Subcellular Localization of Catalase in Sea Urchin (Tetrapigus niger) Gametes: Implications for Peroxisome Biogenesis

Peroxisomes are essential subcellular organelles that appear to be derived from pre-existing organelles. To test the presence of peroxisomes in sea urchin (Tetrapigus niger) sperm and eggs, we performed biochemical and morphological experiments to evaluate the subcellular distribution of catalase as the typical peroxisomal marker. In sea urchin sperm, we found that catalase is localized in the cell cytosol. In contrast, sea urchin eggs contain sedimentable catalase, presumably contained in peroxisome-like structures detected by immunomicroscopy and by cytochemistry. Our results show, for the first time, evidence for the presence of peroxisome-like structures in sea urchin eggs and provide evidence for the peroxisome biogenesis hypothesis by division of pre-existing organelles.     

编辑机器:锥虫RNA编辑复合体(译文)

包括底物前体mRNA和指导RNA（gRNA)在内的核糖核蛋白复合体的装配和解装配是RNA编辑的起始和增殖的关键。本文讨论了这些复合体的组成,以及它们的装配是如何调控RNA编辑的。     

Evolutionary Conservation of the Ribosomal Biogenesis Factor Rbm19/Mrd1: Implications for Function

Ribosome biogenesis in eukaryotes requires coordinated folding and assembly of a pre-rRNA into sequential pre-rRNA-protein complexes in which chemical modifications and RNA cleavages occur. These processes require many small nucleolar RNAs (snoRNAs) and proteins. Rbm19/Mrd1 is one such protein that is built from multiple RNA-binding domains (RBDs). We find that Rbm19/Mrd1 with five RBDs is present in all branches of the eukaryotic phylogenetic tree, except in animals and Choanoflagellates, that instead have a version with six RBDs and Microsporidia which have a minimal Rbm19/Mrd1 protein with four RBDs. Rbm19/Mrd1 therefore evolved as a multi-RBD protein very early in eukaryotes. The linkers between the RBDs have conserved properties; they are disordered, except for linker 3, and position the RBDs at conserved relative distances from each other. All but one of the RBDs have conserved properties for RNA-binding and each RBD has a specific consensus sequence and a conserved position in the protein, suggesting a functionally important modular design. The patterns of evolutionary conservation provide information for experimental analyses of the function of Rbm19/Mrd1. In vivo mutational analysis confirmed that a highly conserved loop 5-β4-strand in RBD6 is essential for function.     

Biogenesis of the cytochrome bc(1) complex and role of assembly factors

The cytochrome bc(1) complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc(1) complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc(1) complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc(1) complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc(1) complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron-sulfur protein and its role in completing the assembly of functional bc(1) complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Trypanosome Letm1 Protein Is Essential for Mitochondrial Potassium Homeostasis

Letm1 is a conserved protein in eukaryotes bearing energized mitochondria. Hemizygous deletion of its gene has been implicated in symptoms of the human disease Wolf-Hirschhorn syndrome. Studies almost exclusively performed in opisthokonts have attributed several roles to Letm1, including maintaining mitochondrial morphology, mediating either calcium or potassium/proton antiport, and facilitating mitochondrial translation. We address the ancestral function of Letm1 in the highly diverged protist and significant pathogen, Trypanosoma brucei. We demonstrate that Letm1 is involved in maintaining mitochondrial volume via potassium/proton exchange across the inner membrane. This role is essential in the vector-dwelling procyclic and mammal-infecting bloodstream stages as well as in Trypanosoma brucei evansi, a form of the latter stage lacking an organellar genome. In the pathogenic bloodstream stage, the mitochondrion consumes ATP to maintain an energized state, whereas that of T. brucei evansi also lacks a conventional proton-driven membrane potential. Thus, Letm1 performs its function in different physiological states, suggesting that ion homeostasis is among the few characterized essential pathways of the mitochondrion at this T. brucei life stage. Interestingly, Letm1 depletion in the procyclic stage can be complemented by exogenous expression of its human counterpart, highlighting the conservation of protein function between highly divergent species. Furthermore, although mitochondrial translation is affected upon Letm1 ablation, it is an indirect consequence of K⁺ accumulation in the matrix.     

Biogenesis and Evolution of Photosynthetic (Thylakoid) Membranes

Work in molecular phylogeny during the past few years has documented that the biogenesis, maintenance, adaptation, and controlled resorption of thylakoid (photosynthetic) membranes are by far more complex than the requirements for maintaining their function, especially in plants (eukaryotic photoautotrophs). Plants, due to their genome compartmentation that originated in a cohabitation of cells (endosymbiotic events), have evolved an exquisite set of regulatory mechanisms for their energy-transducing organelles. These operate in concert with basically ancient regulatory circuits originating in the organelle ancestors. It appears that the biogenesis of thylakoid membranes, as that of chloroplasts in general, cannot be understood without knowledge of the history of the cells.     

