Trivalent Arsenic Inhibits the Functions of Chaperonin Complex

The exact molecular mechanisms by which the environmental pollutant arsenic works in biological systems are not completely understood. Using an unbiased chemogenomics approach in Saccharomyces cerevisiae, we found that mutants of the chaperonin complex TRiC and the functionally related prefoldin complex are all hypersensitive to arsenic compared to a wild-type strain. In contrast, mutants with impaired ribosome functions were highly arsenic resistant. These observations led us to hypothesize that arsenic might inhibit TRiC function, required for folding of actin, tubulin, and other proteins postsynthesis. Consistent with this hypothesis, we found that arsenic treatment distorted morphology of both actin and microtubule filaments. Moreover, arsenic impaired substrate folding by both bovine and archaeal TRiC complexes in vitro. These results together indicate that TRiC is a conserved target of arsenic inhibition in various biological systems.ARSENIC is a ubiquitous environmental pollutant that causes severe health problems in humans. It is also used as an effective therapeutic agent against various diseases and infections. Using advanced genomic tools in the model organism yeast and biochemical experiments, we demonstrated that arsenic disturbs functions of the chaperonin complex required for proper folding and maturation of a large number of proteins. This mechanism of action by arsenic is conserved in various biological systems ranging from archaeal bacteria to mammals. Such an understanding should help in exploring possible ways to overcome toxic effects caused by exposure to arsenic.Trivalent inorganic arsenic is among the most significant environmental hazards affecting the health of millions of people worldwide (Nordstrom 2002). Particularly, inorganic trivalent arsenic [As(III)] in underground drinking water and some mining environments is recognized as the cause of various cancers affecting the skin, lung, urinary tract, bladder, liver, and kidney (Tapio and Grosche 2006), as well as being implicated in several other disorders such as diabetes, hypertension, neuropathy, and vascular diseases (Tseng 2004). Interestingly, As(III) is also an effective therapeutic agent against cancer and human pathogens. A number of models have been proposed to explain the biological effects of As(III), including stimulation of reactive oxygen species (ROS) production (Miller et al. 2002; Tapio and Grosche 2006) and inhibition of tubulin polymerization (Ramirez et al. 1997; Li and Broome 1999). However, exactly how As(III) disturbs biological systems is still not clear.The eukaryotic chaperonin TRiC (TCP1-ring complex, also called CCT) is a ∼900-kDa complex consisting of two apposed heterooligomeric protein rings. Each ring, constituted by eight homologous subunits (encoded by the essential CCT1–CCT8 genes in budding yeast), contains a central cavity in which unfolded polypeptide substrates attain a properly folded state in an ATP-requiring reaction (Bukau and Horwich 1998; Gutsche et al. 1999). TRiC is required for the proper folding of an important subset of cytosolic proteins, including cytoskeleton components, cell cycle regulators, and tumor suppressor proteins (Spiess et al. 2004). Some of these protein substrates are themselves encoded by essential genes; thus TRiC is indispensable for eukaryotic cell survival. Many TRiC substrates are subunits of oligomeric complexes and their assembly into functional multisubunit complexes also requires TRiC (Spiess et al. 2004). Assembly of such macromolecular complexes in some cases eliminates the accumulation of toxic subunits such as free β-tubulin molecules, which can bind to γ-tubulin and thereby disrupt the formation of mitotic spindles in the yeast S. cerevisiae (Archer et al. 1995). Folding of yeast actin, α-tubulin, and β-tubulin and their oligomerization require TRiC and GimC (also known as prefoldin), a nonessential protein complex of six distinct but structurally related subunits of 13–23 kDa (Geiser et al. 1997; Vainberg et al. 1998). Mutational loss of GimC function substantially reduces actin and tubulin folding efficiency although it does not cause obvious growth defects in yeast. However, deletion of various GimC subunits strongly reduces the viability of conditional-lethal alleles of TRiC subunits under permissive conditions (Siegers et al. 1999).To elucidate the mechanisms of inorganic As(III)''s action(s) in a eukaryotic system, we first took an unbiased functional chemogenomics approach in yeast to systematically probe for the genetic determinants of arsenic sensitivity. These genetic and subsequent biochemical results point to the conclusion that As(III) inhibits the yeast TRiC complex. This mechanism of action is apparently conserved because the activities of both a mammalian TRiC complex and an archaeal TRiC-like chaperonin are significantly inhibited by arsenic in vitro. Given that mammalian TRiC and some of its substrates are implicated in tumor suppression, angiogenesis, and neuropathy (Lee et al. 2003; Spiess et al. 2004; Bouhouche et al. 2006), TRiC is likely an important protein mediator of As(III)''s effects on human health.     

