An increase in mitochondrial TOM activates apoptosis to drive retinal neurodegeneration.

Intronic polymorphic TOMM40 variants increasing TOMM40 mRNA expression are strongly correlated to late onset Alzheimer's Disease. The gene product, hTomm40, encoded in the APOE gene cluster, is a core component of TOM, the translocase that imports nascent proteins across the mitochondrial outer membrane. We used Drosophila melanogaster eyes as an in vivo model to investigate the relationship between elevated Tom40 (the Drosophila homologue of hTomm40) expression and neurodegeneration. Here we provide evidence that an overabundance of Tom40 in mitochondria invokes caspase-dependent cell death in a dose-dependent manner, leading to degeneration of the primarily neuronal eye tissue. Degeneration is contingent on the availability of co-assembling TOM components, indicating that an increase in assembled TOM is the factor that triggers apoptosis and degeneration in a neural setting. Eye death is not contingent on inner membrane translocase components, suggesting it is unlikely to be a direct consequence of impaired import. Another effect of heightened Tom40 expression is upregulation and co-association of a mitochondrial oxidative stress biomarker, DmHsp22, implicated in extension of lifespan, providing new insight into the balance between cell survival and death. Activation of regulated death pathways, culminating in eye degeneration, suggests a possible causal route from TOMM40 polymorphisms to neurodegenerative disease.

The suppressor of copper sensitivity protein C from Caulobacter crescentus is a trimeric disulfide isomerase that binds copper(I) with subpicomolar affinity.

The introduction of disulfide bonds into periplasmic proteins is a critical process in many Gram-negative bacteria. The formation and regulation of protein disulfide bonds have been linked to the production of virulence factors. Understanding the different pathways involved in this process is important in the development of strategies to disarm pathogenic bacteria. The well characterized disulfide bond-forming (DSB) proteins play a key role by introducing or isomerizing disulfide bonds between cysteines in substrate proteins. Curiously, the suppressor of copper sensitivity C proteins (ScsCs), which are part of the bacterial copper-resistance response, share structural and functional similarities with DSB oxidase and isomerase proteins, including the presence of a catalytic thioredoxin domain. However, the oxidoreductase activity of ScsC varies with its oligomerization state, which depends on a poorly conserved N-terminal domain. Here, the structure and function of Caulobacter crescentus ScsC (CcScsC) have been characterized. It is shown that CcScsC binds copper in the copper(I) form with subpicomolar affinity and that its isomerase activity is comparable to that of Escherichia coli DsbC, the prototypical dimeric bacterial isomerase. It is also reported that CcScsC functionally complements trimeric Proteus mirabilis ScsC (PmScsC) in vivo, enabling the swarming of P. mirabilis in the presence of copper. Using mass photometry and small-angle X-ray scattering (SAXS) the protein is demonstrated to be trimeric in solution, like PmScsC, and not dimeric like EcDsbC. The crystal structure of CcScsC was also determined at a resolution of 2.6 Å, confirming the trimeric state and indicating that the trimerization results from interactions between the N-terminal α-helical domains of three CcScsC protomers. The SAXS data analysis suggested that the protomers are dynamic, like those of PmScsC, and are able to sample different conformations in solution.

Ion currents through Kir potassium channels are gated by anionic lipids.

Ion currents through potassium channels are gated. Constriction of the ion conduction pathway at the inner helix bundle, the textbook gate of Kir potassium channels, has been shown to be an ineffective permeation control, creating a rift in our understanding of how these channels are gated. Here we present evidence that anionic lipids act as interactive response elements sufficient to gate potassium conduction. We demonstrate the limiting barrier to K+ permeation lies within the ion conduction pathway and show that this gate is operated by the fatty acyl tails of lipids that infiltrate the conduction pathway via fenestrations in the walls of the pore. Acyl tails occupying a surface groove extending from the cytosolic interface to the conduction pathway provide a potential means of relaying cellular signals, mediated by anionic lipid head groups bound at the canonical lipid binding site, to the internal gate.

Identification and characterisation of a phospholipid scramblase in the malaria parasite Plasmodium falciparum.

Recent studies highlight the emerging role of lipids as important messengers in malaria parasite biology. In an attempt to identify interacting proteins and regulators of these dynamic and versatile molecules, we hypothesised the involvement of phospholipid translocases and their substrates in the infection of the host erythrocyte by the malaria parasite Plasmodium spp. Here, using a data base searching approach of the Plasmodium Genomics Resources (www.plasmodb.org), we have identified a putative phospholipid (PL) scramblase in P. falciparum (PfPLSCR) that is conserved across the genus and in closely related unicellular algae. By reconstituting recombinant PfPLSCR into liposomes, we demonstrate metal ion dependent PL translocase activity and substrate preference, confirming PfPLSCR as a bona fide scramblase. We show that PfPLSCR is expressed during asexual and sexual parasite development, localising to different membranous compartments of the parasite throughout the intra-erythrocytic life cycle. Two different gene knockout approaches, however, suggest that PfPLSCR is not essential for erythrocyte invasion and asexual parasite development, pointing towards a possible role in other stages of the parasite life cycle.