Trypanosome mitochondrial 3' terminal uridylyl transferase (TUTase): the key enzyme in U-insertion/deletion RNA editing

A 3' terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei. The enzyme is specific for 3' U-addition in the presence of Mg(2+). TUTase is present in vivo in at least two stable configurations: one contains a approximately 500 kDa TUTase oligomer and the other a approximately 700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect parasite viability.     

Systemic RNA Interference Deficiency-1 (SID-1) Extracellular Domain Selectively Binds Long Double-stranded RNA and Is Required for RNA Transport by SID-1

During systemic RNA interference (RNAi) in Caenorhabditis elegans, RNA spreads across different cells and tissues in a process that requires the systemic RNA interference deficient-1 (sid-1) gene, which encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 cells enables passive uptake of dsRNA and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as an RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1-mediated RNA transport efficiency, providing evidence that dsRNA binding to SID-1 ECD is related to RNA transport. To examine the functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive of dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport.     

Ubiquitin Signaling Regulates RNA Biogenesis,Processing, and Metabolism

The fate of eukaryotic proteins, from their synthesis to destruction, is supervised by the ubiquitin–proteasome system (UPS). The UPS is the primary pathway responsible for selective proteolysis of intracellular proteins, which is guided by covalent attachment of ubiquitin to target proteins by E1 (activating), E2 (conjugating), and E3 (ligating) enzymes in a process known as ubiquitylation. The UPS can also regulate protein synthesis by influencing multiple steps of RNA (ribonucleic acid) metabolism. Here, recent publications concerning the interplay between the UPS and different types of RNA are reviewed. This interplay mainly involves specific RNA-binding E3 ligases that link RNA-dependent processes with protein ubiquitylation. The emerging understanding of their modes of RNA binding, their RNA targets, and their molecular and cellular functions are primarily focused on. It is discussed how the UPS adapted to interact with different types of RNA and how RNA molecules influence the ubiquitin signaling components.     

Experimental Evolution of a Trypanosome Parasite of Bumblebees and its Implications for Infection Success and Host Immune Response

Selection on basic growth properties of parasites may have many consequences for parasite traits, infection outcome, or host responses to infection. It is known that genotypes (strains) of the trypanosome parasite of bumblebees Crithidia bombi vary widely in their growth rates in their natural host, Bombus terrestris, as well as when cultured in medium. To test for changes in growth rates and their consequences, we here experimentally evolved six strains of C. bombi for fast and slow growth under controlled conditions in culture medium. Subsequently, we infected the evolved lines in live host and found that lines selected for slow growth attained higher infection intensity in the live bumblebee than those evolved for fast growth, whilst the immune response of the host was the same to both kinds of lines. These results fit the expectation that attenuation through rapid adaptation to a different environment, the culture medium, makes the parasite less successful in its next host. Selection for fast growth therefore does not necessarily lead to higher parasite success or more transmission. Hence, insect trypanosome pathogens can be attenuated by experimental evolution in the culture; this could inform important aspects of host-parasite evolution and perhaps vaccine development.     

Expression and Subcellular Localization of Arabidopsis thaliana Auxin-Binding Protein 1 (ABP1)

Expression of Arabidopsis thaliana ABP1 (AUXIN-BINDING PROTEIN 1) was studied using a promoter:GUS approach. Two promoter regions were analyzed. The 1585-bp promoter region upstream of the translation start site (P _ABP1 ) showed different activity compared to the promoter region that included, in addition, the first two introns and three exons of the transcribed ABP1 sequence (P _ABP1i1,2), indicating that cis elements were present downstream of the start codon. P _ABP1i1,2-driven β-glucuronidase activity was highest in growing leaves, in the root meristem, in vascular tissues, and in hydathodes. ABP1 promoter activities overlapped largely but not completely with that of DR5, which is a marker for the ARF-AuxRE-dependent auxin response. Subcellular ABP1 localization was studied using a constitutively overexpressed EGFP-ABP1 fusion protein. Results confirmed predominant localization to the endoplasmic reticulum as was concluded previously.     

Reprint of: Biogenesis of the cytochrome bc(1) complex and role of assembly factors

The cytochrome bc(1) complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc(1) complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc(1) complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc(1) complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc(1) complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron-sulfur protein and its role in completing the assembly of functional bc(1) complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.