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The Journal of Biological Chemistry May 2022The human mitochondrial outer membrane is biophysically unique as it is the only membrane possessing transmembrane β-barrel proteins (mitochondrial outer membrane... (Review)
Review
The human mitochondrial outer membrane is biophysically unique as it is the only membrane possessing transmembrane β-barrel proteins (mitochondrial outer membrane proteins, mOMPs) in the cell. The most vital of the three mOMPs is the core protein of the translocase of the outer mitochondrial membrane (TOM) complex. Identified first as MOM38 in Neurospora in 1990, the structure of Tom40, the core 19-stranded β-barrel translocation channel, was solved in 2017, after nearly three decades. Remarkably, the past four years have witnessed an exponential increase in structural and functional studies of yeast and human TOM complexes. In addition to being conserved across all eukaryotes, the TOM complex is the sole ATP-independent import machinery for nearly all of the ∼1000 to 1500 known mitochondrial proteins. Recent cryo-EM structures have provided detailed insight into both possible assembly mechanisms of the TOM core complex and organizational dynamics of the import machinery and now reveal novel regulatory interplay with other mOMPs. Functional characterization of the TOM complex using biochemical and structural approaches has also revealed mechanisms for substrate recognition and at least five defined import pathways for precursor proteins. In this review, we discuss the discovery, recently solved structures, molecular function, and regulation of the TOM complex and its constituents, along with the implications these advances have for alleviating human diseases.
Topics: Humans; Membrane Proteins; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35346689
DOI: 10.1016/j.jbc.2022.101870 -
The Plant Cell Aug 2022Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. In these semi-autonomous organelles, the chloroplast genome encodes...
Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. In these semi-autonomous organelles, the chloroplast genome encodes approximately 100 proteins. The remaining chloroplast proteins, close to 3,000, are encoded by nuclear genes whose products are translated in the cytosol and imported into chloroplasts. However, there is still no consensus on the composition of the protein import machinery including its motor proteins and on how newly imported chloroplast proteins are refolded. In this study, we have examined the function of orf2971, the largest chloroplast gene of Chlamydomonas reinhardtii. The depletion of Orf2971 causes the accumulation of protein precursors, partial proteolysis and aggregation of proteins, increased expression of chaperones and proteases, and autophagy. Orf2971 interacts with the TIC (translocon at the inner chloroplast envelope) complex, catalyzes ATP (adenosine triphosphate) hydrolysis, and associates with chaperones and chaperonins. We propose that Orf2971 is intimately connected to the protein import machinery and plays an important role in chloroplast protein quality control.
Topics: Cell Nucleus; Chloroplast Proteins; Chloroplasts; Molecular Chaperones; Plant Proteins; Protein Transport
PubMed: 35708659
DOI: 10.1093/plcell/koac180 -
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 -
Biological Chemistry May 2020The proteome of the mitochondrial intermembrane space (IMS) contains more than 100 proteins, all of which are synthesized on cytosolic ribosomes and consequently need to... (Review)
Review
The proteome of the mitochondrial intermembrane space (IMS) contains more than 100 proteins, all of which are synthesized on cytosolic ribosomes and consequently need to be imported by dedicated machineries. The mitochondrial disulfide relay is the major import machinery for soluble proteins in the IMS. Its major component, the oxidoreductase MIA40, interacts with incoming substrates, retains them in the IMS, and oxidatively folds them. After this reaction, MIA40 is reoxidized by the sulfhydryl oxidase augmenter of liver regeneration, which couples disulfide formation by this machinery to the activity of the respiratory chain. In this review, we will discuss the import of IMS proteins with a focus on recent findings showing the diversity of disulfide relay substrates, describing the cytosolic control of this import system and highlighting the physiological relevance of the disulfide relay machinery in higher eukaryotes.
Topics: Disulfides; Eukaryotic Cells; Humans; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Models, Molecular
PubMed: 32142475
DOI: 10.1515/hsz-2020-0108 -
Journal of Bacteriology Apr 2021Mycobacteria possess Mce transporters that import lipids and are thought to function analogously to ATP-binding cassette (ABC) transporters. However, whereas ABC...
