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Pancreatic neuroendocrine tumors (pNETs) occur due to the abnormal growth of pancreatic islet cells and predominantly develop in the duodenal-pancreatic region. Somatostatinoma is one of the pNETs associated with tumors of pancreatic δ cells, which produce and secrete somatostatin. Limited information is currently available on the pathogenic mechanisms of somatostatinoma. The large-conductance Ca2+-activated K+ (BK Ca) channel is expressed in several types of cancer cells and regulates cell proliferation, migration, invasion, and metastasis. In the present study, the functional expression of the BK Ca channel was examined in a human somatostatinoma QGP-1 cell line. In QGP-1 cells, outward currents were elicited by membrane depolarization at pCa 6.5 (300 nM) in the pipette solution and inhibited by the specific BK Ca channel blocker, paxilline. Paxilline-sensitive currents were detected, even at pCa 8.0 (10 nM) in the pipette solution, in QGP-1 cells. In addition to the α and β2-4 subunits of the BK Ca channel, the novel regulatory γ1 subunit (BK Ca γ1) was co-localized with the α subunit in QGP-1 cells. Paxilline-sensitive currents at pCa 8.0 in the pipette solution were reduced by the siRNA knockdown of BK Ca γ1. Store-operated Ca2+ entry was smaller in BK Ca γ1 siRNA-treated QGP-1 cells. The proliferation of QGP-1 cells was attenuated by paxilline or the siRNA knockdown of BK Ca γ1. These results strongly suggest that BK Ca γ1 facilitates the proliferation of human somatostatinoma cells. Therefore, BK Ca γ1 may be a novel therapeutic target for somatostatinoma. • Somatostatinoma is a pancreatic neuroendocrine tumor with high malignancy. • Somatostatinoma QGP-1 cells have large-conductance Ca2+-activated K+ (BK Ca) currents. • The γ1 subunit of BK Ca channel (BK Ca γ1) was functionally expressed in QGP-1 cells. • The proliferation of QGP-1 cells was facilitated by the presence of BK Ca γ1. • BK Ca γ1 may be a novel therapeutic target for somatostatinoma. [ABSTRACT FROM AUTHOR]

F 1 is a soluble part of F o F 1 -ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F 1 motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α 3 β 3 hexamer of the F 1 molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure–function relationship of the γ subunit, we prepared a mutant F 1 -ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F 1 -ATP synthase. [ABSTRACT FROM AUTHOR]

Heterotrimeric G proteins are important molecules for regulating plant architecture and transmitting external signals to intracellular target proteins in higher plants and mammals. The rice genome contains one canonical α subunit gene (RGA1), four extra-large GTP-binding protein genes (XLGs), one canonical β subunit gene (RGB1), and five γ subunit genes (tentatively named RGG1, RGG2, RGG3/GS3/Mi/OsGGC1, RGG4/DEP1/DN1/OsGGC3, and RGG5/OsGGC2). RGG1 encodes the canonical γ subunit; RGG2 encodes the plant-specific type of γ subunit with additional amino acid residues at the N-terminus; and the remaining three γ subunit genes encode the atypical γ subunits with cysteine abundance at the C-terminus. We aimed to identify the RGG3/GS3/Mi/OsGGC1 gene product, Gγ3, in rice tissues using the anti-Gγ3 domain antibody. We also analyzed the truncated protein, Gγ3∆Cys, in the RGG3/GS3/Mi/OsGGC1 mutant, Mi, using the anti-Gγ3 domain antibody. Based on nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the immunoprecipitated Gγ3 candidates were confirmed to be Gγ3. Similar to α (Gα) and β subunits (Gβ), Gγ3 was enriched in the plasma membrane fraction, and accumulated in the flower tissues. As RGG3/GS3/Mi/OsGGC1 mutants show the characteristic phenotype in flowers and consequently in seeds, the tissues that accumulated Gγ3 corresponded to the abnormal tissues observed in RGG3/GS3/Mi/OsGGC1 mutants.