The novel F-box protein Mfb1p regulates mitochondrial connectivity and exhibits asymmetric localization in yeast

Although it is clear that mitochondrial morphogenesis is a complex process involving multiple proteins in eukaryotic cells, little is known about regulatory molecules that modulate mitochondrial network formation. Here, we report the identification of a new yeast mitochondrial morphology gene called MFB1 (YDR219C). MFB1 encodes an F-box protein family member, many of which function in Skp1-Cdc53/Cullin-F-box protein (SCF) ubiquitin ligase complexes. F-box proteins also act in non-SCF complexes whose functions are not well understood. Although cells lacking Mfb1p contain abnormally short mitochondrial tubules, Mfb1p is not essential for known pathways that determine mitochondrial morphology and dynamics. Mfb1p is peripherally associated with the mitochondrial surface. Coimmunoprecipitation assays reveal that Mfb1p interacts with Skp1p in an F-box-dependent manner. However, Mfb1p does not coimmunoprecipitate with Cdc53p. The F-box motif is not essential for Mfb1p-mediated mitochondrial network formation. These observations suggest that Mfb1p acts in a complex lacking Cdc53p required for mitochondrial morphogenesis. During budding, Mfb1p asymmetrically localizes to mother cell mitochondria. By contrast, Skp1p accumulates in the daughter cell cytoplasm. Mfb1p mother cell-specific asymmetry depends on the F-box motif, suggesting that Skp1p down-regulates Mfb1p mitochondrial association in buds. We propose that Mfb1p operates in a novel pathway regulating mitochondrial tubular connectivity.     

Domains within the GARP subunit Vps54 confer separate functions in complex assembly and early endosome recognition

Tethering complexes contribute to the specificity of membrane fusion by recognizing organelle features on both donor and acceptor membranes. The Golgi-associated retrograde protein (GARP) complex is required for retrograde traffic from both early and late endosomes to the trans-Golgi network (TGN), presenting a paradox as to how a single complex can interact specifically with vesicles from multiple upstream compartments. We have found that a subunit of the GARP complex, Vps54, can be separated into N- and C-terminal regions that have different functions. Whereas the N-terminus of Vps54 is important for GARP complex assembly and stability, a conserved C-terminal domain mediates localization to an early endocytic compartment. Mutation of this C-terminal domain has no effect on retrograde transport from late endosomes. However, a specific defect in retrieval of Snc1 from early endosomes is observed when recycling from late endosomes to the Golgi is blocked. These data suggest that separate domains recruit tethering complexes to different upstream compartments to regulate individual trafficking pathways.     

Frog population viability under present and future climate conditions: a Bayesian state-space approach

1.?World-wide extinctions of amphibians are at the forefront of the biodiversity crisis, with climate change figuring prominently as a potential driver of continued amphibian decline. As in other taxa, changes in both the mean and variability of climate conditions may affect amphibian populations in complex, unpredictable ways. In western North America, climate models predict a reduced duration and extent of mountain snowpack and increased variability in precipitation, which may have consequences for amphibians inhabiting montane ecosystems. 2.?We used Bayesian capture-recapture methods to estimate survival and transition probabilities in a high-elevation population of the Columbia spotted frog (Rana luteiventris) over 10?years and related these rates to interannual variation in peak snowpack. Then, we forecasted frog population growth and viability under a range of scenarios with varying levels of change in mean and variance in snowpack. 3.?Over a range of future scenarios, changes in mean snowpack had a greater effect on viability than changes in the variance of snowpack, with forecasts largely predicting an increase in population viability. Population models based on snowpack during our study period predicted a declining population. 4.?Although mean conditions were more important for viability than variance, for a given mean snowpack depth, increases in variability could change a population from increasing to decreasing. Therefore, the influence of changing climate variability on populations should be accounted for in predictive models. The Bayesian modelling framework allows for the explicit characterization of uncertainty in parameter estimates and ecological forecasts, and thus provides a natural approach for examining relative contributions of mean and variability in climatic variables to population dynamics. 5.?Longevity and heterogeneous habitat may contribute to the potential for this amphibian species to be resilient to increased climatic variation, and shorter-lived species inhabiting homogeneous ecosystems may be more susceptible to increased variability in climate conditions.     

