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Journal of Experimental Botany Feb 2020The evolution of chloroplasts from the original endosymbiont involved the transfer of thousands of genes from the ancestral bacterial genome to the host nucleus, thereby...
The evolution of chloroplasts from the original endosymbiont involved the transfer of thousands of genes from the ancestral bacterial genome to the host nucleus, thereby combining the two genetic systems to facilitate coordination of gene expression and achieve integration of host and organelle functions. A key element of successful endosymbiosis was the evolution of a unique protein import system to selectively and efficiently target nuclear-encoded proteins to their site of function within the chloroplast after synthesis in the cytoplasm. The chloroplast TOC-TIC (translocon at the outer chloroplast envelope-translocon at the inner chloroplast envelope) general protein import system is conserved across the plant kingdom, and is a system of hybrid origin, with core membrane transport components adapted from bacterial protein targeting systems, and additional components adapted from host genes to confer the specificity and directionality of import. In vascular plants, the TOC-TIC system has diversified to mediate the import of specific, functionally related classes of plastid proteins. This functional diversification occurred as the plastid family expanded to fulfill cell- and tissue-specific functions in terrestrial plants. In addition, there is growing evidence that direct regulation of TOC-TIC activities plays an essential role in the dynamic remodeling of the organelle proteome that is required to coordinate plastid biogenesis with developmental and physiological events.
Topics: Chloroplast Proteins; Chloroplasts; Plant Proteins; Plastids; Protein Transport
PubMed: 31730153
DOI: 10.1093/jxb/erz517 -
Nature Jul 2022Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health. Luminal peroxisomal proteins are imported from the...
Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12). Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.
Topics: Cryoelectron Microscopy; Cytosol; Humans; Membrane Proteins; Peroxins; Peroxisomal Biogenesis Factor 2; Peroxisomes; Polyubiquitin; Protein Transport; RING Finger Domains; Receptors, Cytoplasmic and Nuclear; Ubiquitin-Protein Ligase Complexes
PubMed: 35768507
DOI: 10.1038/s41586-022-04903-x -
ELife Sep 2021The chloroplast proteome contains thousands of different proteins that are encoded by the nuclear genome. These proteins are imported into the chloroplast via the action...
The chloroplast proteome contains thousands of different proteins that are encoded by the nuclear genome. These proteins are imported into the chloroplast via the action of the TOC translocase and associated downstream systems. Our recent work has revealed that the stability of the TOC complex is dynamically regulated by the ubiquitin-dependent chloroplast-associated protein degradation pathway. Here, we demonstrate that the TOC complex is also regulated by the small ubiquitin-like modifier (SUMO) system. mutants representing almost the entire SUMO conjugation pathway can partially suppress the phenotype of , a pale-yellow mutant lacking the Toc33 protein. This suppression is linked to increased abundance of TOC proteins and improvements in chloroplast development. Moreover, data from molecular and biochemical experiments support a model in which the SUMO system directly regulates TOC protein stability. Thus, we have identified a regulatory link between the SUMO system and the chloroplast protein import machinery.
Topics: Arabidopsis; Arabidopsis Proteins; Chloroplast Proteins; Chloroplasts; Membrane Proteins; Protein Transport; SUMO-1 Protein
PubMed: 34473053
DOI: 10.7554/eLife.60960 -
American Journal of Physiology.... Nov 2023The liver plays a crucial role in maintaining systemic iron homeostasis through iron storage, sensing of systemic iron needs, and production of the iron-regulatory...
The liver plays a crucial role in maintaining systemic iron homeostasis through iron storage, sensing of systemic iron needs, and production of the iron-regulatory hormone hepcidin. While mice are commonly used as models for studying human iron homeostasis, their liver structure differs significantly from humans. Since the mouse liver is structured in six separated lobes, often, the analysis of a single defined lobe is preferred due to concerns over data reproducibility between experimental cohorts. In this study, we compared iron-related parameters in distinct liver lobes of C57BL/6 wild-type mice across different ages. We found that the non-heme iron levels, as well as the mRNA and protein expression of iron storage protein Ferritin and the iron importer Transferrin Receptor 1, were similar between liver lobes. Additionally, the mRNA expression of , as well as its regulators, and , and iron importers and were comparable. Minor differences were observed in mRNA levels of 24-wk-old mice; however, this did not correlate with altered iron content. The findings in wild-type mice were reproduced in knock-out mice - a well-established genetic model of the most prevalent form of hemochromatosis. Overall, our results indicate that C57BL/6 mouse liver lobes can be used interchangeably for assessing iron content and expression of iron-related genes. Understanding if these findings are applicable to other mouse developmental stages, strains, or models of (iron-related) disorders will be key to promote reduction of experimental animal numbers and facilitate resource sharing among research groups studying liver iron homeostasis. This study reveals that, despite being structurally separated, liver lobes from C57BL/6 wild-type and iron-overloaded mice can be used interchangeably for the evaluation of iron content and expression of iron-related genes.
