Search

The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a "down" state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an "up" state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme's hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual "open" conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis.

FUS (fused in sarcoma) proteinopathy is a group of neurodegenerative diseases characterized by the formation of inclusion bodies containing the FUS protein, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Previous studies show that mitochondrial damage is an important aspect of FUS proteinopathy. However, the molecular mechanisms by which FUS induces mitochondrial damage remain to be elucidated. Our biochemical and genetic experiments demonstrate that FUS interacts with the catalytic subunit of mitochondrial ATP synthase (ATP5B), disrupts the formation of ATP synthase complexes, and inhibits mitochondrial ATP synthesis. FUS expression activates the mitochondrial unfolded protein response (UPR(mt)). Importantly, down-regulating expression of ATP5B or UPR(mt) genes in FUS transgenic flies ameliorates neurodegenerative phenotypes. Our data show that mitochondrial impairment is a critical early event in FUS proteinopathy, and provide insights into the pathogenic mechanism of FUS-induced neurodegeneration.

ATP synthases are remarkable proteins that regenerate the molecular fuel for cellular processes in all domains of life (1). Embedded into the inner membranes of mitochondria, chloroplasts, and bacteria, FoF1-ATP synthase produces most of the cellular ATP, using ADP and inorganic phosphate as inputs (Fig. 1 A ). This thermodynamically unfavorable reaction is powered by a proton electrochemical gradient across the membrane, which drives the rotation of the c-ring in the Fo part of ATP synthase (2). The asymmetrical central shaft connecting Fo and F1 then deforms the three active sites in the F1 part to catalyze ATP synthesis (1). The proper function of this machine requires efficient rotation of the c-ring in a highly viscous lipid membrane without any proton leaks. This problem is compounded by the unusual structure of the a-subunit facing the c-ring, with its long helices parallel to the membrane distorting the lipid packing around the Fo part (3, 4). Fig. 1. Schematic of FoF1-ATP synthase function. ( A , Top ) Proton translocation mediated by the c-ring of the membrane-embedded Fo part drives rotation. ( A , Bottom ) The asymmetrically shaped rotor axis attached to the c-ring distorts the active sites in the F1 part, where ATP is synthesized from ADP and inorganic phosphate Pi. Simulations by Duncan et al. (5) show that CL, an abundant lipid in the inner mitochondrial membrane, accumulates near the c-ring. The structure is based on Protein Data Bank ID code 5FIK (4). ( B ) CL is an unusual lipid with four acyl chains. In PNAS, Duncan et al. (5) report that an unusual lipid–protein interaction in the Fo part of ATP synthase is central to efficient … [↵][1]1To whom correspondence should be addressed. Email: gerhard.hummer{at}biophys.mpg.de. [1]: #xref-corresp-1-1

The type of metabolic compartmentalization that occurs in red blood cells differs from the types that exist in most eukaryotic cells, such as intracellular organelles. In red blood cells (ghosts), ATP is sequestered within the cytoskeletal–membrane complex. These pools of ATP are known to directly fuel both the Na + /K + and Ca 2+ pumps. ATP can be entrapped within these pools either by incubation with bulk ATP or by operation of the phosphoglycerate kinase and pyruvate kinase reactions to enzymatically generate ATP. When the pool is filled with nascent ATP, metabolic labeling of the Na + /K + or Ca 2+ pump phosphoproteins (E Na -P and E Ca -P, respectively) from bulk [γ- 32 P]-ATP is prevented until the pool is emptied by various means. Importantly, the pool also can be filled with the fluorescent ATP analog trinitrophenol ATP, as well as with a photoactivatable ATP analog, 8-azido-ATP (N 3 -ATP). Using the fluorescent ATP, we show that ATP accumulates and then disappears from the membrane as the ATP pools are filled and subsequently emptied, respectively. By loading N 3 -ATP into the membrane pool, we demonstrate that membrane proteins that contribute to the pool’s architecture can be photolabeled. With the aid of an antibody to N 3 -ATP, we identify these labeled proteins by immunoblotting and characterize their derived peptides by mass spectrometry. These analyses show that the specific peptides that corral the entrapped ATP derive from sequences within β-spectrin, ankyrin, band 3, and GAPDH.

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and acts as a key feedback regulatory component of photosynthesis. Arabidopsis possesses two homologues of the regulatory γ subunit of the ATP synthase, encoded by the ATPC1 and ATPC2 genes. Using a series of mutants, we show that both these subunits can support photosynthetic ATP synthesis in vivo with similar specific activities, but that in wild-type plants, only γ 1 is involved in ATP synthesis in photosynthesis. The γ 1 -containing ATP synthase shows classical light-induced redox regulation, whereas the mutant expressing only γ 2 -ATP synthase (gamma exchange-revised ATP synthase, gamera ) shows equally high ATP synthase activity in the light and dark. In situ redox titrations demonstrate that the regulatory thiol groups on γ 2 -ATP synthase remain reduced under physiological conditions but can be oxidized by the strong oxidant diamide, implying that the redox potential for the thiol/disulphide transition in γ 2 is substantially higher than that for γ 1 . This regulatory difference may be attributed to alterations in the residues near the redox-active thiols. We propose that γ 2 -ATP synthase functions to catalyze ATP hydrolysis-driven proton translocation in nonphotosynthetic plastids, maintaining a sufficient transthylakoid proton gradient to drive protein translocation or other processes. Consistent with this interpretation, ATPC2 is predominantly expressed in the root, whereas modifying its expression results in alteration of root hair development. Phylogenetic analysis suggests that γ 2 originated from ancient gene duplication, resulting in divergent evolution of functionally distinct ATP synthase complexes in dicots and mosses.

ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5′-(β,γ-imido)triphosphate or ADP in conjunction with phosphate analogs , , or , were determined to 2.2- to 2.4-Å resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.

The catalytic domain of the F-ATPase in mitochondria protrudes into the matrix of the organelle, and is attached to the membrane domain by central and peripheral stalks. Energy for the synthesis of ATP from ADP and phosphate is provided by the transmembrane proton-motive-force across the inner membrane, generated by respiration. The proton-motive force is coupled mechanically to ATP synthesis by the rotation at about 100 times per second of the central stalk and an attached ring of c-subunits in the membrane domain. Each c-subunit carries a glutamate exposed around the midpoint of the membrane on the external surface of the ring. The rotation is generated by protonation and deprotonation successively of each glutamate. Each 360° rotation produces three ATP molecules, and requires the translocation of one proton per glutamate by each c-subunit in the ring. In fungi, eubacteria, and plant chloroplasts, ring sizes of c 10 –c 15 subunits have been observed, implying that these enzymes need 3.3–5 protons to make each ATP, but until now no higher eukaryote has been examined. As shown here in the structure of the bovine F 1 -c-ring complex, the c-ring has eight c-subunits. As the sequences of c-subunits are identical throughout almost all vertebrates and are highly conserved in invertebrates, their F-ATPases probably contain c 8 -rings also. Therefore, in about 50,000 vertebrate species, and probably in many or all of the two million invertebrate species, 2.7 protons are required by the F-ATPase to make each ATP molecule.

