Delivery of nascent polypeptides to the mitochondrial surface

AbstractThousands of polypeptides with diverse biochemical properties, some of which are extremely hydrophobic, are targeted from cytoplasmic ribosomes to the surface of mitochondria. Localised synthesis, as well as transient interactions with a wide array of molecular chaperones and other cytoplasmic factors, can promote productive interaction of mitochondrial proteins with the TOM complex to initiate protein import into mitochondria

A comprehensive analysis of the Omp85/TpsB protein superfamily structural diversity, taxonomic occurrence, and evolution

Members of the Omp85/TpsB protein superfamily are ubiquitously distributed in Gram-negative bacteria, and function in protein translocation (e.g., FhaC) or the assembly of outer membrane proteins (e.g., BamA). Several recent findings are suggestive of a further level of variation in the superfamily, including the identification of the novel membrane protein assembly factor TamA and protein translocase PlpD. To investigate the diversity and the causal evolutionary events, we undertook a comprehensive comparative sequence analysis of the Omp85/TpsB proteins. A total of 10 protein subfamilies were apparent, distinguished in their domain structure and sequence signatures. In addition to the proteins FhaC, BamA, and TamA, for which structural and functional information is available, are families of proteins with so far undescribed domain architectures linked to the Omp85 β-barrel domain. This study brings a classification structure to a dynamic protein superfamily of high interest given its essential function for Gram-negative bacteria as well as its diverse domain architecture, and we discuss several scenarios of putative functions of these so far undescribed proteins

Determination of the molecular basis for coprogen import by Gram-negative bacteria

In order to survive in mixed microbial communities, some species of fungi secrete coprogens, siderophores that facilitate capture of the scarce nutrient iron. The TonB-dependent transporter FhuE is integrated in the outer membrane of Gram-negative bacteria and has been reported to scavenge these fungally produced coprogens. In this work, an Escherichia coli strain was engineered that is dependent solely on FhuE for its access to siderophore-sequestered iron. Using this tool, it is shown that while FhuE is highly active in the import of coprogens, it has some level of promiscuity, acting as a low-affinity transporter for related siderophores. The crystal structure of FhuE in complex with coprogen was determined, providing a structural basis to explain this selective promiscuity. The structural data, in combination with functional analysis, presented in this work show that FhuE has evolved to specifically engage with planar siderophores. A potential evolutionary driver, and a critical consequence of this selectivity, is that it allows FhuE to exclude antibiotics that mimic nonplanar hydroxamate siderophores: these toxic molecules could otherwise cross the outer membrane barrier through a Trojan horse mechanism

The structure of a conserved domain of TamB reveals a hydrophobic β taco fold

The translocation and assembly module (TAM) plays a role in the transport and insertion of proteins into the bacterial outer membrane. TamB, a component of this system spans the periplasmic space to engage with its partner protein TamA. Despite efforts to characterize the TAM, the structure and mechanism of action of TamB remained enigmatic. Here we present the crystal structure of TamB amino acids 963-1,138. This region represents half of the conserved DUF490 domain, the defining feature of TamB. TamB963-1138 consists of a concave, taco-shaped β sheet with a hydrophobic interior. This β taco structure is of dimensions capable of accommodating and shielding the hydrophobic side of an amphipathic β strand, potentially allowing TamB to chaperone nascent membrane proteins from the aqueous environment. In addition, sequence analysis suggests that the structure of TamB963-1138 is shared by a large portion of TamB. This architecture could allow TamB to act as a conduit for membrane proteins

Constraints on lateral gene transfer in promoting fimbrial usher protein diversity and function.

Fimbriae are long, adhesive structures widespread throughout members of the family Enterobacteriaceae. They are multimeric extrusions, which are moved out of the bacterial cell through an integral outer membrane protein called usher. The complex folding mechanics of the usher protein were recently revealed to be catalysed by the membrane-embedded translocation and assembly module (TAM). Here, we examine the diversity of usher proteins across a wide range of extraintestinal (ExPEC) and enteropathogenic (EPEC) Escherichia coli, and further focus on a so far undescribed chaperone-usher system, with this usher referred to as UshC. The fimbrial system containing UshC is distributed across a discrete set of EPEC types, including model strains like E2348/67, as well as ExPEC ST131, currently the most prominent multi-drug-resistant uropathogenic E. coli strain worldwide. Deletion of the TAM from a naive strain of E. coli results in a drastic time delay in folding of UshC, which can be observed for a protein from EPEC as well as for two introduced proteins from related organisms, Yersinia and Enterobacter We suggest that this models why the TAM machinery is essential for efficient folding of proteins acquired via lateral gene transfer

The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane

Systems Biology: The Next Frontier for Bioinformatics

Biochemical systems biology augments more traditional disciplines, such as genomics, biochemistry and molecular biology, by championing (i) mathematical and computational modeling; (ii) the application of traditional engineering practices in the analysis of biochemical systems; and in the past decade increasingly (iii) the use of near-comprehensive data sets derived from ‘omics platform technologies, in particular “downstream” technologies relative to genome sequencing, including transcriptomics, proteomics and metabolomics. The future progress in understanding biological principles will increasingly depend on the development of temporal and spatial analytical techniques that will provide high-resolution data for systems analyses. To date, particularly successful were strategies involving (a) quantitative measurements of cellular components at the mRNA, protein and metabolite levels, as well as in vivo metabolic reaction rates, (b) development of mathematical models that integrate biochemical knowledge with the information generated by high-throughput experiments, and (c) applications to microbial organisms. The inevitable role bioinformatics plays in modern systems biology puts mathematical and computational sciences as an equal partner to analytical and experimental biology. Furthermore, mathematical and computational models are expected to become increasingly prevalent representations of our knowledge about specific biochemical systems

Targeting of tail-anchored proteins to yeast mitochondria in vivo

AbstractTail-anchored proteins are inserted into intracellular membranes via a C-terminal transmembrane domain. The topology of the protein is such that insertion must occur post-translationally, since the insertion sequence is not available for membrane insertion until after translation of the tail-anchored polypeptide is completed. Here, we show that the targeting information in one such tail-anchored protein, translocase in the outer mitochondrial membrane 22, is contained in a short region flanking the transmembrane domain. An equivalent region is sufficient to specify the localisation of Bcl2 and SNARE proteins to the secretory membranes. We discuss the targeting process for directing members of this protein family to the secretory and mitochondrial membranes in vivo