Integrated cyto-physiological and proteomic analyses reveal new insight into CMS mechanism in a novel upland cotton CMS line LD6A

Cytoplasmic male sterile (CMS) system has extensively been exploited for hybrid vigor in plant breeding programs. However, its application in many crops is limited due to poor understanding of molecular mechanism of fertility restoration. Using advanced analytical approaches, we elucidated molecular pathways regulating CMS induction and fertility restoration in cotton. Reproductive structures of a novel CMS (LD6A) and its maintainer (LD6B) line were analyzed for physiological and proteomic changes during the development process. Significant differential expression of proteins, such as Abrin, malate dehydrogenase, malic enzyme, isocitrate dehydrogenase, histone acetyltransferase was observed in CMS and its maintainer line. Transmission electron micrographs of anther tapetum showed that inner ridge of CMS mitochondria was relatively indistinct than that of LD6B with narrower membranous space at tetrad stage. Further, relatively higher reactive oxygen species were accumulated in the anther of CMS than its maintainer line at pollen mother cell and tetrad stage. We suggest that abnormal sequence of mitochondrial ribosome gene rps4 and rpl10 and high expression of ribosome-inactivating protein gene Abrin in CMS line damaged mitochondrial membrane and consequently induced pollen sterility. These data provide new insight into CMS mechanism in cotton crops and a tool to develop new CMS germplasm resources.

Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors

Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes. To identify ribosome-associated biogenesis factors for the OXPHOS system, we purified ribosomes and associated proteins from mitochondria. We identified TMEM223 as a ribosome-associated protein involved in complex IV biogenesis. TMEM223 stimulates the translation of COX1 mRNA and is a constituent of early COX1 assembly intermediates. Moreover, we show that SMIM4 together with C12ORF73 interacts with newly synthesized cytochrome b to support initial steps of complex III biogenesis in complex with UQCC1 and UQCC2. Our analyses define the interactome of the human mitochondrial ribosome and reveal novel assembly factors for complex III and IV biogenesis that link early assembly stages to the translation machinery.

Nucleolar targeting in an early-branching eukaryote suggests a general physicochemical mechanism for ribosome protein sorting

The eukaryotic cell targets proteins to the organelles in which they function, both membrane-bound (like the nucleus) and non-membrane-bound (like the nucleolus). Nucleolar targeting relies on positively charged localisation signals, and has received rejuvenated interest since the widespread recognition of liquid-liquid phase separation (LLPS) as a mechanism contributing to nucleolus formation. Here, we exploit a new genome-wide analysis of protein localisation in an early-branching eukaryote, Trypanosoma brucei, to analyse general nucleolar protein properties. T. brucei nucleolar proteins have similar properties to those in common model eukaryotes, specifically basic amino acids. Using protein truncations and addition of candidate targeting sequences to proteins, we show both homopolymer runs and distributed basic amino acids give nucleolar partition, further aided by a nuclear localisation signal (NLS). These findings are consistent with phase separation models of nucleolar formation and protein physical properties being a major contributing mechanism for eukaryotic nucleolar targeting, conserved from the last eukaryotic common ancestor. Importantly, cytoplasmic ribosome proteins in comparison to mitochondrial ribosome proteins followed the same pattern - pointing to adaptation of physicochemical properties to assist segregation.

Investigations of Intercellular Mitochondrial Transfer in Neural Cells by Applied Single Molecule Genotyping