Adenosine 5′-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the ε subunit of the bacterial F o F 1 -ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 μM to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of F o F 1 -ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

Aerobic life is based on a molecular machinery that utilizes oxygen as a terminal electron sink. The membrane-bound cytochrome c oxidase (CcO) catalyzes the reduction of oxygen to water in mitochondria and many bacteria. The energy released in this reaction is conserved by pumping protons across the mitochondrial or bacterial membrane, creating an electrochemical proton gradient that drives production of ATP. A crucial question is how the protons pumped by CcO are prevented from flowing backwards during the process. Here, we show by molecular dynamics simulations that the conserved glutamic acid 242 near the active site of CcO undergoes a protonation state-dependent conformational change, which provides a valve in the pumping mechanism. The valve ensures that at any point in time, the proton pathway across the membrane is effectively discontinuous, thereby preventing thermodynamically favorable proton back-leakage while maintaining an overall high efficiency of proton translocation. Suppression of proton leakage is particularly important in mitochondria under physiological conditions, where production of ATP takes place in the presence of a high electrochemical proton gradient.

The H + /ATP ratio is an important parameter for the energy balance of all cells and for the coupling mechanism between proton transport and ATP synthesis. A straightforward interpretation of rotational catalysis predicts that the H + /ATP coincides with the ratio of the c-subunits to β-subunits, implying that, for the chloroplast and Escherichia coli ATPsynthases, numbers of 4.7 and 3.3 are expected. Here, the energetics described by the chemiosmotic theory was used to determine the H + /ATP ratio for the two enzymes. The isolated complexes were reconstituted into liposomes, and parallel measurements were performed under identical conditions. The internal phase of the liposomes was equilibrated with the acidic medium during reconstitution, allowing to measure the internal pH with a glass electrode. An acid–base transition was carried out and the initial rates of ATP synthesis or ATP hydrolysis were measured with luciferin/luciferase as a function of ΔpH at constant Q = [ATP]/([ADP][P i ]). From the shift of the equilibrium ΔpH as a function of Q the standard Gibbs free energy for phosphorylation, Δ G p 0 ′; and the H + /ATP ratio were determined. It resulted Δ G p 0 ′ = 38 ± 3 kJ·mol −1 and H + /ATP = 4.0 ± 0.2 for the chloroplast and H + /ATP = 4.0 ± 0.3 for the E. coli enzyme, indicating that the thermodynamic H + /ATP ratio is the same for both enzymes and that it is different from the subunit stoichiometric ratio.

The intrinsic chemical reaction of adenosine triphosphate (ATP) hydrolysis catalyzed by myosin is modeled by using a combined quantum mechanics and molecular mechanics (QM/MM) methodology that achieves a near ab initio representation of the entire model. Starting with coordinates derived from the heavy atoms of the crystal structure (Protein Data Bank ID code 1VOM ) in which myosin is bound to the ATP analog ADP·VO 4 − , a minimum-energy path is found for the transformation ATP + H 2 O → ADP + P i that is characterized by two distinct events: ( i ) a low activation-energy cleavage of the P γ O βγ bond and separation of the γ-phosphate from ADP and ( ii ) the formation of the inorganic phosphate as a consequence of proton transfers mediated by two water molecules and assisted by the Glu-459–Arg-238 salt bridge of the protein. The minimum-energy model of the enzyme–substrate complex features a stable hydrogen-bonding network in which the lytic water is positioned favorably for a nucleophilic attack of the ATP γ-phosphate and for the transfer of a proton to stably bound second water. In addition, the P γ O βγ bond has become significantly longer than in the unbound state of the ATP and thus is predisposed to cleavage. The modeled transformation is viewed as the part of the overall hydrolysis reaction occurring in the closed enzyme pocket after ATP is bound tightly to myosin and before conformational changes preceding release of inorganic phosphate.

ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [α- 32 P]8-N 3 -ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N -ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating.

In a rotary motor F o F 1 -ATP synthase that couples H + transport with ATP synthesis/hydrolysis, it is thought that an F o c subunit oligomer ring ( c -ring) in the membrane rotates as protons pass through F o and a 120° rotation produces one ATP at F 1 . Despite several structural studies, the copy number of F o c subunits in the c -ring has not been determined for any functional F o F 1 . Here, we have generated and isolated thermophilic Bacillus F o F 1 , each containing genetically fused 2-mer–14-mer c ( c 2 – c 14 ). Among them, F o F 1 containing c 2 , c 5 , or c 10 showed ATP-synthesis and other activities. When F 1 was removed, F o containing c 10 worked as an H + channel but F o s containing c 9 , c 11 or c 12 did not. Thus, the c -ring of functional F o F 1 of this organism is a decamer. The inevitable consequence of this finding is noninteger ratios of rotation step sizes of F 1 /F o (120°/36°) and of H + /ATP (10:3). This step-mismatch necessitates elastic twisting of the rotor shaft (and/or the side stalk) during rotation and permissive coupling between unit rotations by H + transport at F o and elementary events in catalysis at F 1 .

ATP is the universal energy currency of living cells, and the majority of it is synthesized by the F 1 F 0 ATP synthase. Inhibitors of this enzyme are therefore potentially detrimental for all life forms. Tributyltin chloride (TBT-Cl) inhibits ATP hydrolysis by the Na + -translocating ATP synthase of Ilyobacter tartaricus or the H + -translocating counterpart of Escherichia coli with apparent K i of 200 nM. To target the site of this inhibition, we synthesized a tritium-labeled derivative of TBT-Cl in which one of the butyl groups was replaced by a photoactivatable aryldiazirine residue. Upon illumination, subunit a of the ATP synthase becomes specifically modified, and this labeling is suppressed in the presence of the original inhibitor. In case of the Na + ATP synthase, labeling is also suppressed in the presence of Na + ions, suggesting an interference in Na + or TBT-Cl binding to subunit a. This interference is corroborated by the protection of ATP hydrolysis from TBT-Cl inhibition by 105 mM Na + . TBT-Cl strongly inhibits Na + exchange by the reconstituted I. tartaricus ATP synthase. Taken together these results indicate that the subunit a ion channel is the target site for ATPase inhibition by toxic organotin compounds. An inhibitor interacting specifically with this site has not been reported previously.

