Yeast mitochondria can process de novo designed ß-barrel proteins.

Mitochondrial outer membrane ß-barrel proteins are encoded in the nucleus, translated in the cytosol and then targeted to and imported into the respective organelles. Detailed studies have uncovered the mechanisms involved in the import of these proteins and identified the targeting signals and the cytosolic factors that govern their proper biogenesis. Recently, de novo designed eight-stranded ß-barrel proteins (Tmb2.3 and Tmb2.17) were shown to fold and assemble into lipid membranes. To better understand the general aspects of the biogenesis of ß-barrel proteins, we investigated the fate of these artificial proteins upon their expression in yeast cells. We demonstrate that although these proteins are de novo designed and are not related to bona fide mitochondrial ß-barrel proteins, they were targeted to mitochondria and integrated into the organelle outer membrane. We further studied whether this integration requires components of the yeast mitochondrial import machinery like Tom20, Tom70, Tob55/Sam50 and Mas37/Sam37. Whereas it seems that none of the import receptors was required for the biogenesis of the artificial ß-barrel proteins, we observed a strong dependency on the TOB/SAM complex. Collectively, our findings demonstrate that the mitochondrial outer membrane is the preferential location in yeast cells for any membrane-embedded ß-barrel protein.

The Biogenesis Process of VDAC - From Early Cytosolic Events to Its Final Membrane Integration.

Voltage dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane. It is a membrane embedded ß-barrel protein composed of 19 mostly anti-parallel ß-strands that form a hydrophilic pore. Similar to the vast majority of mitochondrial proteins, VDAC is encoded by nuclear DNA, and synthesized on cytosolic ribosomes. The protein is then targeted to the mitochondria while being maintained in an import competent conformation by specific cytosolic factors. Recent studies, using yeast cells as a model system, have unearthed the long searched for mitochondrial targeting signal for VDAC and the role of cytosolic chaperones and mitochondrial import machineries in its proper biogenesis. In this review, we summarize our current knowledge regarding the early cytosolic stages of the biogenesis of VDAC molecules, the specific targeting of VDAC to the mitochondrial surface, and the subsequent integration of VDAC into the mitochondrial outer membrane by the TOM and TOB/SAM complexes.

Yeast can express and assemble bacterial secretins in the mitochondrial outer membrane.

Secretins form large multimeric pores in the outer membrane (OM) of Gram-negative bacteria. These pores are part of type II and III secretion systems (T2SS and T3SS, respectively) and are crucial for pathogenicity. Recent structural studies indicate that secretins form a structure rich in ß-strands. However, little is known about the mechanism by which secretins assemble into the OM. Based on the conservation of the biogenesis of ß-barrel proteins in bacteria and mitochondria, we used yeast cells as a model system to study the assembly process of secretins. To that end, we analyzed the biogenesis of PulD (T2SS), SsaC (T3SS) and InvG (T3SS) in wild type cells or in cells mutated for known mitochondrial import and assembly factors. Our results suggest that secretins can be expressed in yeast cells, where they are enriched in the mitochondrial fraction. Interestingly, deletion of mitochondrial import receptors like Tom20 and Tom70 reduces the mitochondrial association of PulD but does not affect that of InvG. SsaC shows another dependency pattern and its membrane assembly is enhanced by the absence of Tom70 and compromised in cells lacking Tom20 or the topogenesis of outer membrane ß-barrel proteins (TOB) complex component, Mas37. Collectively, these findings suggest that various secretins can follow different pathways to assemble into the bacterial OM.

Strain-transcending neutralization of malaria parasite by antibodies against Plasmodium falciparum enolase.

BACKGROUND: Plasmodium enolase is a target for the growth neutralizing antibodies. Interestingly, the three invasive stages i.e. sporozoites, merozoites, and ookinetes express this protein on their cell surface. Polyclonal anti-Plasmodium falciparum enolase (Pfeno) antibodies disrupt traversal of ookinete through mosquito mid-gut wall as well as have inhibitory effect on parasite growth at erythrocytic stage. In a recent study, it was observed that immunization with a unique epitope of parasite enolase (EWGWS) could confer partial protection against mouse malaria. Further validation is needed for the protective potential of this unique epitope in otherwise highly conserved enolase. METHODS: In order to investigate the efficacy of growth inhibitory potential of the epitope of P falciparum enolase, a monoclonal antibody specific to EWGWS is generated. In vitro parasite growth inhibition assays and passive immunization of Plasmodium yoelii (or Plasmodium berghei) infected mice were used to assess the parasite growth neutralizing activity of the antibody. RESULTS: Screening a panel of monoclonal antibodies raised against recombinant Pfeno that were specific to EWGWS resulted in isolation of H12E1. This antibody recognized only EWGWS epitope containing enolases. H12E1 strongly inhibited parasite growth in culture. This inhibition was strain transcending. Passive infusion of this antibody in P. yoelii or P. berghei infected mice showed significant reduction in parasitemia as compared to controls (p < 0.001). Surface Plasmon Resonance measurements indicated high affinity binding of H12E1 to P. falciparum enolase (KD ~ 7.6 × 10-9M). CONCLUSIONS: A monoclonal antibody directed against EWGWS epitope of Pfeno was shown to inhibit the growth of blood stage malarial parasites. This inhibition was species/strain transcending and is likely to arise due to blockade of enolase on the surface of merozoites, functionally implicating Pfeno in invasion related events. Presence of enolase on the cell surface of merozoites and ookinetes could potentially result in inhibition of host cell invasions at erythrocytic and transmission stages in the parasite life cycle. It is suggested that antibodies against EWGWS epitope have the potential to confer dual stage, species and strain transcending protection against malaria.

Functions of tryptophan residues in EWGWS insert of Plasmodium falciparum enolase.

Plasmodium falciparum enolase (Pfeno) is a dimeric enzyme with multiple moonlighting functions. This enzyme is thus a potential target for anti-malarial treatments. A unique feature of Pfeno is the presence of a pentapeptide insert 104 EWGWS 108. The functional role of tryptophan residues in this insert was investigated using site-directed mutagenesis. Replacement of these two Trp residues with alanines (or lysines) resulted in a near complete loss of enolase activity and dissociation of the normal dimeric form into monomers. Molecular modeling indicated that 340R forms π-cation bonds with the aromatic rings of 105W and 46Y. Mutation induced changes in the interactions among these three residues were presumably relayed to the inter-subunit interface via a coil formed by 46Y : 59Y, resulting in the disruption of a salt bridge between 11R : 425E and a π-cation interaction between 11R : 59Y. This led to a drop of ~ 4 kcal·mole-1 in the inter-subunit docking energy in the mutant, causing a ~ 103 fold decrease in affinity. Partial restoration of the inter-subunit interactions led to reformation of dimers and also restored a significant fraction of the lost enzyme activity. These results suggested that the perturbations in the conformation of the surface loop containing the insert sequence were relayed to the interface region, causing dimer dissociation that, in turn, disrupted the enzyme's active site. Since Plasmodium enolase is a moonlighting protein with multiple parasite-specific functions, it is likely that these functions may map on to the highly conserved unique insert region of this protein. ENZYMES: Enolase(EC4.2.1.11).