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Current Opinion in Structural Biology Jun 2022The majority of mitochondrial proteins are nuclear-encoded and need to be transported into the mitochondria, including the proteins in the outer mitochondrial membrane.... (Review)
Review
The majority of mitochondrial proteins are nuclear-encoded and need to be transported into the mitochondria, including the proteins in the outer mitochondrial membrane. For β-barrel proteins, the preproteins are initially recognized and imported by the TOM complex, then shuttled to the SAM complex via small Tim proteins. For ⍺-helical proteins, some preproteins are recognized by the TOM complex and imported into the membrane by the MIM complex. In recent years multiple structures of the TOM complex and the SAM complex have been reported, increasing our understanding of the mechanism of protein biogenesis in the outer mitochondrial membrane.
Topics: Mitochondria; Mitochondrial Membranes; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Protein Transport; Saccharomyces cerevisiae Proteins
PubMed: 35504104
DOI: 10.1016/j.sbi.2022.102383 -
Molecular Biology of the Cell Jan 2024What drives nuclear growth? Studying nuclei assembled in egg extract and focusing on importin α/β-mediated nuclear import, we show that, while import is required for...
What drives nuclear growth? Studying nuclei assembled in egg extract and focusing on importin α/β-mediated nuclear import, we show that, while import is required for nuclear growth, nuclear growth and import can be uncoupled when chromatin structure is manipulated. Nuclei treated with micrococcal nuclease to fragment DNA grew slowly despite exhibiting little to no change in import rates. Nuclei assembled around axolotl chromatin with 20-fold more DNA than grew larger but imported more slowly. Treating nuclei with reagents known to alter histone methylation or acetylation caused nuclei to grow less while still importing to a similar extent or to grow larger without significantly increasing import. Nuclear growth but not import was increased in live sea urchin embryos treated with the DNA methylator N-nitrosodimethylamine. These data suggest that nuclear import is not the primary driving force for nuclear growth. Instead, we observed that nuclear blebs expanded preferentially at sites of high chromatin density and lamin addition, whereas small Benzonase-treated nuclei lacking DNA exhibited reduced lamin incorporation into the nuclear envelope. In summary, we report experimental conditions where nuclear import is not sufficient to drive nuclear growth, hypothesizing that this uncoupling is a result of altered chromatin structure.
Topics: Animals; Cell Nucleus; Nuclear Envelope; Chromatin; DNA; Xenopus laevis; Lamins
PubMed: 37903226
DOI: 10.1091/mbc.E23-04-0138 -
Cell Metabolism Oct 2013Most mitochondrial proteins are imported by the translocase of the outer mitochondrial membrane (TOM). Tom22 functions as central receptor and transfers preproteins to...
Most mitochondrial proteins are imported by the translocase of the outer mitochondrial membrane (TOM). Tom22 functions as central receptor and transfers preproteins to the import pore. Casein kinase 2 (CK2) constitutively phosphorylates the cytosolic precursor of Tom22 at Ser44 and Ser46 and, thus, promotes its import. It is unknown whether Tom22 is regulated under different metabolic conditions. We report that CK1, which is involved in glucose-induced signal transduction, is bound to mitochondria. CK1 phosphorylates Tom22 at Thr57 and stimulates the assembly of Tom22 and Tom20. In contrast, protein kinase A (PKA), which is also activated by the addition of glucose, phosphorylates the precursor of Tom22 at Thr76 and impairs its import. Thus, PKA functions in an opposite manner to CK1 and CK2. Our results reveal that three kinases regulate the import and assembly of Tom22, demonstrating that the central receptor is a major target for the posttranslational regulation of mitochondrial protein import.