We show here sodium ion cycling between complex I from Klebsiella pneumoniae and the F 1 F 0 ATP synthase from Ilyobacter tartaricus in a reconstituted proteoliposome system. In the course of NADH oxidation by complex I, an electrochemical sodium ion gradient was established and served as a driving force for the synthesis of ATP from ADP and phosphate. In the opposite direction, the Δμ̃ Na + generated by ATP hydrolysis could be coupled to NADH formation by reversed electron transfer from ubiquinol to NAD. For reverse electron transfer, a transmembrane voltage larger than 30 mV was obligatory. No NADH-driven proton transport into the lumen of proteoliposomes was detected. We conclude that Na + is used as the exclusive coupling ion by the enterobacterial complex I.

The F 1 F o -type ATP synthase is the smallest motor enzyme known. Previous studies had established that the central γ and ɛ subunits of the F 1 part rotate relative to a stator of α 3 β 3 and δ subunits during catalysis. We now show that the ring of c subunits in the F o part moves along with the γ and ɛ subunits. This was demonstrated by linking the three rotor subunits with disulfide bridges between cysteine residues introduced genetically at the interfaces between the γ, ɛ, and c subunits. Essentially complete cross-linking of the γ, ɛ, and c subunits was achieved by using CuCl 2 to induce oxidation. This fixing of the three subunits together had no significant effect on ATP hydrolysis, proton translocation, or ATP synthesis, and each of these functions retained inhibitor sensitivity. These results unequivocally place the c subunit oligomer in the rotor part of this molecular machine.

The rotation of the γ-subunit has been included in the binding-change mechanism of ATP synthesis/hydrolysis by the proton ATP synthase (F O F 1 ). The Escherichia coli ATP synthase was engineered for rotation studies such that its ATP hydrolysis and synthesis activity is similar to that of wild type. A fluorescently labeled actin filament connected to the γ-subunit of the F 1 sector rotated on addition of ATP. This progress enabled us to analyze the γM23K (the γ-subunit Met-23 replaced by Lys) mutant, which is defective in energy coupling between catalysis and proton translocation. We found that the F 1 sector produced essentially the same frictional torque, regardless of the mutation. These results suggest that the γM23K mutant is defective in the transformation of the mechanical work into proton translocation or vice versa.

The epithelial Na + channel (ENaC) is composed of three homologous subunits: α, β and γ. We used gene targeting to disrupt the β subunit gene of ENaC in mice. The βENaC-deficient mice showed normal prenatal development but died within 2 days after birth, most likely of hyperkalemia. In the −/− mice, we found an increased urine Na + concentration despite hyponatremia and a decreased urine K + concentration despite hyperkalemia. Moreover, serum aldosterone levels were increased. In contrast to αENaC-deficient mice, which die because of defective lung liquid clearance, neonatal βENaC deficient mice did not die of respiratory failure and showed only a small increase in wet lung weight that had little, if any, adverse physiologic consequence. The results indicate that, in vivo , the β subunit is required for ENaC function in the renal collecting duct, but, in contrast to the α subunit, the β subunit is not required for the transition from a liquid-filled to an air-filled lung. The phenotype of the βENaC-deficient mice is similar to that of humans with pseudohypoaldosteronism type 1 and may provide a useful model to study the pathogenesis and treatment of this disorder.

During mitochondrial ATP synthesis, F 1 -ATPase—the portion of the ATP synthase that contains the catalytic and regulatory nucleotide binding sites—undergoes a series of concerted conformational changes that couple proton translocation to the synthesis of the high levels of ATP required for cellular function. In the structure of the rat liver F 1 -ATPase, determined to 2.8-Å resolution in the presence of physiological concentrations of nucleotides, all three β subunits contain bound nucleotide and adopt similar conformations. This structure provides the missing configuration of F 1 necessary to define all intermediates in the reaction pathway. Incorporation of this structure suggests a mechanism of ATP synthesis/hydrolysis in which configurations of the enzyme with three bound nucleotides play an essential role.

We report evidence for proton-driven subunit rotation in membrane-bound F o F 1 –ATP synthase during oxidative phosphorylation. A βD380C/γC87 crosslinked hybrid F 1 having epitope-tagged βD380C subunits (β flag ) exclusively in the two noncrosslinked positions was bound to F o in F 1 -depleted membranes. After reduction of the β–γ crosslink, a brief exposure to conditions for ATP synthesis followed by reoxidation resulted in a significant amount of β flag appearing in the β–γ crosslinked product. Such a reorientation of γC87 relative to the three β subunits can only occur through subunit rotation. Rotation was inhibited when proton transport through F o was blocked or when ADP and P i were omitted. These results establish F o F 1 as the second example in nature where proton transport is coupled to subunit rotation.

During oxidative and photo-phosphorylation, F0F1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F0F1. Guided by a recent, high-resolution structure for bovine F1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central gamma subunit relative to the three catalytic beta subunits in soluble F1 from Escherichia coli. In the bovine F1 structure, a specific point of contact between the gamma subunit and one of the three catalytic beta subunits includes positioning of the homolog of E. coli gamma-subunit C87 (gamma C87) close to the beta-subunit 380DELSEED386 sequence. A beta D380C mutation allowed us to induce formation of a specific disulfide bond between beta and gamma C87 in soluble E. coli F1. Formation of the crosslink inactivated beta D380C-F1, and reduction restored full activity. Using a dissociation/reassembly approach with crosslinked beta D380C-F1, we incorporated radiolabeled beta subunits into the two noncrosslinked beta-subunit positions of F1. After reduction of the initial nonradioactive beta-gamma crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled beta subunits with gamma C87 upon reoxidation. The results demonstrate that gamma subunit rotates relative to the beta subunits during catalysis.

Gene targeting was used to create mice with a null mutation of the gene encoding the common beta subunit (beta C) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3; multi-CSF), and interleukin 5 (IL-5) receptor complexes (beta C-/- mice). High-affinity binding of GM-CSF was abolished in beta C-/- bone marrow cells, while cells from heterozygous animals (beta C+/- mice) showed an intermediate number of high-affinity receptors. Binding of IL-3 was unaffected, confirming that the IL-3-specific beta chain remained intact. Eosinophil numbers in peripheral blood and bone marrow of beta C-/- animals were reduced, while other hematological parameters were normal. In clonal cultures of beta C-/- bone marrow cells, even high concentrations of GM-CSF and IL-5 failed to stimulate colony formation, but the cells exhibited normal quantitative responsiveness to stimulation by IL-3 and other growth factors. beta C-/- mice exhibited normal development and survived to young adult life, although they developed pulmonary peribronchovascular lymphoid infiltrates and areas resembling alveolar proteinosis. There was no detectable difference in the systemic clearance and distribution of GM-CSF between beta C-/- and wild-type littermates. The data establish that beta C is normally limiting for high-affinity binding of GM-CSF and demonstrate that systemic clearance of GM-CSF is not mediated via such high-affinity receptor complexes.

