2.9 The Growth of Electron Microscopy in Japan

Fukami, Akira; Adachi, Kôichi; Asakura, Kentaro

doi:10.1016/s1076-5670(08)70048-x

Cited by 20 publications

(26 citation statements)

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“…Grid Preparation-Tubes were transferred by pipetting 5 l from either the surface or the subphase onto 400-mesh copper electron microscope grids (Gilder, Ted Pella) to which a carbon-coated holey film had been applied (29,30) so that the sample was suspended in a thin aqueous layer across the holes. For cryo-preservation of the helical tubes, excess sample was wicked off with Whatman No.…”

Section: Methodsmentioning

confidence: 99%

How a G Protein Binds a Membrane

Zhang

Melia²,

et al. 2004

Journal of Biological Chemistry

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Heterotrimeric G proteins interact with receptors and effectors at the membrane-cytoplasm interface. Structures of soluble forms have not revealed how they interact with membranes. We have used electron crystallography to determine the structure in ice of a helical array of the photoreceptor G protein, transducin, bound to the surface of a tubular lipid bilayer. The protein binds to the membrane with a very small area of contact, restricted to two points, between the surface of the protein and the surface of the lipids. Fitting the x-ray structure into the membrane-bound structure reveals one membrane contact near the lipidated G␥ C terminus and G␣ N terminus, and another near the G␣ C terminus. The narrowness of the tethers to the lipid bilayer provides flexibility for the protein to adopt multiple orientations on the membrane, and leaves most of the G protein surface area available for protein-protein interactions.G proteins mediate signal transduction pathways on the membranes of virtually all eukaryotic cells. In the inactive, GDP-bound, heterotrimeric state most G proteins are themselves tightly membrane associated. Activation takes place when GTP is exchanged for GDP in a complex with an integral membrane G protein-coupled receptor, which produces separated and active G␣-GTP and G␤␥ proteins. These active subunits are then free to move along the membrane and regulate their downstream effectors that may be either peripherally membrane associated (e.g. cGMP phosphodiesterase, phospholipase C) or integral membrane proteins (e.g. adenylyl cyclase and ion channels).There have been numerous studies of G protein-membrane interactions using biochemical, functional, and mutagenic approaches. These have made it clear that membrane association and localization play a critical role in G protein function and have also revealed biochemical features of G␣ and G␥ that are important for membrane binding. All G␣ subunits have one or more fatty acids added at or near their N termini, and both the fatty acids and the amino acids in the N-terminal region are important for membrane binding (1-3). The attachment site is either an N-terminal glycine, which is linked to fatty acid, usually myristic acid, in an amide linkage, or a cysteine at position 2, which is linked to palmitic acid in a thioester linkage. The G␥ subunits are proteolytically processed to leave a C-terminal methyl-esterified cysteine residue linked via a thioether to either a 15-or 20-carbon isoprenyl chain (4 -8).Forms of G␥ without these modifications have been studied, and they show reduced interactions with membranes and defective signaling properties (9 -14).In the crystal structures of G protein heterotrimers (15, 16) these hydrophobic modifications and their immediately adjacent amino acids are missing; however, the resolved portions of the G␥ C terminus and the G␣ N terminus are close to one another in space, suggesting a common site for membrane insertion. The only G protein structure in which a lipid modification is present and visible is the complex o...

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Section: Methodsmentioning

confidence: 99%

How a G Protein Binds a Membrane

Zhang

Melia²,

et al. 2004

Journal of Biological Chemistry

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“…The carbon films were prepared according to the procedure of Fukami and Adachi (24). The hole sizes varied considerably but were in the range of microns.…”

Section: Methodsmentioning

confidence: 99%

Rotational symmetry of the C ring and a mechanism for the flagellar rotary motor

Thomas

Morgan

DeRosier

1999

Proc. Natl. Acad. Sci. U.S.A.

133

112

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FliG, FliM, and FliN, key proteins for torque generation, are located in two rings. The first protein is in the M ring and the last two are in the C ring. The rotational symmetries of the C and M rings have been determined to be about 34 (this paper) and 26 (previous work), respectively. The mechanism proposed here depends on the symmetry mismatch between the rings: the C ring extends 34 levers, of which 26 can bind to the 26 equivalent sites on the M ring. The remaining 8 levers bind to proton-pore complexes (studs) to form 8 torque generators. Movement results from the swapping of stud-bound levers with M ring-bound levers. The model predicts that both the M and C rings rotate in the same direction but at different speeds.The bacterial flagellum [reviewed by Namba and Vorderviszt (1)], the organ of motility in Salmonella typhimurium and many other species of prokaryotes, is powered by a reversible rotary motor. The motor is embedded in the cell envelope, possesses up to eight torque-generating units (2, 3), and is powered by a H ϩ gradient across the cytoplasmic membrane (4, 5). Each revolution consumes Ϸ1,000 H ϩ (6) and requires Ϸ400 steps (7). Initial observations (8, 9) of the cell-proximal end of the flagellum, the site of the motor, revealed an assembly, known as the basal-body complex, composed of several rings surrounding an axial structure. This axial structure extends from the cell to form the flagellar propeller, also known as the filament. Initially, there were four rings in basal-body complexes extracted from S. typhimurium. The L and P rings are associated with the lipopolysaccharide and peptidoglycan layers of the outer membrane, respectively, and are thought to serve as bushings. Two proteins (FlgH and FlgI) make up this structure (10). The S ring sits in the periplasm next to the inner membrane. The M ring crosses the cytoplasmic membrane and serves to transfer torque from the motor to the axial structures on the outside of the cell. A single protein, FliF, gives rise to both the M and S rings (11). Quantitative gel electrophoresis and autoradiography based on 35 Sradiolabeling of basal-body complex proteins (12) and scanning transmission electron microscopy mass measurements of purified basal-body complexes (13) reveal that there are 26 (Ϯ Ϸ2) copies of each of the proteins comprising the L, P, and MS rings. FliG, a protein important in torque generation, binds to FliF, producing a thickened M ring (14, 15). Throughout this paper, when we refer to the M ring, we mean this extended structure of FliG bound to FliF.More recently, a fifth ring has been revealed in preparations of basal-body complexes extracted with less harsh isolation procedures (15-17). This ring is referred to as the C (cytoplasmic) ring and minimally appears to contain FliM and FliN (15) (21) suggest that the stoichiometry of FliN/FliM is more than 1:1. Given the errors in the estimates, the number of subunits of FliM, and hence the rotational symmetry of the C ring, are uncertain.In the membrane surrounding the moto...