Topics: Casein Kinase I; Casein Kinase II; Cyclic AMP-Dependent Protein Kinases; Glucose; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Phosphorylation; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction
PubMed: 24093680
DOI: 10.1016/j.cmet.2013.09.006 -
Open Biology Nov 2015Peroxisomes are capable of importing folded and oligomeric proteins. However, it is a matter of dispute whether oligomer import by peroxisomes is the exception or the... (Review)
Review
Peroxisomes are capable of importing folded and oligomeric proteins. However, it is a matter of dispute whether oligomer import by peroxisomes is the exception or the rule. Here, I argue for a clear distinction between homo-oligomeric proteins that are essentially peroxisomal, and dually localized hetero-oligomers that access the peroxisome by piggyback import, localizing there in limited number, whereas the majority remain in the cytosol. Homo-oligomeric proteins comprise the majority of all peroxisomal matrix proteins. There is evidence that binding by Pex5 in the cytosol can regulate their oligomerization state before import. The hetero-oligomer group is made up of superoxide dismutase and lactate dehydrogenase. These proteins have evolved mechanisms that render import inefficient and retain the majority of proteins in the cytosol.
Topics: Amino Acid Sequence; Animals; Humans; Molecular Sequence Data; Peroxisome-Targeting Signal 1 Receptor; Peroxisomes; Protein Multimerization; Protein Transport; Receptors, Cytoplasmic and Nuclear
PubMed: 26581572
DOI: 10.1098/rsob.150148 -
BMC Systems Biology Feb 2019Iron plays crucial roles in the metabolism of eukaryotic cells. Much iron is trafficked into mitochondria where it is used for iron-sulfur cluster assembly and heme...
BACKGROUND
Iron plays crucial roles in the metabolism of eukaryotic cells. Much iron is trafficked into mitochondria where it is used for iron-sulfur cluster assembly and heme biosynthesis. A yeast strain in which Mrs3/4, the high-affinity iron importers on the mitochondrial inner membrane, are deleted exhibits a slow-growth phenotype when grown under iron-deficient conditions. However, these cells grow at WT rates under iron-sufficient conditions. The object of this study was to develop a mathematical model that could explain this recovery on the molecular level.
RESULTS
A multi-tiered strategy was used to solve an ordinary-differential-equations-based mathematical model of iron import, trafficking, and regulation in growing Saccharomyces cerevisiae cells. At the simplest level of modeling, all iron in the cell was presumed to be a single species and the cell was considered to be a single homogeneous volume. Optimized parameters associated with the rate of iron import and the rate of dilution due to cell growth were determined. At the next level of complexity, the cell was divided into three regions, including cytosol, mitochondria, and vacuoles, each of which was presumed to contain a single form of iron. Optimized parameters associated with import into these regions were determined. At the final level of complexity, nine components were assumed within the same three cellular regions. Parameters obtained at simpler levels of complexity were used to help solve the more complex versions of the model; this was advantageous because the data used for solving the simpler model variants were more reliable and complete relative to those required for the more complex variants. The optimized full-complexity model simulated the observed phenotype of WT and Mrs3/4ΔΔ cells with acceptable fidelity, and the model exhibited some predictive power.
CONCLUSIONS
The developed model highlights the importance of an Fe mitochondrial pool and the necessary exclusion of O in the mitochondrial matrix for eukaryotic iron-sulfur cluster metabolism. Similar multi-tiered strategies could be used for any micronutrient in which concentrations and metabolic forms have been determined in different organelles within a growing eukaryotic cell.