Voltage-gated K+ channels are important modulators of the cardiac action potential. However, the correlation of endogenous myocyte currents with K+ channels cloned from human heart is complicated by the possibility that heterotetrameric alpha-subunit combinations and function-altering beta subunits exist in native tissue. Therefore, a variety of subunit interactions may generate cardiac K+ channel diversity. We report here the cloning of a voltage-gated K+ channel beta subunit, hKv beta 3, from adult human left ventricle that shows 84% and 74% amino acid sequence identity with the previously cloned rat Kv beta 1 and Kv beta 2 subunits, respectively. Together these three Kv beta subunits share > 82% identity in the carboxyl-terminal 329 aa and show low identity in the amino-terminal 79 aa. RNA analysis indicated that hKv beta 3 message is 2-fold more abundant in human ventricle than in atrium and is expressed in both healthy and diseased human hearts. Coinjection of hKv beta 3 with a human cardiac delayed rectifier, hKv1.5, in Xenopus oocytes increased inactivation, induced an 18-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 8.0 msec vs. 35.4 msec at -50 mV). hKv beta 3 was localized to human chromosome 3 by using a human/rodent cell hybrid mapping panel. These data confirm the presence of functionally important K+ channel beta subunits in human heart and indicate that beta-subunit composition must be accounted for when comparing cloned channels with endogenous cardiac currents.

One of the distinguishing features of the gonadotropin and thyrotropin hormone family is their heterodimeric structure, consisting of a common alpha subunit and a hormone-specific beta subunit. Subunit assembly is vital to the function of these hormones: The conformation of the heterodimer is essential for controlling secretion, hormone-specific posttranslational modifications, and signal transduction. To address whether alpha and beta subunits can be synthesized as one chain and also maintain biological activity, a chimera composed of the human chorionic gonadotropin (hCG) beta subunit genetically fused to the alpha subunit was constructed. The resulting polypeptide hCG molecule not only was efficiently secreted but also displayed an increased biological activity in vitro and in vivo. These data show that the alpha and hCG beta subunits encoded as a single chain retain a biologically active conformation similar to that seen in the heterodimer. This approach can be used to investigate structure-function relationships of the glycoprotein hormone family that were previously not tractable because of the absolute dependence on assembly for the biological response. Moreover, other bioactive multisubunit ligands can be engineered where the combination efficiency and specificity of heterodimers and homodimers are otherwise difficult to control.

Target inactivation analysis was used to determine the functional size of the charybdotoxin (ChTX) receptor in aortic and tracheal sarcolemmal membrane vesicles. This receptor has previously been shown to be an integral component of the high-conductance Ca2+-activated K+ (Maxi-K) channel in these smooth muscles. Exposure of either bovine aortic or bovine tracheal sarcolemma to high-energy irradiation results in disappearance of 125I-labeled ChTX binding activity as a monoexponential function of radiation dose; from these functions molecular masses of 88 +/- 10 kDa and 89 +/- 6 kDa, respectively, can be calculated. Similar results were obtained from radiation inactivation studies with the detergent-solubilized ChTX receptor from aortic sarcolemmal membranes. The effect of radiation on 125I-labeled ChTX binding is to decrease the number of functional ChTX receptors without affecting the affinity of receptors for the toxin, indicating that radiation is destroying, rather than altering, the binding site. The validity of the radiation inactivation technique in these membrane preparations is supported by data obtained in parallel experiments in which target sizes of the alpha 1 subunit of the L-type Ca2+ channel and 5'-nucleotidase were measured. The molecular masses determined for these entities are in excellent agreement with those expected from previous studies. The present data are discussed in terms of the recently determined subunit composition of the smooth muscle Maxi-K channel. In light of the target size, a single alpha beta subunit heterodimer complex could serve as the ChTX receptor.

Voltage-dependent cation channels are large heterooligomeric proteins. Heterologous expression of cDNAs encoding the alpha subunits alone of K+, Na+, or Ca2+ channels produces functional multimeric proteins; however, coexpression of those for the latter two with their auxiliary proteins causes dramatic changes in the resultant membrane currents. Fast-activating, voltage-sensitive K+ channels from brain contain four alpha and beta subunits, tightly associated in a 400-kDa complex; although molecular details of the alpha-subunit proteins have been determined, little is known about the beta-subunit constituent. Proteolytic fragments of a beta subunit from bovine alpha-dendrotoxin-sensitive neuronal K+ channels yielded nine different sequences. In the polymerase chain reaction, primers corresponding to two of these peptides amplified a 329-base-pair fragment in a lambda gt10 cDNA library from bovine brain; a full-length clone subsequently isolated encodes a protein of 367 amino acids (M(r) approximately 40,983). It shows no significant homology with any known protein. Unlike the channels' alpha subunits, the hydropathy profile of this sequence failed to reveal transmembrane domains. Several consensus phosphorylation motifs are apparent and, accordingly, the beta subunit could be phosphorylated in the intact K+ channels. These results, including the absence of a leader sequence and N-glycosylation, are consistent with the beta subunit being firmly associated on the inside of the membrane with alpha subunits, as speculated in a simplified model of these authentic K+ channels. Importantly, this first primary structure of a K(+)-channel beta subunit indicates that none of the cloned auxiliary proteins of voltage-dependent cation channels, unlike their alpha subunits, belong to a super-family of genes.

A protocol was designed to obtain a pure fraction of pollen mitochondria from the diploid species Nicotiana sylvestris, the female parent of the allotetraploid Nicotiana tabacum. Most organelles were morphologically intact and able to perform in organello mitochondrial (mt) protein synthesis. As revealed by two-dimensional protein electrophoresis, numerous quantitative differences exist between leaf and pollen mt proteins. Moreover, additional mt polypeptides, named R (for reproductive), encoded by either nuclear or mitochondrial genes, are found in pollen. The most abundant R polypeptide, R1 (M(r) 53,000, pI 5.6), is nuclearly encoded, is membrane bound, and cross-reacts with an antibody directed against the beta subunit of the mt ATP synthase (ATPase). N-terminal microsequence analysis showed that the two ATPase beta subunits present in leaves (beta 1 and beta 2) and the R1 pollen-specific subunit are encoded by distinct genes. A similar additional ATPase beta subunit was observed in pollen mitochondria from Petunia, suggesting that this polypeptide is of general importance for male gametophytic development in Solanaceaes.

