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Biomedical subjects

Trevor Lithgow

Publications and source records attributed to Trevor Lithgow.

At least 19 recordsLinked to original sources
PubMed2006

A mitochondrial protein affects cell morphology, mitochondrial segregation and virulence in Leishmania.

The single mitochondrion of kinetoplastids divides in synchrony with the nucleus and plays a crucial role in cell division. However, despite its importance and potential as a drug target, the mechanism of mitochondrial division and segregation and the molecules involved are only partly understood. In our quest to identify novel mitochondrial proteins in Leishmania, we constructed a hidden Markov model from the targeting motifs of known mitochondrial proteins as a tool to search the Leishmania major genome. We show here that one of the 17 proteins of unknown function that we identified, designated mitochondrial protein X (MIX), is an oligomeric protein probably located in the inner membrane and expressed throughout the Leishmania life cycle. The MIX gene appears to be essential. Moreover, even deletion of one allele from L. major led to abnormalities in cell morphology, mitochondrial segregation and, importantly, to loss of virulence. MIX is unique to kinetoplastids but its heterologous expression in Saccharomyces cerevisiae produced defects in mitochondrial morphology. Our data show that a number of mitochondrial proteins are unique to kinetoplastids and some, like MIX, play a central role in mitochondrial segregation and cell division, as well as virulence.

Amino Acid Sequence↗
PubMed2006

Evolution of the molecular machines for protein import into mitochondria.

In creating mitochondria some 2 billion years ago, the first eukaryotes needed to establish protein import machinery in the membranes of what was a bacterial endosymbiont. Some of the preexisting protein translocation apparatus of the endosymbiont appears to have been commandeered, including molecular chaperones, the signal peptidase, and some components of the protein-targeting machinery. However, the protein translocases that drive protein import into mitochondria have no obvious counterparts in bacteria, making it likely that these machines were created de novo. The presence of similar translocase subunits in all eukaryotic genomes sequenced to date suggests that all eukaryotes can be considered descendants of a single ancestor species that carried an ancestral "protomitochondria."

Bacterial Proteins↗
PubMed2006

Human Bcl-2 cannot directly inhibit the Caenorhabditis elegans Apaf-1 homologue CED-4, but can interact with EGL-1.

Although the anti-apoptotic activity of Bcl-2 has been extensively studied, its mode of action is still incompletely understood. In the nematode Caenorhabditis elegans, 131 of 1090 somatic cells undergo programmed cell death during development. Transgenic expression of human Bcl-2 reduced cell death during nematode development, and partially complemented mutation of ced-9, indicating that Bcl-2 can functionally interact with the nematode cell death machinery. Identification of the nematode target(s) of Bcl-2 inhibition would help clarify the mechanism by which Bcl-2 suppresses apoptosis in mammalian cells. Exploiting yeast-based systems and biochemical assays, we analysed the ability of Bcl-2 to interact with and regulate the activity of nematode apoptosis proteins. Unlike CED-9, Bcl-2 could not directly associate with the caspase-activating adaptor protein CED-4, nor could it inhibit CED-4-dependent yeast death. By contrast, Bcl-2 could bind the C. elegans pro-apoptotic BH3-only Bcl-2 family member EGL-1. These data prompt us to hypothesise that Bcl-2 might suppress nematode cell death by preventing EGL-1 from antagonising CED-9, rather than by inhibiting CED-4.

Animals↗
PubMed2006

The C-terminal TPR domain of Tom70 defines a family of mitochondrial protein import receptors found only in animals and fungi.

In fungi and animals the translocase in the outer mitochondrial membrane (TOM complex) consists of multiple components including the receptor subunit Tom70. Genome sequence analyses suggest no Tom70 receptor subunit exists in plants or protozoans, raising questions about its ancestry, function and the importance of its activity. Here we characterise the relationships within the Tom70 family of proteins. We find that in both fungi and animals, a conserved domain structure exists within the Tom70 family, with a transmembrane segment followed by 11 tetratricopeptide repeat motifs organised in three distinct domains. The C-terminal domain of Tom70 is highly conserved, and crucial for the import of hydrophobic substrate proteins, including those with and those without N-terminal presequences. Tom70 likely arose after fungi and animals diverged from other eukaryote lineages including plants, and subsequent gene duplication gave rise to a paralogue specific to the Saccharomyces group of yeasts. In animals and in fungi, Tom70 plays a fundamental role in the import of precursor proteins, by assisting relatively hydrophobic regions of substrate proteins into the translocation channel in the outer mitochondrial membrane. Proteins that function equivalently to Tom70 may have arisen independently in plants and protists.