Topics: Biological Transport; Cation Transport Proteins; Iron; Kinetics; Mitochondria; Mitochondrial Proteins; Models, Biological; Mutation; Oxygen; Phenotype; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 30791941
DOI: 10.1186/s12918-019-0702-2 -
Biochimica Et Biophysica Acta. Gene... Apr 2018Although tRNAs participate in the essential function of protein translation in the cytoplasm, tRNA transcription and numerous processing steps occur in the nucleus. This... (Review)
Review
Although tRNAs participate in the essential function of protein translation in the cytoplasm, tRNA transcription and numerous processing steps occur in the nucleus. This subcellular separation between tRNA biogenesis and function requires that tRNAs be efficiently delivered to the cytoplasm in a step termed "primary tRNA nuclear export". Surprisingly, tRNA nuclear-cytoplasmic traffic is not unidirectional, but, rather, movement is bidirectional. Cytoplasmic tRNAs are imported back to the nucleus by the "tRNA retrograde nuclear import" step which is conserved from budding yeast to vertebrate cells and has been hijacked by viruses, such as HIV, for nuclear import of the viral reverse transcription complex in human cells. Under appropriate environmental conditions cytoplasmic tRNAs that have been imported into the nucleus return to the cytoplasm via the 3rd nuclear-cytoplasmic shuttling step termed "tRNA nuclear re-export", that again is conserved from budding yeast to vertebrate cells. We describe the 3 steps of tRNA nuclear-cytoplasmic movements and their regulation. There are multiple tRNA nuclear export and import pathways. The different tRNA nuclear exporters appear to possess substrate specificity leading to the tantalizing possibility that the cellular proteome may be regulated at the level of tRNA nuclear export. Moreover, in some organisms, such as budding yeast, the pre-tRNA splicing heterotetrameric endonuclease (SEN), which removes introns from pre-tRNAs, resides on the cytoplasmic surface of the mitochondria. Therefore, we also describe the localization of the SEN complex to mitochondria and splicing of pre-tRNA on mitochondria, which occurs prior to the participation of tRNAs in protein translation. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.
Topics: Animals; Biological Transport; Cell Nucleus; Cytoplasm; Endoribonucleases; Evolution, Molecular; Fungal Proteins; HSP70 Heat-Shock Proteins; Mitochondrial Membranes; Nuclear Pore Complex Proteins; Nucleocytoplasmic Transport Proteins; Plant Proteins; RNA Precursors; RNA Processing, Post-Transcriptional; RNA, Transfer; RNA-Binding Proteins; Saccharomyces cerevisiae Proteins; Transcription, Genetic; Vertebrates; Yeasts
PubMed: 29191733
DOI: 10.1016/j.bbagrm.2017.11.007 -
Biochimica Et Biophysica Acta Jan 2012The recognition of the conserved ATP-binding domains of Pex1p, p97 and NSF led to the discovery of the family of AAA-type ATPases. The biogenesis of peroxisomes... (Review)
Review
The recognition of the conserved ATP-binding domains of Pex1p, p97 and NSF led to the discovery of the family of AAA-type ATPases. The biogenesis of peroxisomes critically depends on the function of two AAA-type ATPases, namely Pex1p and Pex6p, which provide the energy for import of peroxisomal matrix proteins. Peroxisomal matrix proteins are synthesized on free ribosomes in the cytosol and guided to the peroxisomal membrane by specific soluble receptors. At the membrane, the cargo-loaded receptors bind to a docking complex and the receptor-docking complex assembly is thought to form a dynamic pore which enables the transition of the cargo into the organellar lumen. The import cycle is completed by ubiquitination- and ATP-dependent dislocation of the receptor from the membrane to the cytosol, which is performed by the AAA-peroxins. Receptor ubiquitination and dislocation are the only energy-dependent steps in peroxisomal protein import. The export-driven import model suggests that the AAA-peroxins might function as motor proteins in peroxisomal import by coupling ATP-dependent removal of the peroxisomal import receptor and cargo translocation into the organelle.
Topics: ATPases Associated with Diverse Cellular Activities; Adenosine Triphosphatases; Cell Cycle Proteins; Endoplasmic Reticulum-Associated Degradation; Membrane Proteins; Peroxisomes; Protein Multimerization; Protein Structure, Tertiary; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Valosin Containing Protein
PubMed: 21963882
DOI: 10.1016/j.bbamcr.2011.09.005 -
Proceedings of the National Academy of... Jul 1996We report the molecular cloning of import intermediate associated protein (IAP) 100, a 100-kDa protein of the chloroplast protein import machinery of peas. IAP100...