Cotransfection assays were used to show that the members of the GTP-binding protein Gq class of alpha subunits could activate phospholipase C (PLC) beta 2. Similar experiments also demonstrated that G beta 1 gamma 1, G beta 1 gamma 5, and G beta 2 gamma 5 could activate the beta 2 isoform of PLC but not the beta 1 isoform, while G beta 2 gamma 1 did not activate PLC beta 2. To determine which portions of PLC beta 2 are required for activation by G beta gamma or G alpha, a number of PLC beta 2 deletion mutants and chimeras composed of various portions of PLC beta 1 and PLC beta 2 were prepared. We identified the N-terminal segment of PLC beta 2 with amino acid sequence extending to the end of the Y box as the region required for activation by G beta gamma and the C-terminal region as the segment containing amino acid sequences required for activation by G alpha. Furthermore, we found that coexpression of G alpha 16 and G beta 1 gamma 1 but not G beta 1 gamma 5 in COS-7 cells was able to synergistically activate recombinant PLC beta 2. We suggest that G alpha 16 may act together with free G beta 1 gamma 1 to activate PLC beta 2, while G alpha 16 may form heterotrimeric complexes with G beta 1 gamma 5 and be stabilized in an inactive form. We conclude that the regions of PLC beta 2 required for activation by G beta gamma and G alpha are physically separate and that the nature of the G beta subunit may play a role in determining the relative specificity of the G beta gamma complex for effector activation while the nature of the G gamma subunit isoform may be important for determining the affinity of the G beta gamma complex for specific G alpha proteins.

A prominent feature of the Na,K-ATPase reaction is an ATP dependence that suggests high- and low-affinity ATP requirements during the enzymic cycle. As only one ATP-binding domain has been identified in the alpha subunit and none has been identified in the beta subunit, it has seemed likely that the apparent negative cooperativity results from subunit interactions in an (alpha beta)2 diprotomer. To test this possibility, we have examined the behavior of solubilized alpha beta protomers of Na,K-ATPase down to 50 nM [gamma-32P]ATP. Active-enzyme analytical ultracentrifugation shows that the protomer is the active species and that no oligomerization occurs during turnover. However, we find that dual ATP effects can be clearly demonstrated and that nonhydrolyzable ATP analogs can stimulate the Na,K-ATPase activity of the soluble protomer. We conclude that the apparent negative cooperativity is inherent to the alpha beta protomer and that this should explain some of the complexities found with membrane-bound Na,K-ATPase and, perhaps, other P-type cation pumps.

The complete amino acid sequence and location of the disulfide bonds of two-chain botrocetin, which promotes platelet agglutination in the presence of von Willebrand factor, from venom of the snake Bothrops jararaca are presented. Sequences of the alpha and beta subunits were determined by analysis of peptides generated by digestion of the S-pyridylethylated protein with Achromobacter protease I or alpha-chymotrypsin and by chemical cleavage with cyanogen bromide or 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Two-chain botrocetin is a heterodimer composed of the alpha subunit (consisting of 133 amino acid residues) and the beta subunit (consisting of 125 amino acid residues) held together by a disulfide bond. Seven disulfide bonds link half-cystine residues 2 to 13, 30 to 128, and 103 to 120 of the alpha subunit; 2 to 13, 30 to 121, and 98 to 113 of the beta subunit; and 80 of the alpha subunit to 75 of the beta subunit. In terms of amino acid sequence and disulfide bond location, two-chain botrocetin is homologous to echinoidin (a sea urchin lectin) and other C-type (Ca(2+)-dependent) lectins.

Forskolin-resistant mutants derived from Y1 adrenocortical cells display decreased responsiveness both to receptor and postreceptor stimulators of adenylyl cyclase and decreased amounts of the alpha subunits of the GTP-binding proteins (G proteins) that mediate stimulation (Gs) and inhibition (Gi) of adenylyl cyclase--namely, Gs alpha and Gi alpha-2. This phenotype is suggestive of a mutation that affects the processing or plasma membrane incorporation of G protein alpha subunits. Since the membrane attachment of heterotrimeric G proteins has been ascribed in part to the beta gamma subunits, we examined the quantity and functional activity of beta gamma subunits in wild-type Y1 and forskolin-resistant Forsk-10r-9 and Forsk-10r-3 cells. We now show that two assays previously used to examine the activity of purified beta gamma subunits--namely, to support either rhodopsin-catalyzed guanyl nucleotide exchange on Gt alpha or pertussis toxin-catalyzed ADP-ribosylation of Gt alpha--can be used with detergent extracts of cells. In both assays the beta gamma activity in Forsk-10r-9 and Forsk-10r-3 extracts was decreased by 53-76% compared with wild-type Y1 extracts. When normalized for immunoreactive beta subunit, the beta gamma activity in the Forsk-10r-9 samples was decreased by 55-57% compared with the wild-type Y1 samples. These results suggest that a mutation of one of the G protein beta or gamma subunits may result in the multiple defects of adenylyl cyclase activity and apparent loss of G protein alpha subunits seen in the forskolin-resistant mutant cells. The frequency with which these spontaneous mutations arise in the Y1 cell line suggests that they may contribute more generally to genetic abnormalities in signal transduction.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important alpha subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The beta subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1). We report here on expression of the alpha and beta subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodoptera frugiperda insect cells by baculovirus vectors. When the beta subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the alpha subunit was expressed alone, only traces of the alpha subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These alpha subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both alpha- and beta-subunit-producing viruses, an enzyme tetramer was formed, but significant amounts of alpha and beta subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the alpha subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.

Follitropin (FSH) is a pituitary glycoprotein hormone that is essential for the development of ovarian follicles and testicular seminiferous tubules. FSH is used clinically to stimulate follicular maturation for in vitro fertilization and treatment of anovulatory women. One issue regarding the clinical use of FSH is its short half-life in the circulation. To address this point, we constructed chimeric genes containing the sequence encoding the C-terminal peptide of the chorionic gonadotropin beta subunit (CG beta) fused to the translated sequence of the human FSH beta subunit (FSH beta). This region of CG beta is important for maintaining the prolonged plasma half-life of human CG dimer. The presence of the C-terminal peptide sequence did not significantly affect assembly of FSH beta with the alpha subunit or secretion of the dimer. In vitro receptor binding and steroidogenic activity of dimer bearing the FSH beta-C-terminal peptide chimera were the same as wild-type FSH. However, both the in vivo potency and half-life in circulation of the dimer bearing either one or two C-terminal peptide units were enhanced. Dimers containing FSH beta-CG beta chimeras could serve as potent FSH agonists for clinical use, and the present strategy may have wide applications for enhancing the in vivo half-life of diverse proteins.