Changing perspectives on how the permeation pathway through potassium channels is regulated.

The primary means by which ion permeation through potassium channels is controlled, and the key to selective intervention in a range of pathophysiological conditions, is the process by which channels switch between non-conducting and conducting states. Conventionally, this has been explained by a steric mechanism in which the pore alternates between two conformations: a 'closed' state in which the conduction pathway is occluded and an 'open' state in which the pathway is sufficiently wide to accommodate fully hydrated ions. Recently, however, 'non-canonical' mechanisms have been proposed for some classes of K+ channels. The purpose of this review is to illuminate structural and dynamic relationships underpinning permeation control in K+ channels, indicating where additional data might resolve some of the remaining issues.

BAK core dimers bind lipids and can be bridged by them.

BAK and BAX are essential mediators of apoptosis that oligomerize in response to death cues, thereby causing permeabilization of the mitochondrial outer membrane. Their transition from quiescent monomers to pore-forming oligomers involves a well-characterized symmetric dimer intermediate. However, no essential secondary interface that can be disrupted by mutagenesis has been identified. Here we describe crystal structures of human BAK core domain (α2-α5) dimers that reveal preferred binding sites for membrane lipids and detergents. The phospholipid headgroup and one acyl chain (sn2) associate with one core dimer while the other acyl chain (sn1) associates with a neighboring core dimer, suggesting a mechanism by which lipids contribute to the oligomerization of BAK. Our data support a model in which, unlike for other pore-forming proteins whose monomers assemble into oligomers primarily through protein-protein interfaces, the membrane itself plays a role in BAK and BAX oligomerization.

A constricted opening in Kir channels does not impede potassium conduction.

The canonical mechanistic model explaining potassium channel gating is of a conformational change that alternately dilates and constricts a collar-like intracellular entrance to the pore. It is based on the premise that K+ ions maintain a complete hydration shell while passing between the transmembrane cavity and cytosol, which must be accommodated. To put the canonical model to the test, we locked the conformation of a Kir K+ channel to prevent widening of the narrow collar. Unexpectedly, conduction was unimpaired in the locked channels. In parallel, we employed all-atom molecular dynamics to simulate K+ ions moving along the conduction pathway between the lower cavity and cytosol. During simulations, the constriction did not significantly widen. Instead, transient loss of some water molecules facilitated K+ permeation through the collar. The low free energy barrier to partial dehydration in the absence of conformational change indicates Kir channels are not gated by the canonical mechanism.

Engineering protocells: prospects for self-assembly and nanoscale production-lines.

The increasing ease of producing nucleic acids and proteins to specification offers potential for design and fabrication of artificial synthetic "organisms" with a myriad of possible capabilities. The prospects for these synthetic organisms are significant, with potential applications in diverse fields including synthesis of pharmaceuticals, sources of renewable fuel and environmental cleanup. Until now, artificial cell technology has been largely restricted to the modification and metabolic engineering of living unicellular organisms. This review discusses emerging possibilities for developing synthetic protocell "machines" assembled entirely from individual biological components. We describe a host of recent technological advances that could potentially be harnessed in design and construction of synthetic protocells, some of which have already been utilized toward these ends. More elaborate designs include options for building self-assembling machines by incorporating cellular transport and assembly machinery. We also discuss production in miniature, using microfluidic production lines. While there are still many unknowns in the design, engineering and optimization of protocells, current technologies are now tantalizingly close to the capabilities required to build the first prototype protocells with potential real-world applications.

Structure and function of LGR5: an enigmatic G-protein coupled receptor marking stem cells.

G-protein coupled receptors (GPCRs) are an important class of membrane protein that transmit extracellular signals invoked by sensing molecules such as hormones and neurotransmitters. GPCR dysfunction is implicated in many diseases and hence these proteins are of great interest to academia and the pharmaceutical industry. Leucine-rich repeat-containing GPCRs contain a characteristic extracellular domain that is an important modulator of intracellular signaling. One member of this class is the leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), a stem cell marker in intestinal crypts, and mammary glands. LGR5 modulates Wnt signaling in the presence of the ligand R-spondin (RSPO). The mechanism of activation of LGR5 by RSPO is not understood, nor is the intracellular signaling mechanism known. Recently reported structures of the extracellular domain of LGR5 bound to RSPO reveal a horseshoe-shaped architecture made up of consecutive leucine-rich repeats, with RSPO bound on the concave surface. This review discusses the discovery of LGR5 and the impact it is having on our understanding of stem cell and cancer biology of the colon. In addition, it covers functional relationships suggested by sequence homology and structural analyses, as well as some intriguing conundrums with respect to the involvement of LGR5 in Wnt signaling.