<p>Over the past decade and a half, evidence for transfer of whole mitochondria between mammalian cells has emerged in the literature. The notion that mitochondria are restricted to the cell of origin has been overturned by this curious phenomenon, yet the physiological relevance of these transfer events remains unclear. This thesis investigates intercellular mitochondrial transfer in co-cultures of neural cells in vitro, to understand whether neural cells placed under stress demonstrate an enhanced rate of intercellular mitochondrial transfer. This would implicate the phenomenon as a cellular response to stress. Reliable techniques for quantitative study of intercellular mitochondrial transfer are limited so far in this field. To address this, a novel quantitative approach was developed to detect intercellular mitochondrial transfer, based on single molecule genotyping by target-primed rolling circle amplification. This enabled imaging of individual mitochondrial DNA molecules in situ, to detect those molecules which had moved between cells. Through this strategy, intercellular mitochondrial transfer was detected in new in vitro co-culture models. Primary murine pericytes derived from brain microvessels, were found to readily transfer mitochondria to a murine astrocyte cell line in vitro. Cisplatin, a DNA damaging agent; and chloramphenicol, a mitochondrial ribosome inhibitor, used to induce acute cellular injuries in the murine astrocyte cell line. These injuries were characterised and found to induce apoptosis, cause changes in growth characteristics, mitochondrial gene expression, and alter the metabolic phenotype of the cells. A derivative of the astrocyte cell line which completely lacks mitochondrial respiration, was found to model a chronic metabolic injury. As pericytes are prevalent throughout the brain, the pericyte/astrocyte co-culture model was selected to evaluate how the rate of intercellular mitochondrial transfer was altered, when the astrocytes were injured prior to co-culture. Through in situ single molecule genotyping and high throughput confocal microscopy, quantitative data was produced on how the rate of intercellular mitochondrial transfer was altered by injury in these models. The rate of intercellular mitochondrial transfer remained unaltered by chloramphenicol, however both cisplatin and the chronic metabolic injury model demonstrated reduced numbers of pericyte mitochondrial DNAs transferred into the injured astrocytes. These studies demonstrate successful application of a novel approach to study intercellular mitochondrial transfer and enable quantitative studies of this phenomenon.</p>

Investigations of Intercellular Mitochondrial Transfer in Neural Cells by Applied Single Molecule Genotyping

<p>Over the past decade and a half, evidence for transfer of whole mitochondria between mammalian cells has emerged in the literature. The notion that mitochondria are restricted to the cell of origin has been overturned by this curious phenomenon, yet the physiological relevance of these transfer events remains unclear. This thesis investigates intercellular mitochondrial transfer in co-cultures of neural cells in vitro, to understand whether neural cells placed under stress demonstrate an enhanced rate of intercellular mitochondrial transfer. This would implicate the phenomenon as a cellular response to stress. Reliable techniques for quantitative study of intercellular mitochondrial transfer are limited so far in this field. To address this, a novel quantitative approach was developed to detect intercellular mitochondrial transfer, based on single molecule genotyping by target-primed rolling circle amplification. This enabled imaging of individual mitochondrial DNA molecules in situ, to detect those molecules which had moved between cells. Through this strategy, intercellular mitochondrial transfer was detected in new in vitro co-culture models. Primary murine pericytes derived from brain microvessels, were found to readily transfer mitochondria to a murine astrocyte cell line in vitro. Cisplatin, a DNA damaging agent; and chloramphenicol, a mitochondrial ribosome inhibitor, used to induce acute cellular injuries in the murine astrocyte cell line. These injuries were characterised and found to induce apoptosis, cause changes in growth characteristics, mitochondrial gene expression, and alter the metabolic phenotype of the cells. A derivative of the astrocyte cell line which completely lacks mitochondrial respiration, was found to model a chronic metabolic injury. As pericytes are prevalent throughout the brain, the pericyte/astrocyte co-culture model was selected to evaluate how the rate of intercellular mitochondrial transfer was altered, when the astrocytes were injured prior to co-culture. Through in situ single molecule genotyping and high throughput confocal microscopy, quantitative data was produced on how the rate of intercellular mitochondrial transfer was altered by injury in these models. The rate of intercellular mitochondrial transfer remained unaltered by chloramphenicol, however both cisplatin and the chronic metabolic injury model demonstrated reduced numbers of pericyte mitochondrial DNAs transferred into the injured astrocytes. These studies demonstrate successful application of a novel approach to study intercellular mitochondrial transfer and enable quantitative studies of this phenomenon.</p>