Mycobacteria possess Mce transporters that import lipids and are thought to function analogously to ATP-binding cassette (ABC) transporters. However, whereas ABC transporters import substrates using a single solute-binding protein (SBP) to deliver a substrate to permease proteins in the membrane, mycobacterial Mce transporters have a potential for six SBPs (MceA to MceF) working with a pair of permeases (YrbEA and YrbEB), a cytoplasmic ATPase (MceG), and multiple Mce-associated membrane (Mam) and orphaned Mam (Omam) proteins to transport lipids. In this study, we used the model mycobacterium to study the requirement for individual Mce, Mam, and Omam proteins in Mce4 transport of cholesterol. All of the Mce4 and Mam4 proteins we investigated were required for cholesterol uptake. However, not all Omam proteins, which are encoded by genes outside loci, proved to contribute to cholesterol import. OmamA and OmamB were required for cholesterol import, while OmamC, OmamD, OmamE, and OmamF were not. In the absence of any single Mce4, Mam4, or Omam protein that we tested, the abundance of Mce4A and Mce4E declined. This relationship between the levels of Mce4A and Mce4E and these additional proteins suggests a network of interactions that assemble and/or stabilize a multiprotein Mce4 transporter complex. Further support for Mce transporters being multiprotein complexes was obtained by immunoprecipitation-mass spectrometry, in which we identified every single Mce, YrbE, MceG, Mam, and Omam protein with a role in cholesterol transport as associating with Mce4A. This study represents the first time any of these Mce4 transporter proteins has been shown to associate. How lipids travel between membranes of diderm bacteria is a challenging mechanistic question because lipids, which are hydrophobic molecules, must traverse a hydrophilic periplasm. This question is even more complex for mycobacteria, which have a unique cell envelope that is highly impermeable to molecules. A growing body of knowledge identifies Mce transporters as lipid importers for mycobacteria. Here, using protein stability experiments and immunoprecipitation-mass spectrometry, we provide evidence for mycobacterial Mce transporters existing as multiprotein complexes.
Topics: Adenosine Triphosphatases; Bacterial Proteins; Biological Transport; Cholesterol; Membrane Transport Proteins; Multiprotein Complexes; Mycobacterium smegmatis; Operon
PubMed: 33649150
DOI: 10.1128/JB.00685-20 -
Canadian Journal of Microbiology Jan 2016Retroviruses must integrate their cDNA into the host genome to generate proviruses. Viral DNA-protein complexes interact with cellular proteins and produce... (Review)
Review
Retroviruses must integrate their cDNA into the host genome to generate proviruses. Viral DNA-protein complexes interact with cellular proteins and produce pre-integration complexes, which carry the viral genome and cross the nuclear pore channel to enter the nucleus and integrate viral DNA into host chromosomal DNA. If the reverse transcripts fail to integrate, linear or circular DNA species such as 1- and 2-long terminal repeats are generated. Such complexes encounter numerous cellular proteins in the cytoplasm, which restrict viral infection and protect the nucleus. To overcome host cell defenses, the pathogens have evolved several evasion strategies. Viral proteins often contain nuclear localization signals, allowing entry into the nucleus. Among more than 1000 proteins identified as required for HIV infection by RNA interference screening, karyopherins, cleavage and polyadenylation specific factor 6, and nucleoporins have been predominantly studied. This review discusses current opinions about the synergistic relationship between the viral and cellular factors involved in nuclear import, with focus on the unveiled mysteries of the host-pathogen interaction, and highlights novel approaches to pinpoint therapeutic targets.
Topics: Active Transport, Cell Nucleus; Adaptor Proteins, Signal Transducing; Animals; Cell Nucleus; Gene Products, gag; Host-Pathogen Interactions; Humans; Nuclear Pore Complex Proteins; Retroviridae; Transcription Factors; Viral Proteins; Virus Internalization; beta Karyopherins; mRNA Cleavage and Polyadenylation Factors
PubMed: 26553381
DOI: 10.1139/cjm-2015-0350 -
The Journal of Biological Chemistry 2021Fused in sarcoma (FUS) is a predominantly nuclear RNA-binding protein with key functions in RNA processing and DNA damage repair. Defects in nuclear import of FUS have...