The Mitochondrial Fission Adaptors Caf4 and Mdv1 Are Not Functionally Equivalent

Mitochondrial fission in eukaryotes is mediated by protein complexes that encircle and divide mitochondrial tubules. In budding yeast, fission requires the membrane-anchored protein Fis1 and the dynamin-related GTPase Dnm1. Dnm1 is recruited to mitochondria via interactions with the adaptor proteins Caf4 and Mdv1, which bind directly to Fis1. Unlike Mdv1, a function for Caf4 in mitochondrial membrane scission has not been established. In this study, we demonstrate that Caf4 is a bona fide fission adaptor that assembles at sites of mitochondrial division. We also show that fission complexes may contain Caf4 alone or both Caf4 and Mdv1 without compromising fission function. Although there is a correspondence between Caf4 and Mdv1 expression levels and their contribution to fission, the two adaptor proteins are not equivalent. Rather, our functional and phylogenetic analyses indicate that Caf4 mitochondrial fission activity has diverged from that of Mdv1.     

Reversible interruption of Giardia lamblia cyst wall protein transport in a novel regulated secretory pathway

To survive in the environment and infect a new host, Giardia lamblia secretes an extracellular cyst wall using a poorly understood pathway. The two cyst wall proteins (CWPs) form disulphide-bonded heterodimers and are exported via novel encystation-specific secretory vesicles (ESVs). Exposure of eukaryotic cells to dithiothreitol (DTT) blocks the formation of disulphide bonds in nascent proteins that accumulate in the endoplasmic reticulum (ER) and induces an unfolded protein response (UPR). Proteins that have exited the ER are not susceptible. Exposure to DTT inhibits ESV formation by > 85%. Addition of DTT to encysting cells causes rapid ( t _1/2 < 10 min), reversible disappearance of ESVs, correlated with reduction of CWPs to monomers and reformation of CWP oligomers upon removal of DTT. Neither CWPs nor ESVs are affected by mercaptoethanesulphonic acid, a strong reducing agent that does not penetrate cells. DTT does not inhibit the overall protein secretory pathway, and recovery does not require new protein synthesis. We found evidence of protein disulphide isomerases in the ESV and the surface of encysting cells, in which they may catalyse initial CWP folding and recovery from DTT. This is the first suggestion of non-CWP proteins in ESVs and of enzymes on the giardial surface. DTT treatment did not stimulate a UPR, suggesting that Giardia may have diverged before the advent of this conserved form of ER quality control.     

Extreme endurance flights by landbirds crossing the Pacific Ocean: ecological corridor rather than barrier?

Mountain ranges, deserts, ice fields and oceans generally act as barriers to the movement of land-dependent animals, often profoundly shaping migration routes. We used satellite telemetry to track the southward flights of bar-tailed godwits (Limosa lapponica baueri), shorebirds whose breeding and non-breeding areas are separated by the vast central Pacific Ocean. Seven females with surgically implanted transmitters flew non-stop 8,117-11,680 km (10153+/-1043 s.d.) directly across the Pacific Ocean; two males with external transmitters flew non-stop along the same corridor for 7,008-7,390 km. Flight duration ranged from 6.0 to 9.4 days (7.8+/-1.3 s.d.) for birds with implants and 5.0 to 6.6 days for birds with externally attached transmitters. These extraordinary non-stop flights establish new extremes for avian flight performance, have profound implications for understanding the physiological capabilities of vertebrates and how birds navigate, and challenge current physiological paradigms on topics such as sleep, dehydration and phenotypic flexibility. Predicted changes in climatic systems may affect survival rates if weather conditions at their departure hub or along the migration corridor should change. We propose that this transoceanic route may function as an ecological corridor rather than a barrier, providing a wind-assisted passage relatively free of pathogens and predators.     

Mitofusins and OPA1 Mediate Sequential Steps in Mitochondrial Membrane Fusion

Mitochondrial fusion requires the coordinated fusion of the outer and inner membranes. Three large GTPases—OPA1 and the mitofusins Mfn1 and Mfn2—are essential for the fusion of mammalian mitochondria. OPA1 is mutated in dominant optic atrophy, a neurodegenerative disease of the optic nerve. In yeast, the OPA1 ortholog Mgm1 is required for inner membrane fusion in vitro; nevertheless, yeast lacking Mgm1 show neither outer nor inner membrane fusion in vivo, because of the tight coupling between these two processes. We find that outer membrane fusion can be readily visualized in OPA1-null mouse cells in vivo, but these events do not progress to inner membrane fusion. Similar defects are found in cells lacking prohibitins, which are required for proper OPA1 processing. In contrast, double Mfn-null cells show neither outer nor inner membrane fusion. Mitochondria in OPA1-null cells often contain multiple matrix compartments bounded together by a single outer membrane, consistent with uncoupling of outer versus inner membrane fusion. In addition, unlike mitofusins and yeast Mgm1, OPA1 is not required on adjacent mitochondria to mediate membrane fusion. These results indicate that mammalian mitofusins and OPA1 mediate distinct sequential fusion steps that are readily uncoupled, in contrast to the situation in yeast.