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Cloud computing has drastically changed the delivery and consumption of live streaming content. The designs, challenges, and possible uses of cloud computing for live streaming are studied. A comprehensive overview of the technical and business issues surrounding cloud‐based live streaming is provided, including the benefits of cloud computing, the various live streaming architectures, and the challenges that live streaming service providers face in delivering high‐quality, real‐time services. The different techniques used to improve the performance of video streaming, such as adaptive bit‐rate streaming, multicast distribution, and edge computing are discussed and the necessity of low‐latency and high‐quality video transmission in cloud‐based live streaming is underlined. Issues such as improving user experience and live streaming service performance using cutting‐edge technology, like artificial intelligence and machine learning are discussed. In addition, the legal and regulatory implications of cloud‐based live streaming, including issues with network neutrality, data privacy, and content moderation are addressed. The future of cloud computing for live streaming is examined in the section that follows, and it looks at the most likely new developments in terms of trends and technology. For technology vendors, live streaming service providers, and regulators, the findings have major policy‐relevant implications. Suggestions on how stakeholders should address these concerns and take advantage of the potential presented by this rapidly evolving sector, as well as insights into the key challenges and opportunities associated with cloud‐based live streaming are provided. [ABSTRACT FROM AUTHOR]

In contrast to most bacteria, the mycobacterial F1FO‐ATP synthase (α3:β3:γ:δ:ε:a:b:b':c9) does not perform ATP hydrolysis‐driven proton translocation. Although subunits α, γ and ε of the catalytic F1‐ATPase component α3:β3:γ:ε have all been implicated in the suppression of the enzyme's ATPase activity, the mechanism remains poorly defined. Here, we brought the central stalk subunit ε into focus by generating the recombinant Mycobacterium smegmatis F1‐ATPase (MsF1‐ATPase), whose 3D low‐resolution structure is presented, and its ε‐free form MsF1αβγ, which showed an eightfold ATP hydrolysis increase and provided a defined system to systematically study the segments of mycobacterial ε's suppression of ATPase activity. Deletion of four amino acids at ε's N terminus, mutant MsF1αβγεΔ2‐5, revealed similar ATP hydrolysis as MsF1αβγ. Together with biochemical and NMR solution studies of a single, double, triple and quadruple N‐terminal ε‐mutants, the importance of the first N‐terminal residues of mycobacterial ε in structure stability and latency is described. Engineering ε's C‐terminal mutant MsF1αβγεΔ121 and MsF1αβγεΔ103‐121 with deletion of the C‐terminal residue D121 and the two C‐terminal ɑ‐helices, respectively, revealed the requirement of the very C terminus for communication with the catalytic α3β3‐headpiece and its function in ATP hydrolysis inhibition. Finally, we applied the tools developed during the study for an in silico screen to identify a novel subunit ε‐targeting F‐ATP synthase inhibitor. [ABSTRACT FROM AUTHOR]

The Na+translocating F1FO ATP synthase from Acetobacterium woodii shows a subunit stoichiometry of α3:β3:γ:δ:ε:a:b2:(c2/3)9:c1 and reveals an evolutionary path between synthases and pumps involving adaptations in the rotor c‐ring, which is composed of F‐ and vacuolar‐type c subunits in a stoichiometry of 9 : 1. This hybrid turbine couples rotation with Na+ translocation in the FO part and rotation of the central stalk subunits γ‐ε to drive ATP synthesis in the catalytic α3:β3 headpiece. Here, we isolated a highly pure recombinant A. woodii F‐ATP synthase and present the first projected structure of this hybrid engine as determined by negative‐stain electron microscopy and single‐particle analysis. The uniqueness of the A. woodii F‐ATP synthase is also reflected by an extra 17 amino acid residues loop (195TSGKVKITEETKEEKSK211) in subunit γ. Deleting the loop‐encoding DNA sequence (γΔ195–211) and purifying the recombinant F‐ATP synthase γΔ195–211 mutant provided a platform to study its effect in enzyme stability and activity. The recombinant F‐ATP synthase γΔ195–211 mutant revealed the same subunit composition as the wild‐type enzyme and a minor reduction in ATP hydrolysis. When reconstituted into proteoliposomes ATP synthesis and Na+ transport were diminished, demonstrating the importance of the γ195–211 loop in both enzymatic processes. Based on a structural model, a coupling mechanism for this enzyme is proposed, highlighting the role of the γ‐loop. Finally, the γ195–211 loop of A. woodii is discussed in comparison with the extra γ‐loops of mycobacterial and chloroplasts F‐ATP synthases described to be involved in species‐specific regulatory mechanisms. [ABSTRACT FROM AUTHOR]