Topics: Mice; Humans; Animals; Hepcidins; Hemochromatosis Protein; Histocompatibility Antigens Class I; Reproducibility of Results; Mice, Inbred C57BL; Liver; Hemochromatosis; Iron; RNA, Messenger; Mice, Knockout; Homeostasis
PubMed: 37667844
DOI: 10.1152/ajpgi.00085.2023 -
Biochemical Society Transactions Oct 2019Mitochondria are essential eukaryotic organelles responsible for primary cellular energy production. Biogenesis, maintenance, and functions of mitochondria require... (Review)
Review
Mitochondria are essential eukaryotic organelles responsible for primary cellular energy production. Biogenesis, maintenance, and functions of mitochondria require correct assembly of resident proteins and lipids, which require their transport into and within mitochondria. Mitochondrial normal functions also require an exchange of small metabolites between the cytosol and mitochondria, which is primarily mediated by a metabolite channel of the outer membrane (OM) called porin or voltage-dependent anion channel. Here, we describe recently revealed novel roles of porin in the mitochondrial protein and lipid transport. First, porin regulates the formation of the mitochondrial protein import gate in the OM, the translocase of the outer membrane (TOM) complex, and its dynamic exchange between the major form of a trimer and the minor form of a dimer. The TOM complex dimer lacks a core subunit Tom22 and mediates the import of a subset of mitochondrial proteins while the TOM complex trimer facilitates the import of most other mitochondrial proteins. Second, porin interacts with both a translocating inner membrane (IM) protein like a carrier protein accumulated at the small TIM chaperones in the intermembrane space and the TIM22 complex, a downstream translocator in the IM for the carrier protein import. Porin thereby facilitates the efficient transfer of carrier proteins to the IM during their import. Third, porin facilitates the transfer of lipids between the OM and IM and promotes a back-up pathway for the cardiolipin synthesis in mitochondria. Thus, porin has roles more than the metabolite transport in the protein and lipid transport into and within mitochondria, which is likely conserved from yeast to human.
Topics: Biological Transport; Cell Cycle; Lipid Metabolism; Mitochondria; Mitochondrial Proteins; Porins
PubMed: 31670371
DOI: 10.1042/BST20190153 -
Biochemistry Nov 2021Iron is an essential nutrient for virtually every living organism, especially pathogenic prokaryotes. Despite its importance, however, both the acquisition and the... (Review)
Review
Iron is an essential nutrient for virtually every living organism, especially pathogenic prokaryotes. Despite its importance, however, both the acquisition and the export of this element require dedicated pathways that are dependent on oxidation state. Due to its solubility and kinetic lability, reduced ferrous iron (Fe) is useful to bacteria for import, chaperoning, and efflux. Once imported, ferrous iron may be loaded into apo and nascent enzymes and even sequestered into storage proteins under certain conditions. However, excess labile ferrous iron can impart toxicity as it may spuriously catalyze Fenton chemistry, thereby generating reactive oxygen species and leading to cellular damage. In response, it is becoming increasingly evident that bacteria have evolved Fe efflux pumps to deal with conditions of ferrous iron excess and to prevent intracellular oxidative stress. In this work, we highlight recent structural and mechanistic advancements in our understanding of prokaryotic ferrous iron import and export systems, with a focus on the connection of these essential transport systems to pathogenesis. Given the connection of these pathways to the virulence of many increasingly antibiotic resistant bacterial strains, a greater understanding of the mechanistic details of ferrous iron cycling in pathogens could illuminate new pathways for future therapeutic developments.
Topics: Anti-Bacterial Agents; Bacteria; Biological Transport; Catalysis; Homeostasis; Ion Transport; Iron; Kinetics; Membrane Proteins; Membrane Transport Proteins; Oxidation-Reduction; Oxidative Stress; Prokaryotic Cells; Reactive Oxygen Species; Solubility; Virulence
PubMed: 34670078
DOI: 10.1021/acs.biochem.1c00586 -
Current Genetics Jun 2020Mitochondrial dysregulation is a pivotal hallmark of aging-related disorders. Although there is a considerable understanding of the molecular counteracting responses... (Review)
Review
Mitochondrial dysregulation is a pivotal hallmark of aging-related disorders. Although there is a considerable understanding of the molecular counteracting responses toward damaged mitochondria, the molecular underpinnings connecting the abnormal aggregation of mitochondrial precursor protein fragments and abrogation of mitochondrial import machinery are far from clear. Recently, proteasomal-dependent degradation was unveiled as a pivotal fine-tuner of TOM machinery-dependent mitochondrial import. Herein, the role of proteasomal-mediated degradation in regulating fidelity of TOM-dependent import is briefly discussed and analyzed. The insights obtained from the characterization of this process may be applied to targeting mitochondrial import dysfunction in some neurodegenerative disorders.
