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Fish is a popular human food. Over two-and-a-half billion people globally obtain their daily nutrient intake from fish. In India, it is a major dietary component for over 50 percent, and is a particularly important nutrition source for the poor. Mercury is a deadly environmental pollutant, both in its elemental form and in combination with other chemicals. When released into the environment mercury is transformed into methylmercury through microbial action. Methylmercury is the most pernicious form of mercury. It bioaccumulates in fish and enters human body with the consumption of contaminated fish. Fish in polluted water bodies accumulate methylmercury – a toxic pollutant of high potency that crosses the blood brain barrier and placental barrier, making it an intergenerational toxin. It enters the food chain both from point and non-point sources. Effluent pipes from industrial processes often contain mercury or mercury compounds. Emissions and ash from coal-fired power plants also contain mercury. It is well known that mercury circulates globally and deposits in water, bioaccumulating in the food chain through algae and fish. The higher the pecking order of a fish in the food chain, greater is the amount of mercury it is likely to contain. Advisories on fish consumption are quite common in developed countries, especially for pregnant women. Human exposure to such toxins therefore assumes significance. Contamination of this vital food is a key issue. In developing countries, issues like food contamination rarely draw attention. Mere availability of food is argued to be of foremost concern. In this scenario of poverty and hunger, system of industrial production has largely remained unaccountable to society and the environmental pollution it causes.

Detailed molecular information on G-actin assembly into filaments (F-actin), and their structure, dynamics, and interactions, is essential for understanding their cellular functions. Previous studies indicate that a flexible DNase I binding loop (D-loop, residues 40-50) plays a major role in actin's conformational dynamics. Phalloidin, a ``gold standard{''} for actin filament staining, stabilizes them and affects the D-loop. Using disulfide crosslinking in yeast actin D-loop mutant Q41C/V45C, light-scattering measurements, and cryoelectron microscopy reconstructions, we probed the constraints of D-loop dynamics and its contribution to F-actin formation/stability. Our data support a model of residues 41-45 distances that facilitate G- to F-actin transition. We report also a 3.3-angstrom resolution structure of phalloidin-bound F-actin in the ADP-Pi-like (ADP-BeFx) state. This shows the phalloidin-binding site on F-actin and how the relative movement between its two protofilaments is restricted by it. Together, our results provide molecular details of F-actin structure and D-loop dynamics.

Summary Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains at opposite ends. The dimers display significant conformational heterogeneity, with a preference for compact to intermediate conformations. Phosphoinositide-dependent membrane recruitment is mediated by one PH domain at a time and alters the conformational dynamics to prime allosteric activation by Arf-GTP. A structural model for membrane targeting and allosteric activation of full-length cytohesin dimers is discussed.

Summary Membrane dynamic processes including vesicle biogenesis depend on Arf GTPase activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane targeting module such as a PH domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization and conformational dynamics of Cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site., eTOC Blurb Arf GTPases control membrane dynamic processes following activation by guanine nucleotide exchange factors (GEFs). Malaby et al. investigate the role of structural dynamics in allosteric activation of Cytohesin family Arf GEFs. Hinge dynamics expose the allosteric site for activator binding whereas conformational entropy drives accessibility of the substrate site.

A recent study revealed new roles for the Rab11 GTPase during mitosis. Rab11 is involved in recycling endosome localization to mitotic spindle poles via dynein-mediated transport. This process is in contrast to Golgi membranes, which disperse in mitosis and do not appear to directly contribute to mitotic functions. Rab11-depletion prevents recycling endosome organization at spindle poles, delays mitotic progression, and disrupts spindle pole protein recruitment, astral microtubule organization, and mitotic spindle orientation. However, Rab11 is not the only endocytic and/or trafficking protein that regulates mitotic progression. Clathrin and two small GTPases (Rab6A', Rab5) play key roles in spindle organization and function. In this commentary, we discuss the roles of all these canonical endocytic and membrane trafficking proteins during mitosis and speculate on possible cross-communication between them and their molecular pathways that ensure faithful progression through mitosis.