Oligomerization at the membrane: potassium channel structure and function.

Cell membranes present a naturally impervious barrier to aqueous solutes, such that the physiochemical environment on either side of the lipid bilayer can substantially differ. Integral membrane proteins are embedded in this heterogeneous lipid environment, wherein the juxtaposition of apolar and polar molecular surfaces defines factors such as transverse orientation, the surface area available for oligomerisation and the symmetry of resultant assemblies. This chapter focuses on potassium channels -representative molecular pores that play a critical role in electrical signalling by enabling selective transport of K(+) ions across cell membranes. Oligomerization is central to K(+) channel action; individual subunits are nonfunctional and conduction, selectivity and gating involve manipulation of the common subunit interface of the tetramer. Regulation of channel activity can be viewed from the perspective that the pore of K(+) channels has coopted other proteins, utilizing a process of hetero-oligomerisation to absorb new functions that both enable the pore to respond to extrinsic signals and provide an electrical signature.

Translocation of a Bak C-terminus mutant from cytosol to mitochondria to mediate cytochrome C release: implications for Bak and Bax apoptotic function.

BACKGROUND: One of two proapoptotic Bcl-2 proteins, Bak or Bax, is required to permeabilize the mitochondrial outer membrane during apoptosis. While Bax is mostly cytosolic and translocates to mitochondria following an apoptotic stimulus, Bak is constitutively integrated within the outer membrane. Membrane anchorage occurs via a C-terminal transmembrane domain that has been studied in Bax but not in Bak, therefore what governs their distinct subcellular distribution is uncertain. In addition, whether the distinct subcellular distributions of Bak and Bax contributes to their differential regulation during apoptosis remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: To gain insight into Bak and Bax targeting to mitochondria, elements of the Bak C-terminus were mutated, or swapped with those of Bax. Truncation of the C-terminal six residues (C-segment) or substitution of three basic residues within the C-segment destabilized Bak. Replacing the Bak C-segment with that from Bax rescued stability and function, but unexpectedly resulted in a semi-cytosolic protein, termed Bak/BaxCS. When in the cytosol, both Bax and Bak/BaxCS sequestered their hydrophobic transmembrane domains in their hydrophobic surface groove. Upon apoptotic signalling, Bak/BaxCS translocated to the mitochondrial outer membrane, inserted its transmembrane domain, oligomerized, and released cytochrome c. Despite this Bax-like subcellular distribution, Bak/BaxCS retained Bak-like regulation following targeting of Mcl-1. CONCLUSIONS/SIGNIFICANCE: Residues in the C-segment of Bak and of Bax contribute to their distinct subcellular localizations. That a semi-cytosolic form of Bak, Bak/BaxCS, could translocate to mitochondria and release cytochrome c indicates that Bak and Bax share a conserved mode of activation. In addition, the differential regulation of Bak and Bax by Mcl-1 is predominantly independent of the initial subcellular localizations of Bak and Bax.

Minimal requirements for actin filament disassembly revealed by structural analysis of malaria parasite actin-depolymerizing factor 1.

Malaria parasite cell motility is a process that is dependent on the dynamic turnover of parasite-derived actin filaments. Despite its central role, actin's polymerization state is controlled by a set of identifiable regulators that is markedly reduced compared with those of other eukaryotic cells. In Plasmodium falciparum, the most virulent species that affects humans, this minimal repertoire includes two members of the actin-depolymerizing factor/cofilin (AC) family of proteins, P. falciparum actin-depolymerizing factor 1 (PfADF1) and P. falciparum actin-depolymerizing factor 2. This essential class of actin regulator is involved in the control of filament dynamics at multiple levels, from monomer binding through to filament depolymerization and severing. Previous biochemical analyses have suggested that PfADF1 sequesters monomeric actin but, unlike most eukaryotic counterparts, has limited potential to bind or depolymerize filaments. The molecular basis for these unusual properties and implications for parasite cell motility have not been established. Here we present the crystal structure of an apicomplexan AC protein, PfADF1. We show that PfADF1 lacks critical residues previously implicated as essential for AC-mediated actin filament binding and disassembly, having a substantially reduced filament-binding loop and C-terminal α4 helix. Despite this divergence in structure, we demonstrate that PfADF1 is capable of efficient actin filament severing. Furthermore, this severing occurs despite PfADF1's low binding affinity for filaments. Comparative structural analysis along with biochemical and microscopy evidence establishes that severing is reliant on the availability of an exposed basic residue in the filament-binding loop, a conserved minimal requirement that defines AC-mediated filament disassembly across eukaryotic cells.