Mitochondrial ribosomal stress in lung diseases

Mitochondria are involved in a variety of critical cellular functions, and their impairment drives cell injury. The mitochondrial ribosome (mitoribosome) is responsible for the protein synthesis of mitochondrial DNA encoded genes. These proteins are involved in oxidative phosphorylation, respiration, and ATP production required in the cell. Mitoribosome components originate from both mitochondrial and nuclear genomes. Their dysfunction can be caused by impaired mitochondrial protein synthesis or mitoribosome misassembly, leading to a decline in mitochondrial translation. This decrease can trigger mitochondrial ribosomal stress and contribute to pulmonary cell injury, death, and diseases. This review focuses on the contribution of the impaired mitoribosome structural components and function to respiratory disease pathophysiology. We present recent findings in the fields of lung cancer, chronic obstructive pulmonary disease, interstitial lung disease, and asthma. We also include reports on the mitoribosome dysfunction in pulmonary hypertension, high altitude pulmonary edema, bacterial and viral infections. Studies of the mitoribosome alterations in respiratory diseases can lead to novel therapeutic targets.

NME6 is a phosphotransfer-inactive, monomeric NME/NDPK family member and functions in complexes at the interface of mitochondrial inner membrane and matrix

Background NME6 is a member of the nucleoside diphosphate kinase (NDPK/NME/Nm23) family which has key roles in nucleotide homeostasis, signal transduction, membrane remodeling and metastasis suppression. The well-studied NME1-NME4 proteins are hexameric and catalyze, via a phospho-histidine intermediate, the transfer of the terminal phosphate from (d)NTPs to (d)NDPs (NDP kinase) or proteins (protein histidine kinase). For the NME6, a gene/protein that emerged early in eukaryotic evolution, only scarce and partially inconsistent data are available. Here we aim to clarify and extend our knowledge on the human NME6. Results We show that NME6 is mostly expressed as a 186 amino acid protein, but that a second albeit much less abundant isoform exists. The recombinant NME6 remains monomeric, and does not assemble into homo-oligomers or hetero-oligomers with NME1-NME4. Consequently, NME6 is unable to catalyze phosphotransfer: it does not generate the phospho-histidine intermediate, and no NDPK activity can be detected. In cells, we could resolve and extend existing contradictory reports by localizing NME6 within mitochondria, largely associated with the mitochondrial inner membrane and matrix space. Overexpressing NME6 reduces ADP-stimulated mitochondrial respiration and complex III abundance, thus linking NME6 to dysfunctional oxidative phosphorylation. However, it did not alter mitochondrial membrane potential, mass, or network characteristics. Our screen for NME6 protein partners revealed its association with NME4 and OPA1, but a direct interaction was observed only with RCC1L, a protein involved in mitochondrial ribosome assembly and mitochondrial translation, and identified as essential for oxidative phosphorylation. Conclusions NME6, RCC1L and mitoribosomes localize together at the inner membrane/matrix space where NME6, in concert with RCC1L, may be involved in regulation of the mitochondrial translation of essential oxidative phosphorylation subunits. Our findings suggest new functions for NME6, independent of the classical phosphotransfer activity associated with NME proteins.

Visualizing formation of the active site in the mitochondrial ribosome

Ribosome assembly is an essential and conserved process that is regulated at each step by specific factors. Using cryo-electron microscopy (cryo-EM), we visualize the formation of the conserved peptidyl transferase center (PTC) of the human mitochondrial ribosome. The conserved GTPase GTPBP7 regulates the correct folding of 16S ribosomal RNA (rRNA) helices and ensures 2ʹ-O-methylation of the PTC base U3039. GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which sequester H68-71 of the 16S rRNA and allow biogenesis factors to access the maturing PTC. Mutations that disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activate mitochondrial stress and cause viability, development, and sterility defects. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4, but not its enzymatic activity, is indispensable for mitochondrial protein synthesis.