Affinity grids (AG) are specialized EM grids that bind macromolecular complexes containing tagged proteins to obtain maximum occupancy for structural analysis through single-particle EM. In this study, utilizing AG, we show that His-tagged activated PKC βII binds to the small ribosomal subunit (40S). We reconstructed a cryo-EM map which shows that PKC βII interacts with RACK1, a seven-bladed β-propeller protein present on the 40S and binds in two different regions close to blades 3 and 4 of RACK1. This study is a first step in understanding the molecular framework of PKC βII/RACK1 interaction and its role in translation.

An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors1–4. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs alone5,6,7. Here, we addressed the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA16,7 recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harboring Rab5. Surprisingly, structural analysis revealed that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers we confirmed that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measured its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induced prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle toward the target membrane to initiate docking and fusion.

Eukaryotic translation termination results from the complex functional interplay between two eukaryotic release factors, eRF1 and eRF3, and the ribosome, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, using cryo-electron microscopy (cryo-EM) and flexible fitting, we determined the structure of eRF1–eRF3–guanosine 5′-[β,γ-imido]triphosphate (GMPPNP)-bound ribosomal pretermination complex (pre-TC), which corresponds to the initial, pre-GTP hydrolysis stage of factor attachment. Our results show that eukaryotic translation termination involves a network of interactions between the two release factors and the ribosome. Our structure provides mechanistic insight into the coordination between GTP hydrolysis by eRF3 and subsequent peptide release by eRF1.

The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and the protein-conducting channel and by undergoing ATP-driven conformation cycles that drive this process. There are conflicting reports about whether SecA functions as a monomer or dimer during this dynamic process. Here we reexamined the roles of the amino and carboxyl termini of SecA in promoting its dimerization and functional state by examining three secA mutants and the corresponding proteins: SecAΔ8 lacking residues 2 to 8, SecAΔ11 lacking residues 2 to 11, and SecAΔ11/N95 lacking both residues 2 to 11 and the carboxyl-terminal 70 residues. We demonstrated that whether SecAΔ11 or SecAΔ11/N95 was functional for promoting cell growth depended solely on the vivo level of the protein, which appeared to govern residual dimerization. All three SecA mutant proteins were defective for promoting cell growth unless they were highly overproduced. Cell fractionation revealed that SecAΔ11 and SecAΔ11/N95 were proficient in membrane association, although the formation of integral membrane SecA was reduced. The presence of a modestly higher level of SecAΔ11/N95 in the membrane and the ability of this protein to form dimers, as detected by chemical cross-linking, were consistent with the higher level of secA expression and better growth of the SecAΔ11/N95 mutant than of the SecAΔ11 mutant. Biochemical studies showed that SecAΔ11 and SecAΔ11/N95 had identical dimerization defects, while SecAΔ8 was intermediate between these proteins and wild-type SecA in terms of dimer formation. Furthermore, both SecAΔ11 and SecAΔ11/N95 were equally defective in translocation ATPase specific activity. Our studies showed that the nonessential carboxyl-terminal 70 residues of SecA play no role in its dimerization, while increasing the truncation of the amino-terminal region of SecA from 8 to 11 residues results in increased defects in SecA dimerization and poor in vivo function unless the protein is highly overexpressed. They also clarified a number of conflicting previous reports and support the essential nature of the SecA dimer.

Plasmodium falciparum, the mosquito-transmitted Apicomplexan parasite, causes the most severe form of human malaria. In the asexual blood-stage, the parasite resides within erythrocytes where it proliferates, multiplies and finally spreads to new erythrocytes. Development of drugs targeting the ribosome, the site of protein synthesis, requires specific knowledge of its structure and work cycle, and, critically, the ways they differ from those in the human host. Here, we present five cryo-electron microscopy (cryo-EM) reconstructions of ribosomes purified from P. falciparum blood-stage schizonts at sub-nanometer resolution. Atomic models were built from these density maps by flexible fitting. Significantly, our study has taken advantage of new capabilities of cryo-EM, in visualizing several structures co-existing in the sample at once, at a resolution sufficient for building atomic models. We have discovered structural and dynamic features that differentiate the ribosomes of P. falciparum from those of mammalian system. Prompted by the absence of RACK1 on the ribosome in our and an earlier study we confirmed that RACK1 does not specifically co-purify with the 80S fraction in schizonts. More extensive studies, using cryo-EM methodology, of translation in the parasite will provide structural knowledge that may lead to development of novel anti-malarials.