Animals↗
PubMed2006

Convergent evolution of receptors for protein import into mitochondria.

BACKGROUND: Mitochondria evolved from intracellular bacterial symbionts. Establishing mitochondria as organelles required a molecular machine to import proteins across the mitochondrial outer membrane. This machinery, the TOM complex, is composed of at least seven component parts, and its creation and evolution represented a sizeable challenge. Although there is good evidence that a core TOM complex, composed of three subunits, was established in the protomitochondria, we suggest that the receptor component of the TOM complex arose later in the evolution of this machine. RESULTS: We have solved by nuclear magnetic resonance the structure of the presequence binding receptor from the TOM complex of the plant Arabidopsis thaliana. The protein fold suggests that this protein, AtTom20, belongs to the tetratricopeptide repeat (TPR) superfamily, but it is unusual in that it contains insertions lengthening the helices of each TPR motif. Peptide titrations map the presequence binding site to a groove of the concave surface of the receptor. In vitro functional assays and peptide titrations suggest that the plant Tom20 is functionally equivalent to fungal and animal Tom20s. CONCLUSIONS: Comparison of the sequence and structure of Tom20 from plants and animals suggests that these two presequence binding receptors evolved from two distinct ancestral genes following the split of the animal and plant lineages. The need to bind equivalent mitochondrial targeting sequences and to make similar interactions within an equivalent protein translocation machine has driven the convergent evolution of two distinct proteins to a common structure and function.

Amino Acid Sequence↗
PubMed2006

Integral membrane proteins in the mitochondrial outer membrane of Saccharomyces cerevisiae.

Mitochondria evolved from a bacterial endosymbiont ancestor in which the integral outer membrane proteins would have been beta-barrel structured within the plane of the membrane. Initial proteomics on the outer membrane from yeast mitochondria suggest that while most of the protein components are integral in the membrane, most of these mitochondrial proteins behave as if they have alpha-helical transmembrane domains, rather than beta-barrels. These proteins are usually predicted to have a single alpha-helical transmembrane segment at either the N- or C-terminus, however, more complex topologies are also seen. We purified the novel outer membrane protein Om14 and show it is encoded in the gene YBR230c. Protein sequencing revealed an intron is spliced from the transcript, and both transcription from the YBR230c gene and steady-state level of the Om14 protein is dramatically less in cells grown on glucose than in cells grown on nonfermentable carbon sources. Hydropathy predictions together with data from limited protease digestion show three alpha-helical transmembrane segments in Om14. The alpha-helical outer membrane proteins provide functions derived after the endosymbiotic event, and require the translocase in the outer mitochondrial membrane complex for insertion into the outer membrane.

Amino Acid Sequence↗
PubMed2005

Giardia mitosomes and trichomonad hydrogenosomes share a common mode of protein targeting.

Mitochondria are archetypal organelles of endosymbiotic origin in eukaryotic cells. Some unicellular eukaryotes (protists) were considered to be primarily amitochondrial organisms that diverged from the eukaryotic lineage before the acquisition of the premitochondrial endosymbiont, but their amitochondrial status was recently challenged by the discovery of mitochondria-like double membrane-bound organelles called mitosomes. Here, we report that proteins targeted into mitosomes of Giardia intestinalis have targeting signals necessary and sufficient to be recognized by the mitosomal protein import machinery. Expression of these mitosomal proteins in Trichomonas vaginalis results in targeting to hydrogenosomes, a hydrogen-producing form of mitochondria. We identify, in Giardia and Trichomonas, proteins related to the component of the translocase in the inner membrane from mitochondria and the processing peptidase. A shared mode of protein targeting supports the hypothesis that mitosomes, hydrogenosomes, and mitochondria represent different forms of the same fundamental organelle having evolved under distinct selection pressures.

Amino Acid Sequence↗
PubMed2005

Protein targeting: entropy, energetics and modular machines.

New light is being shed on the mechanism of protein import into mitochondria. The inner membrane translocase can switch between modes of translocation, and assists what might be an entropic device to drive the initial entry of substrate proteins across the outer membrane.

Entropy↗
PubMed2005

Mature DIABLO/Smac is produced by the IMP protease complex on the mitochondrial inner membrane.

DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX(5)P in Imp1 and NX(5)S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space.

Amino Acid Motifs↗
PubMed2005

Patterns that define the four domains conserved in known and novel isoforms of the protein import receptor Tom20.