Domain reorientation and rotation of an intracellular assembly regulate conduction in Kir potassium channels.

Potassium channels embedded in cell membranes employ gates to regulate K+ current. While a specific constriction in the permeation pathway has historically been implicated in gating, recent reports suggest that the signature ion selectivity filter located in the outer membrane leaflet may be equally important. Inwardly rectifying K+ channels also control the directionality of flow, using intracellular polyamines to stem ion efflux by a valve-like action. This study presents crystallographic evidence of interdependent gates in the conduction pathway and reveals the mechanism of polyamine block. Reorientation of the intracellular domains, concomitant with activation, instigates polyamine release from intracellular binding sites to block the permeation pathway. Conformational adjustments of the slide helices, achieved by rotation of the cytoplasmic assembly relative to the pore, are directly correlated to the ion configuration in the selectivity filter. Ion redistribution occurs irrespective of the constriction, suggesting a more expansive role of the selectivity filter in gating than previously appreciated.

Structural and functional requirements for activity of the Tim9-Tim10 complex in mitochondrial protein import.

The Tim9-Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9-Tim10 hexameric assembly determined to 2.5 A and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.

Mitochondrial protein-import machinery: correlating structure with function.

Most mitochondrial proteins are synthesized in the cytosol, translocated into the organelle and directed along specific sorting pathways. Over the past 20 years, >30 proteins have been identified as having key roles in mitochondrial protein import. Recently, the elucidation of the structures of several import components has provided fresh insight into the import process. Here, we review the different pathways involved in sorting proteins into mitochondrial subcompartments. Along the way, we highlight the available structural information about the protein-import machinery and discuss how these structures correlate with previously ascribed functions. Future challenges for the cell biologists will be to use this structural information to test specific hypotheses addressing the molecular mechanisms of mitochondrial protein import.

A structural viral mimic of prosurvival Bcl-2: a pivotal role for sequestering proapoptotic Bax and Bak.

Many viruses express antiapoptotic proteins to counter host defense mechanisms that would otherwise trigger the rapid clearance of infected cells. For example, adenoviruses and some gamma-herpesviruses express homologs of prosurvival Bcl-2 to subvert the host's apoptotic machinery. Myxoma virus, a double-stranded DNA virus of the pox family, harbors antiapoptotic M11L, its virulence factor. Analysis of its three-dimensional structure reveals that despite lacking any primary sequence similarity to Bcl-2, it adopts a virtually identical protein fold. This allows it to associate with BH3 domains, especially those of Bax and Bak. We found that M11L acts primarily by sequestering Bax and Bak, thereby blocking the killing action of these essential cell-death mediators. These findings expand the family of protein sequences that act like Bcl-2 to block apoptosis and support the conclusion that the prosurvival action of these proteins critically depends on their ability to bind and antagonize Bax and/or Bak.

Crystal structure of the mitochondrial chaperone TIM9.10 reveals a six-bladed alpha-propeller.

Import of proteins into mitochondria occurs by coordinated actions of preprotein translocases in the outer and inner membranes. Tim9 and Tim10 are translocase components of the intermembrane space, related to deafness-dystonia peptide 1 (DDP1). They coassemble into a hexamer, TIM9.10, which captures and chaperones precursors of inner membrane metabolite carriers as they exit the TOM channel in the outer membrane. The crystal structure of TIM9.10 reveals a previously undescribed alpha-propeller topology in which helical "blades" radiate from a narrow central pore lined with polar residues. The propeller blades are reminiscent of "tentacles" in chaperones Skp and prefoldin. In each TIM9.10 subunit, a signature "twin CX3C" motif forms two intramolecular disulfides. There is no obvious binding pocket for precursors, which we suggest employ the chaperone-like tentacles of TIM9.10 as surrogate lipid contacts. The first reported crystal structure of a mitochondrial translocase assembly provides insights into selectivity and regulation of precursor import.

Potassium channel structures: do they conform?

Potassium channels are signalling elements vital to vertebrate neurotransmission, and cardiac and renal function. Two inherent qualities equip them for their role in the interconversion of chemical and electrical messages: high selectivity for potassium ions and the ability to open (gate) on cue. The crystal structure of KcsA, published in 1998, explained much about potassium selectivity and high ion flux. The enormous diversity of potassium channels (some hundreds of genes in humans) may have hampered similar progress in understanding gating processes. The recent determination of several representative structures has provided us with a valuable reference for discriminating between features that are utilized in gating across the potassium channel genre and features that determine responsiveness to family-specific gating cues.