The two major components of the Eubacteria Sec-dependent protein translocation system are the heterotrimeric channel-forming component SecYEG and its binding partner, the SecA ATPase nanomotor. Once bound to SecYEG, the preprotein substrate, and ATP, SecA undergoes ATP-hydrolytic cycles that drive the stepwise translocation of proteins. Although a previous site-directed in vivo photocross-linking study (Mori, H., and Ito, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 16159–16164) elucidated residues of SecY needed for interaction with SecA, no reciprocal study for SecA protein has been reported to date. In the present study we mapped residues of SecA that interact with SecY or SecG utilizing this approach. Our results show that distinct domains of SecA on two halves of the molecule interact with two corresponding SecY partners as well as with the central cytoplasmic domain of SecG. Our data support the in vivo relevance of the Thermotoga maritima SecA·SecYEG crystal structure that visualized SecYEG interaction for only one-half of SecA as well as previous studies indicating that SecA normally binds two molecules of SecYEG.

The SecA nanomotor promotes protein translocation in Eubacteria by binding both protein cargo and the protein-conducting channel and undergoing ATP-driven conformation cycles that drive this process. Conflicting reports exist as to whether SecA functions as a monomer or dimer during this dynamic process.Here we re-examine the role of amino and carboxyl termini of SecA in promoting its dimerization and functional state by examining three secA mutants and their respective proteins: SecAD8 lacking residues 2-8, SecAD11 lacking residues 2-11, and SecAD11/N95 lacking both residues 2-11 and its carboxyl-terminal 70 residues. We demonstrate that whether or not SecAD11 or SecAD11/N95 was functional for promoting cell growth depends solely on their vivo levels that appear to govern residual dimerization. Cell fractionation revealed that SecAD11 and SecAD11/N95 were still proficient in membrane association, although they were reduced in the formation of integral membrane SecA. The presence of a modestly higher level of SecAD11/N95 in the membrane and its ability to form dimers as detected by chemical crosslinking were consistent with the higher secA expression level and better growth property of this mutant compared to secAD11. Biochemical studies showed that SecAD11 and SecAD11/N95 displayed identical dimerization defects, while SecAD8 was intermediate between these proteins and wildtype SecA. Furthermore, both SecAD11 and SecAD11/N95 were equally defective in their translocation ATPase specific activity. Our studies show that the non-essential carboxyl-terminal 70 residues of SecA play no role in its dimerization, while increasing truncation of the amino-terminal region of SecA from 8 to 11 residues results in an increasing defect in SecA dimerization and poor in vivo function unless highly overexpressed and also clarify a number of conflicting reports in the literature and support the essential nature of the SecA dimer.

In Escherichia coli (E. coli) the major secretory pathway for proteins is the “general secretion pathway” or “Sec-dependent pathway”. Its major components include the SecA ATPase, the SecB chaperone, and SecYEG: the channel-forming complex. Once bound to SecYEG, the preprotein substrate, and ATP, SecA undergoes an ATP-driven conformational cycle that drives the step-wise translocation of proteins. While cytosolic SecA specifically recognizes and binds SecYEG, the specific residues by which these two proteins interact are not known. A study from Mori and Ito (2006) analyzed SecY-SecA interaction by using an in vivo site-directed cross-linking technique developed by Schultz and co-workers. In this experimental system, an amber suppressor tRNA and a tyrosyl-tRNA synthetase from Methanococcus jannaschii are genetically engineered to allow for the charging of the tRNA with p-benzoyl-phenylalanine (pBpa), a photo-reactive phenylalanine derivative. They showed that pBpa residues introduced into the second, fourth, fifth, and sixth cytoplasmic domains of SecY could be crosslinked to SecA.We have chosen a similar approach to Mori and Ito, but we have designed sites of in vivo pBpa incorporation into SecA based on an in vivo SecA membrane topology study performed by Jilaveanu et al. (2006). The amber positions in SecA were chosen based on residues of SecA that were accessible to small molecule labeling from the exterior side of the membrane-indicating proximity to the SecYEG channel. Our results demarcate novel sites of SecA interaction with SecY. Our data provide in vivo support for the biological relevance of the recent SecA-SecYEG X-ray structure (Zimmer et al., 2008), but they also demonstrate the importance of both the SecA N-domain as well as its C-domain in stabilizing the interaction in vivo.