Felicity Alcock

Minor modifications and major adaptations: The evolution of molecular machines driving mitochondrial protein import

Bacterial endosymbionts gave rise to mitochondria in a process that depended on the acquisition of protein import pathways. Modification and in some cases major re-tooling of the endosymbionts cellular machinery produced these pathways, establishing mitochondria as organelles common to all eukaryotic cells. The legacy of this evolutionary tinkering can be seen in the homologies and structural similarities between mitochondrial protein import machinery and modern day bacterial proteins. Comparative analysis of these systems is revealing both possible routes for the evolution of the mitochondrial membrane translocases and a greater understanding of the mechanisms behind mitochondrial protein import. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

Significance The twin-arginine translocation (Tat) pathway transports folded proteins across a membrane without significant ion leakage. The mechanism by which Tat is able to carry out this challenging feat is unclear. We used direct imaging of fluorescent protein-tagged Tat components in bacterial cells to show that the TatA element of the Tat system undergoes substrate- and proton motive force-dependent oligomerization. Thus the Tat transporter element is assembled on demand, avoiding the need to seal the transporter between translocation events. The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. It has been inferred that the Tat translocation site is assembled on demand by substrate-induced association of the protein TatA. We tested this model by imaging YFP-tagged TatA expressed at native levels in living Escherichia coli cells in the presence of low levels of the TatA paralogue TatE. Under these conditions the TatA-YFP fusion supports full physiological Tat transport activity. In agreement with the TatA association model, raising the number of transport-competent substrate proteins within the cell leads to an increase in the number of large TatA complexes present. Formation of these complexes requires both a functional TatBC substrate receptor and the transmembrane proton motive force (PMF). Removing the PMF causes TatA complexes to dissociate, except in strains with impaired Tat transport activity. Based on these observations we propose that TatA assembly reaches a critical point at which oligomerization can be reversed only by substrate transport. In contrast to TatA-YFP, the oligomeric states of TatB-YFP and TatC-YFP fusions are not affected by substrate or the PMF, although TatB-YFP oligomerization does require TatC.

Mammalian OS-9 is upregulated in response to endoplasmic reticulum stress and facilitates ubiquitination of misfolded glycoproteins.

Proteins that fail to fold or assemble with partner subunits are selectively removed from the endoplasmic reticulum (ER) via the ER-associated degradation (ERAD) pathway. Proteins selected for ERAD are polyubiquitinated and retrotranslocated into the cytosol for degradation by the proteasome. Although it is unclear how proteins are initially identified by the ERAD system in mammalian cells, OS-9 was recently proposed to play a key role in this process. Here we show that OS-9 is upregulated in response to ER stress and is associated both with components of the ERAD machinery and with ERAD substrates. Using RNA interference, we show that OS-9 is required for efficient ubquitination of glycosylated ERAD substrates, suggesting that it helps transfer misfolded proteins to the ubiquitination machinery. We also find that OS-9 binds to a misfolded nonglycosylated protein destined for ERAD, but not to the properly folded wild-type protein. Surprisingly, however, OS-9 is not required for ubiquitination or degradation of this nonglycosylated ERAD substrate. We propose a model in which OS-9 recognises terminally misfolded proteins via polypeptide-based rather than glycan-based signals, but is only required for transferring those bearing N-glycans to the ubiquitination machinery.

Tinkering Inside the Organelle

Debate about eukaryote evolution includes alternate views on the processes that gave rise to mitochondria. Among the questions about the evolution of eukaryotes is the debate over how they acquired the membrane-bound organelle, mitochondria. Mitochondria produce energy in nearly all eukaryotic cells (1) and regulate cell metabolism by controlling the flow of factors such as ions, amino acids, and carbohydrates between themselves and the cytoplasm. Mitochondria evolved from a bacterial endosymbiont (an α-proteobacterium), and this process depended on the establishment of new pathways that facilitated the import of proteins into and across the double membrane (inner and outer) of the ancestral endosymbiont. Herein lies a debate: How did the process of protein import in mitochondria—which facilitated the evolution of this organelle, and thus, eukaryotic cell evolution—arise? Was the process driven by the ancestral host cell or by the prokaryotic endosymbiont, or by both?