We report the molecular cloning of import intermediate associated protein (IAP) 100, a 100-kDa protein of the chloroplast protein import machinery of peas. IAP100 contains two potential alpha-helical transmembrane segments and also behaves like an integral membrane protein. It was localized to the inner chloroplast envelope membrane. Immunoprecipitation experiments using monospecific anti-IAP100 antibodies and a nonionic detergent-generated chloroplast lysate gave the following results. (i) The four integral membrane proteins of the outer chloroplast import machinery were not coprecipitated with IAP100 indicating that the inner and outer membrane import machineries are not coupled in isolated chloroplasts. (ii) the major protein that coprecipitated with IAP100 was identified as stromal chaperonin 60 (cpn60); the association of IAP100 and cpn60 was specific and was abolished when immunoprecipitation was carried out in the presence of ATP. (iii) In a lysate from chloroplasts that had been preincubated for various lengths of time in an import reaction with radiolabeled precursor (pS) of the small subunit of Rubisco, we detected coimmunoprecipitation of IAP100, cpn60, and the imported mature form (S) of precursor. Relative to the time course of import, coprecipitation of S first increased and then decreased, consistent with a transient association of the newly imported S with the chaperonin bound to IAP100. These data suggest that IAP100 serves in recruiting chaperonin for folding of newly imported proteins.
Topics: Amino Acid Sequence; Autoradiography; Base Sequence; Chaperonin 60; Chloroplasts; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Kinetics; Membrane Proteins; Molecular Sequence Data; Oligodeoxyribonucleotides; Pisum sativum; Protein Binding; Protein Folding; Protein Processing, Post-Translational; Recombinant Proteins; Sulfur Radioisotopes
PubMed: 8755536
DOI: 10.1073/pnas.93.15.7684 -
Molecular and Cellular Biology Oct 2009The PTS1-dependent peroxisomal matrix protein import is facilitated by the receptor protein Pex5 and can be divided into cargo recognition in the cytosol, membrane...
The PTS1-dependent peroxisomal matrix protein import is facilitated by the receptor protein Pex5 and can be divided into cargo recognition in the cytosol, membrane docking of the cargo-receptor complex, cargo release, and recycling of the receptor. The final step is controlled by the ubiquitination status of Pex5. While polyubiquitinated Pex5 is degraded by the proteasome, monoubiquitinated Pex5 is destined for a new round of the receptor cycle. Recently, the ubiquitin-conjugating enzymes involved in Pex5 ubiquitination were identified as Ubc4 and Pex4 (Ubc10), whereas the identity of the corresponding protein-ubiquitin ligases remained unknown. Here we report on the identification of the protein-ubiquitin ligases that are responsible for the ubiquitination of the peroxisomal protein import receptor Pex5. It is demonstrated that each of the three RING peroxins Pex2, Pex10, and Pex12 exhibits ubiquitin-protein isopeptide ligase activity. Our results show that Pex2 mediates the Ubc4-dependent polyubiquitination whereas Pex12 facilitates the Pex4-dependent monoubiquitination of Pex5.
Topics: Membrane Proteins; Membrane Transport Proteins; Peroxins; Peroxisome-Targeting Signal 1 Receptor; Peroxisomes; Protein Transport; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 19687296
DOI: 10.1128/MCB.00388-09 -
Cell Aug 2009Most mitochondrial proteins are synthesized on cytosolic ribosomes and must be imported across one or both mitochondrial membranes. There is an amazingly versatile set... (Review)
Review
Most mitochondrial proteins are synthesized on cytosolic ribosomes and must be imported across one or both mitochondrial membranes. There is an amazingly versatile set of machineries and mechanisms, and at least four different pathways, for the importing and sorting of mitochondrial precursor proteins. The translocases that catalyze these processes are highly dynamic machines driven by the membrane potential, ATP, or redox reactions, and they cooperate with molecular chaperones and assembly complexes to direct mitochondrial proteins to their correct destinations. Here, we discuss recent insights into the importing and sorting of mitochondrial proteins and their contributions to mitochondrial biogenesis.
Topics: Adenosine Triphosphate; Animals; Humans; Mitochondria; Mitochondrial Proteins; Protein Sorting Signals; Protein Transport
PubMed: 19703392
DOI: 10.1016/j.cell.2009.08.005