Tom20 is the master receptor for protein import into mitochondria. Analysis of motifs present in Tom20 sequences from fungi and animals found several highly conserved regions, including features of the transmembrane segment, the ligand-binding domain and functionally important flexible segments at the N terminus and the C terminus of the protein. Hidden Markov model searches of genome sequence data revealed novel isoforms of Tom20 in vertebrate and invertebrate animals. A three-dimensional comparative model of the novel type I Tom20, based on the structurally characterized type II isoform, shows important differences in the amino acid residues lining the ligand-binding groove, where the type I protein from animals is more similar to the fungal form of Tom20. Given that the two receptor types from mouse interact with the same set of precursor protein substrates, comparative analysis of the substrate-binding site provides unique insight into the mechanism of substrate recognition. No Tom20-related protein was found in genome sequence data from plants or protozoans, suggesting the receptor Tom20 evolved after the split of animals and fungi from the main lineage of eukaryotes.

Amino Acid Sequence↗
PubMed2005

Molecular architecture and function of the Omp85 family of proteins.

Omp85 is a protein found in Gram-negative bacteria where it serves to integrate proteins into the bacterial outer membrane. Members of the Omp85 family of proteins are defined by the presence of two domains: an N-terminal, periplasmic domain rich in POTRA repeats and a C-terminal beta-barrel domain embedded in the outer membrane. The widespread distribution of Omp85 family members together with their fundamental role in outer membrane assembly suggests the ancestral Omp85 arose early in the evolution of prokaryotic cells. Mitochondria, derived from an ancestral bacterial endosymbiont, also use a member of the Omp85 family to assemble proteins in their outer membranes. More distant relationships are seen between the Omp85 family and both the core proteins in two-partner secretion systems and the Toc75 family of protein translocases found in plastid outer envelopes. Aspects of the ancestry and molecular architecture of the Omp85 family of proteins is providing insight into the mechanism by which proteins might be integrated and assembled into bacterial outer membranes.

Bacterial Outer Membrane Proteins↗
PubMed2004

Mitochondrial release of pro-apoptotic proteins: electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes.

A key step in the initiation of apoptosis is the release from the mitochondrial intermembrane space of cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO, Omi/HtrA2, apoptosis-inducing factor (AIF), and endonuclease G (EndoG). Discrepancies have arisen, however, as to whether all these proteins are released in different systems. Our results suggest that failure to observe cytochrome c release may be due to the use of different buffers because after permeabilization by caspase-8 cleaved human Bid (tBid), cytochrome c dissociation from mitochondria was highly dependent on ionic strength and required 50-80 mm KCl, NaCl, or LiCl. In addition, mitochondria isolated from apoptotic cells using low ionic strength buffer bound a greater proportion of endogenous cytochrome c. In contrast to cytochrome c, Smac/DIABLO and Omi/HtrA2 were released independent of ionic strength, and AIF and EndoG behaved as if they are exposed to the intermembrane space but tethered to or within the inner membrane. AIF and EndoG were also not released by active caspases, which suggests their involvement in apoptosis may be limited. In summary, whereas tBid permeabilizes the outer membrane to cytochrome c, Smac/DIABLO, and Omi/HtrA2, the release of cytochrome c during apoptosis will be underestimated unless sufficient ionic strength is maintained to overcome the electrostatic association of cytochrome c with membranes.

Apoptosis↗
PubMed2004

Zim17, a novel zinc finger protein essential for protein import into mitochondria.

Translocation of precursor proteins across the mitochondrial membranes requires the coordinated action of multisubunit translocases in the outer and inner membrane, and the driving force for translocation across the inner membrane is provided by the matrix-located heat shock protein 70 (mtHsp70). The central components of the protein import machinery are essential. Here we describe Zim17, an essential protein with a zinc finger motif involved in protein import into mitochondria. Comparative genomics suggested a correction to the open reading frame of YNL310c, the gene encoding Zim17 in Saccharomyces cerevisiae. The revised open reading frame codes for a classic mitochondrial targeting signal, which is processed from Zim17 in the mitochondrial matrix. Loss of Zim17 selectively diminishes import of proteins into the matrix of mitochondria, but this loss of Zim17 is partially suppressed by overexpression of the J-protein Pam18/Tim14. We propose that Zim17 functions as an example of a "fractured" J-protein, where a protein like Zim17 contributes a zinc finger domain to Type III J-proteins, in toto providing for substrate loading onto Hsp70.

Amino Acid Sequence↗
PubMed2004

Distinct roles for the Hsp40 and Hsp90 molecular chaperones during cystic fibrosis transmembrane conductance regulator degradation in yeast.