The beta subunit of the cGMP phosphodiesterase (PDE) gene has been identified as the candidate gene for retinal degeneration in the rd mouse. To study the molecular mechanisms underlying degeneration and the potential for gene repair, we have expressed a functional bovine cGMP PDE beta subunit in transgenic rd mice. One transgenic mouse line showed complete photoreceptor rescue across the entire span of the retina. A second independently derived line showed partial rescue in which photoreceptors in the superior but not the inferior hemisphere of the retina were rescued. In the latter animals, intermediate stages of degeneration were observed in the transition zone between rescued and diseased photoreceptors. Pathologic changes in the retina ranged from vesiculation of the basalmost outer segment discs in otherwise structurally intact rod cells to photoreceptors with highly disorganized outer segments and intact inner segments. Totally or partially rescued retinas showed a corresponding restoration of cGMP PDE activity, whereas nonrescued retinas had minimal enzyme activity, characteristic of the rd phenotype. These transgenic animals provide models for studying the molecular basis of retinal degenerative disease and conclusively demonstrate that the phenotype of rd mice is produced by a defect in the beta subunit of cGMP PDE.

The extracellular and intracellular domains of the human thyrotropin receptor were expressed in Escherichia coli and the proteins were used to produce monoclonal anti-receptor antibodies. Immunoblot studies and immunoaffinity purification showed that the receptor is composed of two subunits linked by disulfide bridges and probably derived by proteolytic cleavage of a single 90-kDa precursor. The extracellular alpha subunit (hormone binding) had an apparent molecular mass of 53 kDa (35 kDa after deglycosylation with N-glycosidase F). The membrane-spanning beta subunit seemed heterogeneous and had an apparent molecular mass of 33-42 kDa. Human thyroid membranes contained a 2.5- to 3-fold excess of beta subunits over alpha subunits. Immunocytochemistry showed the presence of both subunits in all the follicular thyroid cells, and both subunits were restricted to the basolateral region of the cell membrane.

The active Na+/K+ pump is composed of an alpha and a beta subunit. Until now, three putative isoforms of the beta subunit have been identified that share sequence similarity. We have expressed the beta 1 and beta 3 isoforms of Xenopus laevis Na+/K(+)-ATPase in Xenopus oocytes to compare functional properties of the Na+/K+ pump, including either of these two isoforms. Na+/K+ pump current, estimated as K(+)-induced outward current in voltage-clamped oocytes, was doubled by coexpression of alpha 1 subunits with either isoform of the beta subunit compared to expression of alpha 1 subunits alone. The kinetics of activation by external K+ and the voltage dependence of the electrogenic activity of the Na+/K+ pump were similar with both beta isoforms, indicating that both beta 1 and beta 3 isoforms can support expression at the oocyte surface of an active Na+/K+ pump with similar functional properties.

We have used in situ hybridization histochemistry to analyze the subcellular distribution of mRNAs encoding Na,K-ATPase alpha- and beta-subunit isoforms in the rat central nervous system. Substantial differences in the cell-specific pattern of expression were found for the genes encoding three isoforms of the alpha subunit. Transcripts of alpha 1-subunit gene were detected in virtually all cell types and structures examined. Expression of alpha 2-subunit mRNA was characteristic of glia, whereas alpha 3-subunit transcripts were predominant in neurons. Transcripts encoding the beta 1 subunit were detected in neurons, whereas beta 2-subunit mRNA expression was characteristic of glia. mRNA encoding both beta-subunit isoforms was present in choroidal epithelial cells. The distribution pattern of alpha- and beta-subunit mRNAs in structures throughout the central nervous system is consistent with the possibility of six structurally distinct Na+,K(+)-ATPase isoenzymes.

Classical insulin and insulin-like growth factor I (IGF-I) receptors exist as well defined alpha 2 beta 2 heterotetrameric complexes that are assembled from two identical alpha beta heterodimeric half-receptor precursors. Recent evidence suggests that insulin and IGF-I half-receptors can heterologously assemble to form alpha 2 beta 2 insulin/IGF-I hybrid receptor complexes in vivo and in vitro. We have utilized hybrid receptor complexes to examine ligand-stimulated transmembrane signaling of wild-type insulin (alpha beta INS.WT) or IGF-I (alpha beta IGF.WT) half-receptors assembled with a kinase-defective insulin half-receptor mutant (alpha beta INS.A/K). In vitro assembly of either (alpha beta)IGF.WT/(alpha beta)INS.A/K or (alpha beta)INS.WT/(alpha beta)INS.A/K hybrid receptors resulted in decreased substrate protein kinase activity. The degree of protein kinase inactivation directly correlated with the amount of immunologically cross-reactive hybrid receptors formed. In contrast to substrate kinase activity, insulin-stimulated autophosphorylation of the (alpha beta)INS.WT/(alpha beta)INS.A/K hybrid receptor complex was completely unaffected in comparison to the wild-type (alpha beta)INS.WT/(alpha beta)INS.WT receptor. To assess a molecular basis for this difference, autophosphorylation of a hybrid receptor composed of a truncated beta-subunit insulin half-receptor with the kinase-defective half-receptor, (alpha beta)INS. delta CT/(alpha beta)INS.A/K, demonstrated the exclusive autophosphorylation of the (alpha beta)INS.A/K half-receptor beta subunit. These results demonstrate that ligand-dependent substrate phosphorylation by insulin and IGF-I holoreceptors requires interactions between two functional beta subunits within the alpha 2 beta 2 heterotetrameric complex and occurs through an intramolecular trans-phosphorylation reaction.

We have previously described the isolation of several genes expressed exclusively in the nervous system of adult Xenopus laevis and activated in the embryo shortly after neural induction. The sequence of one of these cDNAs, 24-15, identifies the corresponding protein as an isotype of the beta subunit of Na+,K(+)-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting); EC 3.6.1.37]. This form is distinct from the previously described beta 1 subunit of Xenopus, and the protein sequence comparison suggests that it is not the frog homolog of the mammalian beta 2 subunit; therefore, we refer to the 24-15 protein as the beta 3 subunit of Na+,K(+)-ATPase of Xenopus. Antisera directed against beta 3-subunit fusion protein detected a protein in adult brain extracts with the size and properties expected for a Na+,K(+)-ATPase beta subunit. In Xenopus the beta 1 and beta 3 subunits are expressed as maternal mRNAs at similar levels; during embryogenesis rapid accumulation of beta 3 mRNA begins at stage 14 (early neurula), and the rapid accumulation of beta 1 mRNA begins at stage 23/24. In situ hybridization of antisense RNA probes to tadpole brain sections indicates that beta 3 subunit is expressed throughout the developing brain. We suggest that beta 3 is a major Na+,K(+)-ATPase beta subunit present during early nervous system development in the frog.

