Transmembrane topology of the sulfonylurea receptor SUR1

Sulfonylurea receptors (SURx) are multi-spanning transmembrane proteins of the ATP-binding cassette (ABC) family, which associate with Kir6.x to form ATP-sensitive potassium channels. Two models, with 13-17 transmembrane segments, have been proposed for SURx topologies. Recently, we demonstrated that the amino-terminal region of SUR1 contains 5 transmembrane segments, supporting the 17-transmembrane model. To investigate the topology of the complete full-length SUR1, two strategies were employed. Topology was probed by accessibility of introduced cysteines to a membrane-impermeable biotinylating reagent, biotin maleimide. Amino acid positions 6/26, 99, 159, 337, 567, 1051, and 1274 were accessible, therefore extracellular, whereas many endogenous and some introduced cysteines were inaccessible, thus likely cytoplasmic or intramembrane. These sites correspond to extracellular loops 1-3, 5-6, and 8 and the NH2 terminus, and intracellular loops 3-8 and COOH terminus in the 17-transmembrane model. Immunofluorescence was used to determine accessibility of epitope-tagged SUR1 in intact and permeabilized cells. Epitopes at positions 337 and 1050 (putative external loops 3 and 6) were labeled in intact cells, therefore external, whereas positions 485 and 1119 (putative internal loops 5 and 7) only were accessible after permeabilization and therefore internal. These results are compatible with the 17-transmembrane model with two pairs of transmembrane segments as possible reentrant loops.     

Capturing cold-stress-related sequence diversity from a wild relative of common bean (Phaseolus angustissimus)

One restriction to the cultivation of common bean, Phaseolus vulgaris L., is its limited tolerance to low temperatures. In the present study, subtraction suppression hybridization was employed to enrich for stress responsive genes in both a chilling-susceptible common bean and a relatively more chilling-tolerant wild bean species, Phaseolus angustissimus. For each species, approximately 11 000 expressed sequence tags were generated. Comparative sequence analysis of the EST collection with the available annotated genome sequences of the model Fabaceae species Medicago truncatula and Glycine max identified protein homologues for approximately 65% and 80% of the Phaseolus sequences, respectively. This difference reflects the closer phylogenetic relationship between the genera Phaseolus and Glycine compared with Medicago. Annotation of the Phaseolus sequences was facilitated through this comparative analysis and indicated that several heat shock proteins, cytochrome P450s, and DNA binding factors were uniquely found among the sequences from the wild species P. angustissimus. The Phaseolus sequences have been made available on a GBrowse implementation using M. truncatula as the reference genome, providing rapid access to the sequence data and associated comparative genome data.     

Lamellar Gel (Lβ) Phases of Ternary Lipid Composition Containing Ceramide and Cholesterol

Lipid lateral segregation into specific domains in cellular membranes is associated with cell signaling and metabolic regulation. This phenomenon partially arises as a consequence of the very distinct bilayer-associated lipid physico-chemical properties that give rise to defined phase states at a given temperature. Until now lamellar gel (L_β) phases have been described in detail in single or two-lipid systems. Using x-ray scattering, differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy, we have characterized phases of ternary lipid compositions in the presence of saturated phospholipids, cholesterol, and palmitoyl ceramide mixtures. These phases stabilized by direct cholesterol-ceramide interaction can exist either with palmitoyl sphingomyelin or with dipalmitoyl phosphatidylcholine and present intermediate properties between raft-associated phospholipid-cholesterol liquid-ordered and phospholipid-ceramide L_β phases. The present data provide novel, to our knowledge, evidence of a chemically defined, multicomponent lipid system that could cooperate in building heterogeneous segregated platforms in cell membranes.     

Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels

The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking.     

Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice

Bax and Bak are critical effectors of apoptosis. Although both are widely expressed and usually functionally redundant, recent studies suggest that Bak has particular importance in certain cell types. Genetic and biochemical studies indicate that Bak activation is prevented primarily by Mcl-1 and Bcl-x_L, whereas Bax is held in check by all pro-survival Bcl-2 homologues, including Bcl-2 itself. In this study, we have investigated whether loss of Bak or elevated Mcl-1 modulates haemopoietic abnormalities provoked by overexpression of Bcl-2. The Mcl-1 transgene had little impact, probably because the expression level was insufficient to effectively reduce Bak activation. However, loss of Bak enhanced lymphocytosis in vavP-BCL-2 transgenic mice and increased resistance of their thymocytes to some cytotoxic agents, implying that Bak-specific signals can be triggered in certain lymphoid populations. Nevertheless, lack of Bak had no significant impact on thymic abnormalities in vavP-BCL-2tg mice, which kinetic analysis suggested was due to accumulation of self-reactive thymocytes that resist deletion. Intriguingly, although Bak^−/− mice have elevated platelet counts, Bak^−/−vavP-BCL-2 mice, like vavP-BCL-2 littermates, were thrombocytopaenic. To clarify why, the vavP-BCL-2 platelet phenotype was scrutinised more closely. Platelet life span was found to be elevated in vavP-BCL-2 mice, which should have provoked thrombocytosis, as in Bak^−/− mice. Analysis of bone marrow chimaeric mice suggested the low platelet phenotype was due principally to extrinsic factors. Following splenectomy, blood platelets remained lower in vavP-BCL-2 than wild-type mice. However, in Rag1^−/− BCL-2tg mice, platelet levels were normal, implying that elevated lymphocytes are primarily responsible for BCL-2tg-induced thrombocytopaenia.     

Alanine: Glyoxylate aminotransferase 1 is required for mobilization and utilization of triglycerides during infection process of the rice blast pathogen,Magnaporthe oryzae

The rice blast pathogen, Magnaporthe oryzae has been widely used as a model pathogen to study plant infection-related fungal morphogenesis, such as penetration via appressorium and plant-microbe interactions at the molecular level. Previously, we identified a gene encoding peroxisomal alanine: glyoxylate aminotransferase 1 (AGT1) in M. oryzae and demonstrated that the AGT1 was indispensable for pathogenicity. The AGT1 knockout mutants were unable to penetrate the host plants, such as rice and barley, and therefore were non-pathogenic. The inability of ∆Moagt1 mutants to penetrate the susceptible plants was likely due to the disruption in coordination of the β-oxidation and the glyoxylate cycle resulted from a blockage in lipid droplet mobilization and eventually utilization during conidial germination and appressorium morphogenesis, respectively. Here, we further demonstrate the role of AGT1 in lipid mobilization by in vitro germination assays and confocal microscopy.     

Slips, leaks and channels in glutamate transporters

Glutamate transporters are unusual proteins in that they can function as both a transporter and a chloride channel. With the recent determination of the crystal structure of an archaeal aspartate transporter it is now possible to begin to put together a physical picture of how these proteins are able to carry out their dual functions. In this review we shall discuss our current understanding of the functional states of glutamate transporters and how they may arise. We will also discuss some of the alternate conducting states of glutamate transporters and provide definitions of the various states.     

Interactions Among the Aphid Diuraphis noxia , the Entomopathogenic Fungus Paecilomyces fumosoroseus and the Coccinellid Hippodamia convergens

Paecilomyces fumosoroseus (Wize) Brown & Smith is under development as a mycoinsecticide for control of the Russian wheat aphid, Diuraphis noxia Kurdjumov. Interactions with other natural enemies within the agro-ecosystem, such as the coccinellid Hippodamia convergens Guerin, require evaluation before its potential can be realized. In laboratory bioassays H. convergens adults were sprayed with suspensions of P. fumosoroseus conidia at different concentrations (including potential field rates) and mortality assessed. Although a proportion of coccinellids succumbed to infection (the greatest proportion was 22% when the ladybirds had suffered stress) it is unlikely that they would be at risk from infection as a direct result of a spray application unless there were prolonged periods of high humidity or the coccinellids were stressed. When provided with uninfected or P. fumosoroseus -infected D. noxia cadavers as prey, coccinellids consumed more uninfected aphids. The predators never consumed aphid cadavers from which the fungus was erupting or sporulating. Hippodamia convergens is, therefore, unlikely to be a significant intraguild predator of P. fumosoroseus . Predators contaminated with conidia of P. fumosoroseus using different methods (sprayed coccinellids, coccinellids foraging on sprayed aphids, and those foraging in the presence of sporulating D. noxia cadavers) were able to transfer conidia to healthy D. noxia populations and initiate infection in a proportion of those aphids. The proportion of aphids becoming infected was greatest when the coccinellids became contaminated when foraging amongst sporulating cadavers. Some coccinellids also succumbed to infection under these conditions.