Aberrant secreted proteins can be destroyed by ER-associated protein degradation (ERAD), and a prominent, medically relevant ERAD substrate is the cystic fibrosis transmembrane conductance regulator (CFTR). To better define the chaperone requirements during CFTR maturation, the protein was expressed in yeast. Because Hsp70 function impacts CFTR biogenesis in yeast and mammals, we first sought ER-associated Hsp40 cochaperones involved in CFTR maturation. Ydj1p and Hlj1p enhanced Hsp70 ATP hydrolysis but CFTR degradation was slowed only in yeast mutated for both YDJ1 and HLJ1, suggesting functional redundancy. In contrast, CFTR degradation was accelerated in an Hsp90 mutant strain, suggesting that Hsp90 preserves CFTR in a folded state, and consistent with this hypothesis, Hsp90 maintained the solubility of an aggregation-prone domain (NBD1) in CFTR. Soluble ERAD substrate degradation was unaffected in the Hsp90 or the Ydj1p/Hlj1p mutants, and surprisingly CFTR degradation was unaffected in yeast mutated for Hsp90 cochaperones. These results indicate that Hsp90, but not the Hsp90 complex, maintains CFTR structural integrity, whereas Ydj1p/Hlj1p catalyze CFTR degradation.

Amino Acid Sequence↗
PubMed2004

A biophysical analysis of the tetratricopeptide repeat-rich mitochondrial import receptor, Tom70, reveals an elongated monomer that is inherently flexible, unstable, and unfolds via a multistate pathway.

Proteins destined for all submitochondrial compartments are translocated across the outer mitochondrial membrane by the TOM (translocase of the outer membrane) complex, which consists of a number of specialized receptor subunits that bind mitochondrial precursor proteins for delivery into the translocation channel. One receptor, Tom70, binds large, hydrophobic mitochondrial precursors. The current model of Tom70-mediated import involves multiple dimers of the receptor recognizing a single molecule of substrate. Here we show via a battery of biophysical and spectroscopic techniques that the cytosolic domain of Tom70 is an elongated monomer. Thermal and urea-induced denaturation revealed that the receptor, which unfolds via a multistate pathway, is a relatively unstable molecule undergoing major conformational change at physiological temperatures. The data suggest that the malleability of the monomeric Tom70 receptor is an important factor in mitochondrial import.

Amino Acid Motifs↗
PubMed2004

Tom22', an 8-kDa trans-site receptor in plants and protozoans, is a conserved feature of the TOM complex that appeared early in the evolution of eukaryotes.

One of the earliest events in the evolution of mitochondria was the development a means to translocate proteins made in the cytosol into the "protomitochondrion." How this was achieved remains uncertain, and the nature of the earliest version of the protein translocation machinery is not known. Comparative sequence analysis suggests three subunits, Tom40, Tom7, and Tom22 as common elements of the protein translocase in the mitochondrial outer membrane in diverse extant eukaryotes. Tom22, the 22-kDa subunit, plays a critical role in the function of this complex in fungi and animals, and we show that an 8-kDa subunit of the plant translocase is a truncated form of Tom22. It has a single transmembrane segment conforming in sequence to the same region of Tom22 from other eukaryotic lineages and a short carboxy-terminal trans domain located in the mitochondrial intermembrane space. The trans domain from the Arabidopsis thaliana protein functions in yeast lacking their own Tom22 by complementing protein import defects and restoring cell growth. Moreover, we have identified orthologs of Tom22, Tom7, and Tom40 in diverse eukaryotes such as the diatom Phaeodactylum tricornutum, the amoebic slime Dictyostelium discoideum, and the protozoan parasite Plasmodium falciparum. This finding strongly suggests these subunits as the core of the protein translocase in the earliest mitochondria.

Amino Acid Sequence↗
PubMed2004

Bacterial proteins predisposed for targeting to mitochondria.

Mitochondria evolved from an endosymbiotic proteobacterium in a process that required the transfer of genes from the bacterium to the host cell nucleus, and the translocation of proteins thereby made in the host cell cytosol into the internal compartments of the organelle. According to current models for this evolution, two highly improbable events are required to occur simultaneously: creation of a protein translocation machinery to import proteins back into the endosymbiont and creation of targeting sequences on the protein substrates themselves. Using a combination of two independent prediction methods, validated through tests on simulated genomes, we show that at least 5% of proteins encoded by an extant proteobacterium are predisposed for targeting to mitochondria, and propose we that mitochondrial targeting information was preexisting for many proteins of the endosymbiont. We analyzed a family of proteins whose members exist both in bacteria and in mitochondria of eukaryotes and show that the amino-terminal extensions occasionally found in bacterial family members can function as a crude import sequence when the protein is presented to isolated mitochondria. This activity leaves the development of a primitive translocation channel in the outer membrane of the endosymbiont as a single hurdle to initiating the evolution of mitochondria.

Amino Acid Sequence↗