Topics: Animals; Carrier Proteins; Humans; Membrane Transport Proteins; Mitochondria; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Proteolysis
PubMed: 32060627
DOI: 10.1007/s00294-020-01056-0 -
Brain, Behavior, and Immunity Feb 2024Neuroinflammation and microglial iron load are significant hallmarks found in several neurodegenerative diseases. In in vitro systems, microglia preferentially...
Neuroinflammation and microglial iron load are significant hallmarks found in several neurodegenerative diseases. In in vitro systems, microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and it has been shown that iron can augment cellular inflammation, suggesting a feed-forward loop between mechanisms involved in iron import and inflammatory signaling. However, it is not understood how microglial iron import mechanisms contribute to inflammation in vivo, or whether altering a microglial iron-related gene affects the inflammatory response. These studies aimed to determine the effect of knocking down microglial iron import gene Slc11a2 on the inflammatory response in vivo. We generated a novel model of tamoxifen-inducible, microglial-specific Slc11a2 knockdown using Cx3cr1 mice. Transgenic male and female mice were administered intraperitoneal saline or lipopolysaccharide (LPS) and assessed for sickness behavior post-injection. Plasma cytokines and microglial bulk RNA sequencing (RNASeq) analyses were performed at 4 h post-LPS, and microglia were collected for gene expression analysis after 24 h. A subset of mice was assessed in a behavioral test battery following LPS-induced sickness recovery. Control male, but not female, mice significantly upregulated microglial Slc11a2 at 4 and 24 h following LPS. In Slc11a2 knockdown mice, we observed an improvement in the acute behavioral sickness response post-LPS in male, but not female, animals. Microglia from male, but not female, knockdown animals exhibited a significant decrease in LPS-provoked pro-inflammatory cytokine expression after 24 h. RNASeq data from male knockdown microglia 4 h post-LPS revealed a robust downregulation in inflammatory genes including Il6, Tnfα, and Il1β, and an increase in anti-inflammatory and homeostatic markers (e.g., Tgfbr1, Cx3cr1, and Trem2). This corresponded with a profound decrease in plasma pro-inflammatory cytokines 4 h post-LPS. At 4 h, male knockdown microglia also upregulated expression of markers of iron export, iron recycling, and iron homeostasis and decreased iron storage and import genes, along with pro-oxidant markers such as Cybb, Nos2, and Hif1α. Overall, this work elucidates how manipulating a specific gene involved in iron import in microglia alters acute inflammatory signaling and overall cell activation state in male mice. These data highlight a sex-specific link between a microglial iron import gene and the pro-inflammatory response to LPS in vivo, providing further insight into the mechanisms driving neuroinflammatory disease.
Topics: Animals; Female; Male; Mice; Cytokines; Inflammation; Iron; Lipopolysaccharides; Membrane Glycoproteins; Mice, Inbred C57BL; Microglia; Receptors, Immunologic
PubMed: 38141840
DOI: 10.1016/j.bbi.2023.12.020 -
Biological Chemistry May 2020Mitochondrial precursor proteins with amino-terminal presequences are imported via the presequence pathway, utilizing the TIM23 complex for inner membrane translocation.... (Review)
Review
Mitochondrial precursor proteins with amino-terminal presequences are imported via the presequence pathway, utilizing the TIM23 complex for inner membrane translocation. Initially, the precursors pass the outer membrane through the TOM complex and are handed over to the TIM23 complex where they are sorted into the inner membrane or translocated into the matrix. This handover process depends on the receptor proteins at the inner membrane, Tim50 and Tim23, which are critical for efficient import. In this review, we summarize key findings that shaped the current concepts of protein translocation along the presequence import pathway, with a particular focus on the precursor handover process from TOM to the TIM23 complex. In addition, we discuss functions of the human TIM23 pathway and the recently uncovered pathogenic mutations in TIM50.
Topics: Carrier Proteins; Humans; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Precursor Protein Import Complex Proteins; Protein Transport
PubMed: 32074073
DOI: 10.1515/hsz-2020-0101 -
ELife Jun 2022Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM...
Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner membrane (TIM23-complex) imports proteins with cleavable pre-sequences. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how ∆ψ and ATP hydrolysis, the two main driving forces for import into the matrix, contribute to the transport of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the outer membrane and initiation of inner membrane transport by the pre-sequence - the rates of which are influenced by PCP size and net charge. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel - presumably rapid success or failure of import is critical for maintenance of mitochondrial fitness.
Topics: Carrier Proteins; Luciferases; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35674314
DOI: 10.7554/eLife.75426