When incorporated into lipid vesicles, the purified inositol 1,4,5-trisphosphate (IP3) receptor protein mediates 45Ca2+ flux. We observe a potent, selective allosteric regulation by ATP of IP3 actions on Ca2+ flux. The action of ATP is selective for adenine nucleotides with ADP and AMP less potent and GTP inactive. At 1-10 microM, ATP increases maximal IP3-induced flux by 50% with no change in IP3 potency. The enhancing effect of ATP diminishes between 0.1 and 1 mM. Concentration-response curves are steep for both the increasing and the decreasing effects of ATP on IP3 actions, suggesting a physiological regulatory role of ATP in IP3-induced Ca2+ release. Diminishing local ATP concentrations coincident with filling of Ca2+ stores by the Ca2(+)-ATPase may enhance IP3 release of Ca2+, an effect that would decline as ATP returns to physiological levels. ATP regulation of Ca2+ release may also play a role in oscillations of intracellular Ca2+ concentration.

The signal-transducing guanine nucleotide-binding regulatory (G) proteins are heterotrimers composed of three subunits--alpha, beta, and gamma. Although multiple distinctive forms of the alpha subunit have been described, only two forms of the beta subunits of the G proteins have been identified. To investigate further the structural diversity of the beta subunits, we screened bovine and human retina cDNA libraries and isolated clones encoding three distinct types of G protein beta subunit. One form was identical to previously isolated beta 1-subunit cDNA clones that encode the 36-kDa form of the beta subunit, whereas a second form was identical to previously described beta 2 cDNAs that encode the 35-kDa beta isoform. In addition, we identified another species, designated beta 3 subunit, which encodes a third distinct form of the beta subunit. The beta 3-subunit cDNA corresponds to a 2.0-kilobase mRNA expressed in all tissues and clonal cell lines examined. Nucleotide sequence analysis indicates that the encoded peptide consists of 340-amino acid residues with a Mr of 37,221. The amino acid sequences of the three beta subunits are closely related: 83% identity between beta 1 and beta 3 subunits and 81% identity between beta 2 and beta 3 subunits. By contrast, the 3'-untranslated regions of the three cDNAs show no significant homology. Our data support the hypothesis that a family of beta-subunit polypeptides exists and extend understanding of beta-subunit structure.

ATP-binding cassette (ABC) transporters are probably the most common as well as the most wide-spread active transport systems. This family includes many bacterial transporters that either export complex molecules (lipids, polysaccharides, or proteins), or import small nutrients in cooperation with the soluble solute-binding proteins (e.g., the maltose and histidine transport systems of Escherichia coli), as well as such human proteins as the cystic fibrosis transmembrane conductance regulator and the P-glycoprotein that pumps out many anticancer agents. Each transport complex usually contains four domains or subunits, two transmembrane domains each typically containing six transmembrane helices, and two ATPase domains (Fig. 1). The sequence of ABC or ATPase domains always contains the characteristic Walker A and B motifs that are involved in ATP binding (1), but the intervening section between these two motifs is usually longer here than in other ATP-binding enzymes. It ends in a unique motif called LSGGQ motif, C motif, or “signature motif,” because it is present in all ABC subunits, but usually not in other ATPases. Although ATP hydrolysis is clearly the driving force for active transport by ABC transporters, it was not clear how it was coupled to the transmembrane transport of solutes. In this issue of PNAS, Fetsch and Davidson (2) report results that indicate how the two ATPase domains collaborate in ATP hydrolysis, and suggest how this is likely to be coupled to solute transport.

This study examines the mechanism of guanine nucleotide-binding protein (G protein) coupling of receptors to phospholipase C. The Xenopus oocyte has a muscarinic receptor-activated Cl- current that is mediated by inositol 1,4,5-trisphosphate. Modulation of the muscarinic receptor-evoked Cl- current was examined under voltage clamp in oocytes injected with resolved G-protein subunits. The presence of an alpha subunit of G proteins in oocytes was shown by pertussis toxin-labeling of a 41-kDa band in oocyte membranes. The presence of the beta subunit of G proteins was demonstrated by immunoblotting experiments with an antiserum (U-49) that is specific for the beta subunit. Pertussis toxin treatment of oocytes resulted in the uncoupling of muscarinic receptors from activation of the Cl- current. Cells microinjected with 1.5 ng of human erythrocyte beta gamma-subunit complex or 1.0 ng of bovine brain beta gamma-subunit complex showed approximately a 95% reduction in the evoked Cl- current. Cells injected with equal volumes of protein storage vehicle showed no change in response. Cells injected with boiled beta gamma subunits, bovine serum albumin, or resolved alpha subunits also showed no reduction in response. Cells injected with various concentrations of beta gamma subunits showed a concentration dependence with half-maximal inhibition of the muscarinic activated Cl- current at about 10 nM. Cells injected with 1.0 ng of bovine brain beta gamma subunits could not respond to bath-applied agonist but could generate the Cl- current on intracellular injection of inositol 1,4,5-trisphosphate. These observations suggest that there is a G protein responsible for muscarinic receptor-mediated signal transduction through phospholipase C and that it is an alpha beta gamma heterotrimer. It appears that the mode of action of the G protein in the phospholipase C system may be similar to that of the hormone-activated adenylyl cyclase.

125I-Labeled human choriogonadotropin (125I-hCG) bound to rat ovarian receptor was solubilized in Triton X-100. By using increasing concentrations of nine different antisera specific for the individual subunits of human choriogonadotropin (hCG), free 125I-hCG or 125I-hCG-receptor complex was precipitated by double-antibody technique. The ability of any antiserum to bind to the hormone-specific beta subunit was not affected by hCG binding to receptor, suggesting that this subunit is not directly involved with the receptor in the final state of the hormone-receptor complex. In contrast, every antiserum specific for the alpha subunit was dramatically inhibited in binding to the solubilized 125I-hCG-receptor complex. These results suggest that the alpha subunit directly interacts with the receptor, thereby masking immunoreactive sites normally available on the free hormone. Because a number of reports describe binding activity of high concentrations of immunopurified beta subunits of hCG, we propose a two-step model for the binding of hCG to receptor and postulate separate and distinct roles for the subunits. We propose that the binding of hCG to the receptor involves a specific low-affinity initial interaction of the beta subunit with the receptor that activates a second site for the high-affinity binding of alpha subunit and stabilization of the hormone-receptor complex.