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“…Hexyl-agarose-purified G t was ϳ95% pure by Coomassie staining on SDS-polyacrylamide gel electrophoresis gels. For G␣ t -GTP␥S, either G t was extracted from ROS with Low salt 1 ϩ GTP␥S, or hexyl-agarose-purified G t was activated with a catalytic amount of urea-washed ROS in the presence of GTP␥S and a trace amount of GTP-␥- 35 S. G␣ t -GTP␥S was separated from G␤␥ by loading onto a Reactive Blue 2-Sepharose CL-6B column (Sigma) and eluted in increasing [KCl]. In some cases, G t or G␣ t -GTP␥S were further purified by high performance liquid chromatography anion-exchange, as described (12).…”

Section: Methodsmentioning

confidence: 99%

“…When G␣ t -GTP␥S concentrations determined by this assay were compared with those determined by radioactivity of purified protein prepared using GTP-␥- 35 S, the ratio was found to be 1.04. Preparation of Vesicles-Lipids were mixed in organic solvent and dried down under a stream of N 2 .…”

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Enhancement of Phototransduction Protein Interactions by Lipid Surfaces

Melia¹,

Malinski²,

et al. 2000

Journal of Biological Chemistry

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The G protein cascade of vision depends on two peripheral membrane proteins: the G protein, transducin (G t ), and cGMP phosphodiesterase (PDE). Each has covalently attached lipids, and interacts with transduction components on the membrane surface. We have found that their surface interactions are critically dependent on the nature of the lipid. Membranes enhance their protein-protein interactions, especially if electrostatic attraction is introduced with positively charged lipids. These interactions are less enhanced on highly curved surfaces, but are most enhanced by unsaturated or bulky acyl chains. On positively charged membranes, G t assembles at a high enough density to form two-dimensional arrays with short-range crystalline order. Cationic membranes also support extremely efficient activation of PDE by the GTP␥S (guanosine 5-O-(thiotriphosphate)) form of G␣ t (G␣ t -GTP␥S), minimizing functional heterogeneity of transducin and allowing activation with nanomolar G␣ t -GTP␥S. Quantification of PDE activation and of the amount of G␣ t -GTP␥S bound to PDE indicated that G t activates PDE maximally when bound in a 1:1 molar ratio. No cooperativity was observed, even at nanomolar concentrations. Thus, under these conditions, the one binding site for G␣ t -GTP␥S on PDE that stimulates catalysis must be of higher affinity than one or more additional sites which are silent with respect to activation of PDE.Transduction of extracellular signals into intracellular responses by heterotrimeric G proteins (reviewed in Ref. 1) is dominated by protein-protein interactions occurring on the cytoplasmic surfaces of membranes containing heptahelical receptors. G protein subunits convey information between transmembrane receptors and effectors that are also typically either integral or peripheral membrane proteins. Regulators of transduction, such as receptor kinases, arrestin, and its homologues, and GTPase accelerating proteins, are also often localized to the signaling domains of cell membranes. Localization of multiple cascade components may be important for achieving specificity of interactions, and rapid tightly coupled responses.The G protein cascade of vision has served as a useful model system for studying the role of lipid membranes in signal transduction. Both the G protein transducin (G t ) 1 and its effector, cGMP phosphodiesterase (PDE) are normally associated with the surface of disc membranes in rod outer segments, and each contains two covalently attached lipids: farnesyl and geranylgeranyl groups on PDE␣ and PDE␤, respectively (2), and NH 2 -terminal fatty acid (3-6) and COOH-terminal farnesyl (7, 8) on G␣ t and G␥ t , respectively. However, these lipids confer only modest membrane affinity, and both PDE and G t can be selectively extracted without use of detergents, and purified by chromatography. Interactions between activated forms of the ␣ subunit of the G protein, e.g. G␣ t -GTP␥S, and PDE can be reconstituted using the purified proteins, and show a strong membrane dependence (9 -13).The utility of th...

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2.9 The Growth of Electron Microscopy in Japan

Cited by 20 publications

References 0 publications

How a G Protein Binds a Membrane

How a G Protein Binds a Membrane

Rotational symmetry of the C ring and a mechanism for the flagellar rotary motor

Enhancement of Phototransduction Protein Interactions by Lipid Surfaces

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