Fused in sarcoma (FUS) is a predominantly nuclear RNA-binding protein with key functions in RNA processing and DNA damage repair. Defects in nuclear import of FUS have been linked to severe neurodegenerative diseases; hence, it is of great interest to understand this process and how it is dysregulated in disease. Transportin-1 (TNPO1) and the closely related transportin-2 have been identified as major nuclear import receptors of FUS. They bind to the C-terminal nuclear localization signal of FUS and mediate the protein's nuclear import and at the same time also suppress aberrant phase transitions of FUS in the cytoplasm. Whether FUS can utilize other nuclear transport receptors for the purpose of import and chaperoning has not been examined so far. Here, we show that FUS directly binds to different import receptors in vitro. FUS formed stable complexes not only with TNPO1 but also with transportin-3, importin β, importin 7, or the importin β/7 heterodimer. Binding of these alternative import receptors required arginine residues within FUS-RG/RGG motifs and was weakened by arginine methylation. Interaction with these importins suppressed FUS phase separation and reduced its sequestration into stress granules. In a permeabilized cell system, we further showed that transportin-3 had the capacity to import FUS into the nucleus, albeit with lower efficiency than TNPO1. Our data suggest that aggregation-prone RNA-binding proteins such as FUS may utilize a network of importins for chaperoning and import, similar to histones and ribosomal proteins.
Topics: Cell Nucleus; HeLa Cells; Humans; Karyopherins; Molecular Chaperones; Nuclear Localization Signals; Protein Binding; RNA-Binding Protein FUS; Receptors, Cytoplasmic and Nuclear; beta Karyopherins
PubMed: 33857479
DOI: 10.1016/j.jbc.2021.100659 -
Trends in Immunology Jun 2023Upon activation by double-stranded DNA (dsDNA), the cytosolic dsDNA sensor cyclic GMP-AMP synthase (cGAS) synthesizes the diffusible cyclic dinucleotide 2'3'-cGAMP... (Review)
Review
Upon activation by double-stranded DNA (dsDNA), the cytosolic dsDNA sensor cyclic GMP-AMP synthase (cGAS) synthesizes the diffusible cyclic dinucleotide 2'3'-cGAMP (cyclic GMP-AMP), which subsequently binds to the adaptor STING, triggering a cascade of events leading to an inflammatory response. Recent studies have highlighted the role of 2'3'-cGAMP as an 'immunotransmitter' between cells, a process facilitated by gap junctions as well as by specialized membrane-spanning importer and exporter channels. This review highlights recent advances from a structural perspective of intercellular trafficking of 2'3'-cGAMP, with particular emphasis on the binding of importer SLC19A1 to 2'3'-cGAMP, as well as the significance of associated folate nutrients and antifolate therapeutics. This provides a path forward for structure-guided understanding of the transport cycle in immunology, as well as for candidate targeting approaches towards therapeutic intervention in inflammation.
Topics: Humans; Inflammation; Membrane Proteins; Nucleotides, Cyclic; Nucleotidyltransferases
PubMed: 37147228
DOI: 10.1016/j.it.2023.04.006 -
Biological Chemistry Feb 2023Peroxisomal integrity and function are highly dependent on its membrane and soluble (matrix) components. Matrix enzymes are imported post-translationally in a folded or... (Review)
Review
Peroxisomal integrity and function are highly dependent on its membrane and soluble (matrix) components. Matrix enzymes are imported post-translationally in a folded or even oligomeric state, via a still mysterious protein translocation mechanism. They are guided to peroxisomes via the Peroxisomal Targeting Signal (PTS) sequences which are recognized by specific cytosolic receptors, Pex5, Pex7 and Pex9. Subsequently, cargo-loaded receptors bind to the docking complex in an initial step, followed by channel formation, cargo-release, receptor-recycling and -quality control. The docking complexes of different species share Pex14 as their core component but differ in composition and oligomeric state of Pex14. Here we review and highlight the latest insights on the structure and function of the peroxisomal docking complex. We summarize differences between yeast and mammals and then we integrate this knowledge into our current understanding of the import machinery.
Topics: Animals; Membrane Proteins; Peroxisomes; Protein Transport; Carrier Proteins; Saccharomyces cerevisiae; Mammals
PubMed: 36117327
DOI: 10.1515/hsz-2022-0161