Intracellular ATP levels in whole cells of Methanococcus voltae respond to electron transfer coupled to methanogenesis. ATP synthesis can also be induced by an artificially imposed transmembrane electrical potential [formed by electrogenic movement outwards of potassium (induced by valinomycin) or of protons (induced by an uncoupler], or by a pH gradient (acid outside). These results implicate the existence of a reversible ATPase coupled to electrogenic movement of an ion(s) other than potassium or proton, and that ionophores are competent to catalyze ion movement across the cytoplasmic membrane of this organism (which is the sole membrane structure in this species). ATP synthesis driven by electron transfer is, however, insensitive to the addition of such ionophores. These results indicate that although cells possess an ion-translocating ATPase (possibly involved in the maintenance of internal ionic composition homeostasis), methanogenesis-driven ATP synthesis does not involve the intermediacy of a transmembrane ion gradient. Primarily because methane formation has been previously demonstrated to involve true electron transfer, substrate-level phosphorylation (at least in analogy to other systems) has been generally ruled out. The results presented here suggest that at least one methanogenic bacterium may use a direct linkage of ATP synthesis to electron transfer.

The question of whether the two subunits of the glycoprotein hormone human chorionic gonadotropin (hCG) are handled separately or as a unit by target cells was addressed by using a dual-labeling procedure. The individual alpha and beta subunits of hCG were labeled with 131I or 125I, recombined, chromatographed, and injected intravenously into hormonally primed immature rats. The ovaries of these rats contained large numbers of antral follicles, the granulosa cells of which possess high concentrations of receptor for hCG. Comparisons of the distribution of the different radioisotopic labels in various tissues over time indicated the activity associated with the beta subunit was preferentially retained by the ovarian granulosa cells in a hormone-specific manner, while the activity associated with the alpha subunit was preferentially lost. This contrasted with other tissues, including other ovarian cells with receptor for hCG, in which both radiolabels were either handled nondifferentially or handled differentially in a hormonally nonspecific manner.

Gangliosides inhibit 125I-labeled thyrotropin binding to the thyrotropin receptors on bovine thyroid plasma membranes, on guinea pig retro-orbital tissue plasma membranes, and on human adipocyte membranes. This inhibition by gangliosides is critically altered by the number and location of the sialic acid residues within the ganglioside structure, the efficacy of inhibition having the following order: GD1b greater than GT1 greater than GM1 greater than GM2 = GM3 greater than GD1a. The inhibition results from the interaction of thyrotropin and gangliosides, rather than the interaction of membrane and gangliosides. Fluorescence studies show that the inhibition is associated with a distinct conformational change of the thyrotropin molecule and that the progression from a "noninhibitory conformation" to an "inhibitory conformation" parallels exactly the order of effectiveness in inhibiting 125I-labeled thyrotropin binding. The ganglioside inhibition of 125I-labeled thyrotropin binding appears to be hormonally specific in that it is not affected by albumin, glucagon, insulin, prolactin, follicle-stimulating hormone, growth hormone, or corticotropin. The possibility that a ganglioside or ganglioside-like structure is a component of the thyrotropin receptor is suggested by the finding that gangliosides more complex than N-acetylneuraminylgalactosylglucosylceramide are present in bovine thyroid membranes in much higher quantities than have been previously found in extraneural tissue. The finding that the B component of cholera toxin, which also interacts with gangliosides, has a peptide sequence in common with the beta subunit of thyrotropin, suggests that thyrotropin and cholera toxin may be analogous in their mode of action on the membrane.

A 9S mRNA, purified from mouse thyrotropic pituitary tumors by sucrose density gradient centrifugation of poly(A)-enriched mRNA, directed the synthesis of only alpha and beta subunits of thyrotropin in the reticulocyte lysate translation system. Analysis of radioiodinated 9S mRNA, repurified by oligo(dT)-cellulose chromatography and sucrose gradient centrifugation, yielded two species of RNA on urea/polyacrylamide gel electrophoresis. The major RNA species contained 620 nucleotides, and the minor RNA species contained 560 nucleotides. Unlabeled 9S mRNA was further purified by urea/polyacrylamide gel electrophoresis; the mRNAs were separately eluted from slices of the gel containing material migrating with an apparent length of 620 and 560 nucleotides. Translation of these mRNAs in the reticulocyte lysate showed that the longer mRNA coded for the alpha subunit and the shorter mRNA coded for the beta subunit of mouse thyrotropin. Because more alpha than beta subunit of thyrotropin was consistently synthesized, unbalanced amounts of thyrotropin subunits appear to be synthesized by translation of unbalanced amounts of individual mRNAs. We have demonstrated that the synthesis of thyrotropin is directed by two separate mRNA molecules, each coding for a different subunit of the hormone.

A temperature-sensitive mutant having a lethal mutation in the gene for the beta subunit of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) exhibits an apparent 2- to 3-fold decrease in the rates of both beta and beta' subunit synthesis at the non-permissive temperature, relative to total protein. In contrast, a temperature-sensitive mutant with a lethal mutation in the gene encoding beta' has a 5- to 6-fold increase in the rates of beta and beta' synthesis at 42 degrees. These beta and beta' mutants also exhibit rapid degradation of both subunits at the high temperature.

It has been shown that a photoactivable derivative of human choriogonadotropin (hCG) labels the lutropin receptor on porcine granulosa cells [Ji, I. & Ji, T. H. (1980) Proc. Natl. Acad. Sci. USA 77, 7167-7170]. In an attempt to identify which of the hCG subunits labeled the receptor, three sets of different hCG derivatives were prepared. In the first set, hCG was coupled to the N-hydroxysuccinimide ester of 4-azidobenzoylglycine and radioiodinated. In the second set, only one of the subunits was radioiodinated, but both subunits were allowed to react with the reagent. In the third set, both the reagent and [125I]iodine were coupled to only one of the subunits. The binding activity of each hormone derivative was comparable to that of 125I-labeled hCG. After binding of these hormone derivatives to the granulosa cell surface, they were photolyzed. After solubilization, autoradiographs of sodium dodecyl sulfate/polyacrylamide gels of each sample revealed a number of labeled bands; the hCG derivatives containing 125I-labeled alpha subunit produced four bands (molecular weights 120,000 +/- 6,000, 96,000 +/- 5,000, 76,000 +/- 4,000, and 73,000 +/- 4,000) and those containing 125I-labeled beta subunit produced three bands (molecular weights 106,000 +/- 6,000, 88,000 +/- 5,000, and 83,000 +/- 4,000). Results were the same when the hormone-receptor complexes were solubilized in 0.5% Triton X-100 and then photolyzed or when the hormone was derivatized with a family of reagents having arms of various lengths. We conclude that both the alpha subunit and the beta subunit of hCG photoaffinity labeled certain membrane polypeptides and that these polypeptides are related to the hormone receptor.