Conserved substrate binding by chaperones in the bacterial periplasm and the mitochondrial intermembrane space.

Mitochondria were derived from intracellular bacteria and the mitochondrial intermembrane space is topologically equivalent to the bacterial periplasm. Both compartments contain ATP-independent chaperones involved in the transport of hydrophobic membrane proteins. The mitochondrial TIM (translocase of the mitochondrial inner membrane) 10 complex and the periplasmic chaperone SurA were examined in terms of evolutionary relation, structural similarity, substrate binding specificity and their function in transporting polypeptides for insertion into membranes. The two chaperones are evolutionarily unrelated; structurally, they are also distinct both in their characteristics, as determined by SAXS (small-angle X-ray scattering), and in pairwise structural comparison using the distance matrix alignment (DALILite server). Despite their structural differences, SurA and the TIM10 complex share a common binding specificity in Pepscan assays of substrate proteins. Comprehensive analysis of the binding on a total of 1407 immobilized 13-mer peptides revealed that the TIM10 complex, like SurA, does not bind hydrophobic peptides generally, but that both chaperones display selectivity for peptides rich in aromatic residues and with net positive charge. This common binding specificity was not sufficient for SurA to completely replace TIM10 in yeast cells in vivo. In yeast cells lacking TIM10, when SurA is targeted to the intermembrane space of mitochondria, it binds translocating substrate proteins, but fails to completely transfer the substrate to the translocase in the mitochondrial inner membrane. We suggest that SurA was incapable of presenting substrates effectively to the primitive TOM (translocase of the mitochondrial outer membrane) and TIM complexes in early mitochondria, and was replaced by the more effective small Tim chaperone.

Assembling the Tat protein translocase

The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes. DOI: http://dx.doi.org/10.7554/eLife.20718.001

The TatC component of the twin-arginine protein translocase functions as an obligate oligomer

The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate‐bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild‐type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue‐native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild‐type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate‐induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.

A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase

Significance The twin-arginine translocation (Tat) system transports folded proteins across the prokaryotic inner membrane and the thylakoid membrane of plant chloroplasts. Proteins are targeted to the Tat system by signal peptides containing a highly conserved twin arginine motif. We isolated suppressors in the TatB component that allowed a Tat substrate with a defective twin arginine motif to be transported. The strongest of these suppressors, TatB F13Y, resulted in the constitutive assembly of the Tat translocase in the absence of signal peptide binding. These results show that Tat signal peptides have two separable roles: they target their passenger proteins to the Tat machinery but they also trigger the assembly of the active Tat transporter. The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system of Escherichia coli is made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA–YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase.

A Small Tim Homohexamer in the Relict Mitochondrion of Cryptosporidium

The apicomplexan parasite Cryptosporidium parvum possesses a mitosome, a relict mitochondrion with a greatly reduced metabolic capability. This mitosome houses a mitochondrial-type protein import apparatus, but elements of the protein import pathway have been reduced, and even lost, through evolution. The small Tim protein family is a case in point. The genomes of C. parvum and related species of Cryptosporidium each encode just one small Tim protein, CpTimS. This observation challenged the tenet that small Tim proteins are always found in pairs as α3β3 hexamers. We show that the atypical CpTimS exists as a relatively unstable homohexamer, shedding light both on the early evolution of the small Tim protein family and on small Tim hexamer formation in contemporary eukaryotes.

Mitochondrial ATP-independent chaperones.

Mitochondria possess a dedicated‐chaperone system in the intermembrane space, the small Tims that are ubiquitous in all eukaryotes from yeast to man. They escort membrane proteins to the outer or the inner membrane for proper insertion. These mitochondrial chaperones do not require external energy to perform their function and have structural similarities to other ATP‐independent chaperones. Here, we discuss their structural properties and how these relate to their chaperoning function in the mitochondrial intermembrane space.