Abstract: We have analysed 385 mitochondrial and 567 chloroplastic signal sequences of proteins found in the organellar proteomes of Arabidopsis thaliana. Despite overall similarities, the first 16 residues of transit peptides differ remarkably. To test the hypothesis that the N-terminally truncated transit peptides would redirect chloroplastic precursor proteins to mitochondria, we studied import of the N-terminal deletion mutants of ELIP, PetC and Lhcb2.1. The results show that the deletion mutants were neither imported into chloroplasts nor miss-targeted to mitochondria in vitro and in vivo, showing that the entire transit peptide is necessary for correct targeting as well as miss-sorting. [Copyright &y& Elsevier]

Presequence protease PreP is a novel protease that degrades targeting peptides as well as other unstructured peptides in both mitochondria and chloroplasts. The first structure of PreP from Arabidopsis thaliana refined at 2.1 Å resolution shows how the 995-residue polypeptide forms a unique proteolytic chamber of more than 10 000 Å3 in which the active site resides. Although there is no visible opening to the chamber, a peptide is bound to the active site. The closed conformation places previously unidentified residues from the C-terminal domain at the active site, separated by almost 800 residues in sequence to active site residues located in the N-terminal domain. Based on the structure, a novel mechanism for proteolysis is proposed involving hinge-bending motions that cause the protease to open and close in response to substrate binding. In support of this model, cysteine double mutants designed to keep the chamber covalently locked show no activity under oxidizing conditions. The manner in which substrates are processed inside the chamber is reminiscent of the proteasome; therefore, we refer to this protein as a peptidasome. [ABSTRACT FROM AUTHOR]

We have recently isolated and identified a novel mitochondrial metalloprotease, pre-sequence protease (PreP) from potato and shown that it degrades mitochondrial pre-sequences. PreP belongs to the pitrilysin protease family and contains an inverted zinc-binding motif. To further investigate the degradation of targeting peptides, we have overexpressed the Arabidopsis thaliana homologue of PreP, zinc metalloprotease (Zn-MP), in Escherichia coli. We have characterized the recombinant Zn-MP with respect to its catalytic site, substrate specificity and intracellular localization. Mutagenesis studies of the residues involved in metal binding identified the histidines and the proximal glutamate as essential residues for the proteolytic activity. Substrate specificity studies showed that the Zn-MP has the ability to degrade both mitochondrial pre-sequences and chloroplastic transit peptides, as well as other unstructured peptides. The Zn-MP does not recognize an amino acid sequence per se. Immunological studies and proteolytic activity measurements in isolated mitochondria and chloroplasts revealed the presence of the Zn-MP in both organelles. Furthermore, the Zn-MP was found to be dually imported to both mitochondria and chloroplasts in vitro. In summary, our data show that the Zn-MP is present and serves the same function in chloroplasts as in mitochondria – degradation of targeting peptides. [ABSTRACT FROM AUTHOR]

Here we show, using the green fluorescent protein (GFP) fusion system, that an Arabidopsis thaliana zinc-metalloprotease (AtZn- MP) is targeted to both mitochondria and chloroplasts. A deletion mutant lacking the amino-terminal 28 residues, with translation initiation at the second methionine residue, was imported into chloroplasts only. However, a mutated form of the full-length targeting peptide, in which the second methionine residue is changed to leucine, was imported to both organelles. No GFP fluorescence was detected when a frame-shift mutation was introduced between the first and second ATG codons of the Zn-MP-GFP construct, suggesting no alternative translational initiation. Our results show that the dual targeting of the Zn-MP is due to an ambiguous targeting peptide. Furthermore, we show that the recombinant AtZn-MP degrades mitochondrial and chloroplastic targeting peptides, indicating its function as a signal peptide degrading protease in both mitochondria and chloroplasts. [ABSTRACT FROM AUTHOR]