to the plasma membrane is a key mechanism to regulate synaptic function. In non-neuronal cells, proteins recycle to...
Trafficking of cell-surface proteins from endosomes to the plasma membrane is a key mechanism to regulate synaptic function. In non-neuronal cells, proteins recycle to the plasma membrane either via the SNX27-Retromer-WASH pathway or via the recently discovered SNX17-Retriever-CCC-WASH pathway. While SNX27 is responsible for the recycling of key neuronal receptors, the roles of SNX17 in neurons are less understood. Here, using cultured hippocampal neurons, we demonstrate that the SNX17 pathway regulates synaptic function and plasticity. Disruption of this pathway results in a loss of excitatory synapses and prevents structural plasticity during chemical long-term potentiation (cLTP). cLTP drives SNX17 recruitment to synapses, where its roles are in part mediated by regulating the surface expression of β1-integrin. SNX17 recruitment relies on NMDAR activation, CaMKII signaling, and requires binding to the Retriever and PI(3)P. Together, these findings provide molecular insights into the regulation of SNX17 at synapses and define key roles for SNX17 in synaptic maintenance and in regulating enduring forms of synaptic plasticity.
Topics: Cell Membrane; Long-Term Potentiation; Membrane Proteins; Neuronal Plasticity; Protein Transport; Synapses; Sorting Nexins; Cells, Cultured; Neurons
PubMed: 37141105
DOI: 10.1083/jcb.202207025 -
Journal of Cell Science Sep 2023Chloroplasts conduct photosynthesis and numerous metabolic and signalling processes that enable plant growth and development. Most of the ∼3000 proteins in...
Chloroplasts conduct photosynthesis and numerous metabolic and signalling processes that enable plant growth and development. Most of the ∼3000 proteins in chloroplasts are nucleus encoded and must be imported from the cytosol. Thus, the protein import machinery of the organelle (the TOC-TIC apparatus) is of fundamental importance for chloroplast biogenesis and operation. Cytosolic factors target chloroplast precursor proteins to the TOC-TIC apparatus, which drives protein import across the envelope membranes into the organelle, before various internal systems mediate downstream routing to different suborganellar compartments. The protein import system is proteolytically regulated by the ubiquitin-proteasome system (UPS), enabling centralized control over the organellar proteome. In addition, the UPS targets a range of chloroplast proteins directly. In this Cell Science at a Glance article and the accompanying poster, we present mechanistic details of these different chloroplast protein targeting and translocation events, and of the UPS systems that regulate chloroplast proteins.
Topics: Ubiquitin; Chloroplasts; Photosynthesis; Proteasome Endopeptidase Complex; Chloroplast Proteins; Protein Transport
PubMed: 37732520
DOI: 10.1242/jcs.241125 -
Nature Sep 2023The presequence translocase of the mitochondrial inner membrane (TIM23) represents the major route for the import of nuclear-encoded proteins into mitochondria. About...
The presequence translocase of the mitochondrial inner membrane (TIM23) represents the major route for the import of nuclear-encoded proteins into mitochondria. About 60% of more than 1,000 different mitochondrial proteins are synthesized with amino-terminal targeting signals, termed presequences, which form positively charged amphiphilic α-helices. TIM23 sorts the presequence proteins into the inner membrane or matrix. Various views, including regulatory and coupling functions, have been reported on the essential TIM23 subunit Tim17 (refs. ). Here we mapped the interaction of Tim17 with matrix-targeted and inner membrane-sorted preproteins during translocation in the native membrane environment. We show that Tim17 contains conserved negative charges close to the intermembrane space side of the bilayer, which are essential to initiate presequence protein translocation along a distinct transmembrane cavity of Tim17 for both classes of preproteins. The amphiphilic character of mitochondrial presequences directly matches this Tim17-dependent translocation mechanism. This mechanism permits direct lateral release of transmembrane segments of inner membrane-sorted precursors into the inner membrane.
Topics: Mitochondria; Mitochondrial Membranes; Mitochondrial Precursor Protein Import Complex Proteins; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37527780
DOI: 10.1038/s41586-023-06477-8 -
Applied Microbiology and Biotechnology Dec 2024Application of filamentous fungi for the production of commercial enzymes such as amylase, cellulase, or xylanase is on the rise due to the increasing demand to degrade... (Review)
Review
Application of filamentous fungi for the production of commercial enzymes such as amylase, cellulase, or xylanase is on the rise due to the increasing demand to degrade several complex carbohydrates as raw material for biotechnological processes. Also, protein production by fungi for food and feed gains importance. In any case, the protein production involves both cellular synthesis and secretion outside of the cell. Unfortunately, the secretion of proteins or enzymes can be hampered due to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) as a result of too high synthesis of enzymes or (heterologous) protein expression. To cope with this ER stress, the cell generates a response known as unfolded protein response (UPR). Even though this mechanism should re-establish the protein homeostasis equivalent to a cell under non-stress conditions, the enzyme expression might still suffer from repression under secretory stress (RESS). Among eukaryotes, Saccharomyces cerevisiae is the only fungus, which is studied quite extensively to unravel the UPR pathway. Several homologs of the proteins involved in this signal transduction cascade are also found in filamentous fungi. Since RESS seems to be absent in S. cerevisiae and was only reported in Trichoderma reesei in the presence of folding and glycosylation inhibitors such as dithiothreitol and tunicamycin, more in-depth study about this mechanism, specifically in filamentous fungi, is the need of the hour. Hence, this review article gives an overview on both, protein secretion and associated stress responses in fungi. KEY POINTS: • Enzymes produced by filamentous fungi are crucial in industrial processes • UPR mechanism is conserved among many fungi, but mediated by different proteins • RESS is not fully understood or studied in industrially relevant filamentous fungi.
Topics: Saccharomyces cerevisiae; Protein Transport; Fungi; Biological Transport; Proteostasis
PubMed: 38204136
DOI: 10.1007/s00253-023-12985-4 -
Developmental Cell Apr 2024Control of protein stoichiometry is essential for cell function. Mitochondrial oxidative phosphorylation (OXPHOS) presents a complex stoichiometric challenge as the...
Control of protein stoichiometry is essential for cell function. Mitochondrial oxidative phosphorylation (OXPHOS) presents a complex stoichiometric challenge as the ratio of the electron transport chain (ETC) and ATP synthase must be tightly controlled, and assembly requires coordinated integration of proteins encoded in the nuclear and mitochondrial genome. How correct OXPHOS stoichiometry is achieved is unknown. We identify the Mitochondrial Regulatory hub for respiratory Assembly (MiRA) platform, which synchronizes ETC and ATP synthase biogenesis in yeast. Molecularly, this is achieved by a stop-and-go mechanism: the uncharacterized protein Mra1 stalls complex IV assembly. Two "Go" signals are required for assembly progression: binding of the complex IV assembly factor Rcf2 and Mra1 interaction with an Atp9-translating mitoribosome induce Mra1 degradation, allowing synchronized maturation of complex IV and the ATP synthase. Failure of the stop-and-go mechanism results in cell death. MiRA controls OXPHOS assembly, ensuring correct stoichiometry of protein machineries encoded by two different genomes.
Topics: Oxidative Phosphorylation; Saccharomyces cerevisiae; Mitochondria; Saccharomyces cerevisiae Proteins; Mitochondrial Proton-Translocating ATPases; Electron Transport Complex IV; Mitochondrial Proteins
PubMed: 38508182
DOI: 10.1016/j.devcel.2024.02.011