Phycoerythrins (PE) in phycobilisomes from Polysiphonia urceolata were studied in this research. Dissociative products of phycobilisomes were analyzed by sucrose density gradient centrifugation and native-PAGE. At least three types of PEs were found in the dissociative products of phycobilisomes. According to their molecular weights, absorption spectra and subunit components, they should be PE hexamer containing γ 1 subunit, PE hexamer containing γ 2 subunit and PE monomer containing no γ subunit. PEs bigger than hexmer were also found in the dissociative products of phycobilisomes in 200 mM phosphate buffer when dissociated phycobilisomes were analyzed by sucrose density gradient centrifugation. PE trimers containing no γ subunits were also found in products of dissociated phycobilisomes in deionized water when dissociated phycobilisomes were analyzed by native-PAGE. This is the first time that pure PE hexamers containing γ 2 subunits were isolated from P. urceolata . The PE monomers containing no γ subunits should come from PE trimers or hexamers containing no γ subunits in the “rod” of phycobilisomes. It can be concluded that there are three types of PEs in “rod” of phycobilisomes from P. urceolata : PE containing γ 1 subunit, PE containing γ 2 subunit and PE containing no γ subunit. [ABSTRACT FROM AUTHOR]

Heterotrimeric G proteins (αβγ subunits) that are activated by G protein-coupled receptors (GPCRs) mediate the biological responses of eukaryotic cells to extracellular signals. The α subunits and the tightly bound βγ subunit complex of G proteins have been extensively studied and shown to control the activity of effector molecules. In contrast, the potential roles of the large family of γ subunits have been less studied. In this review, we focus on present knowledge about these proteins. Induced loss of individual γ subunit types in animal and plant models result in strikingly distinct phenotypes indicating that γ subtypes play important and specific roles. Consistent with these findings, downregulation or upregulation of particular γ subunit types result in various types of cancers. Clues about the mechanistic basis of γ subunit function have emerged from imaging the dynamic behavior of G protein subunits in living cells. This shows that in the basal state, G proteins are not constrained to the plasma membrane but shuttle between membranes and on receptor activation βγ complexes translocate reversibly to internal membranes. The translocation kinetics of βγ complexes varies widely and is determined by the membrane affinity of the associated γ subtype. On translocating, some βγ complexes act on effectors in internal membranes. The variation in translocation kinetics determines differential sensitivity and adaptation of cells to external signals. Membrane affinity of γ subunits is thus a parsimonious and elegant mechanism that controls information flow to internal cell membranes while modulating signaling responses., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Highlights: [•] Two bands (about 40kDa and 50kDa) in SDS-PAGE gels of R-PE were studied. [•] The 42.1kDa and 53.7kDa bands were complexes of αβ and βγ1, respectively. [•] Formation of the bands should be related to PUB. [•] γ subunits of R-PE play roles in structural stability of R-PE aggregates. [•] Interactions between β subunits and γ subunit may be the main forces of γ subunit to maintain the stability of R-PE. [Copyright &y& Elsevier]

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy-sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant Sn RK1 function is still limited. In addition to a family of γ-like proteins, plants also encode a hybrid βγ protein that combines the Four-Cystathionine β-synthase ( CBS)-domain ( FCD) structure in γ subunits with a glycogen-binding domain ( GBD), typically found in β subunits. We used integrated functional analyses by ectopic Sn RK1 complex reconstitution, yeast mutant complementation, in-depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast-containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre- CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/ SNF/Sn RK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the Sn RK1 energy sensor and novel FCD gene functions. [ABSTRACT FROM AUTHOR]

Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo. [ABSTRACT FROM AUTHOR]

G protein plays an important role in signaling pathways and is involved in various signal transduction systems in plant. A full-length cDNA encoding a putative G protein γ subunit (Gγ), designated as BnGG2, was isolated from Brassica napus L. BnGG2 is predicted to encode a precursor protein of 100 amino acid residues. The expression of BnGG2 in different B. napus tissues and developmental stages was analyzed by using real-time PCR. The results showed that BnGG2 expression level was high at the 7-day-old seedling stage, the bolting stage, and the fruiting stage. Moreover, BnGG2 was analyzed under four different plant hormones. All of the four tested hormones, abscisic acid (ABA), gibberellins acid 3 (GA), brassinosteroid (BR) and indole-3-acetic acid (IAA), triggered an induction of BnGG2 expression at different hormone concentrations. The expression of BnGG2 was significantly induced by the high concentrations of ABA, BR, and IAA. The expression of BnGG2 was also induced by low GA concentrations and inhibited by high GA concentrations. In addition, BnGG2 was responsive to different abiotic stresses. The BnGG2 was up-regulated in salt and drought stress and down-regulated in heat and cold stress. These results suggested that BnGG2 plays an important role in plant hormone signaling pathways and may be also involved in plant defense systems against environmental stresses in B. napus. [ABSTRACT FROM AUTHOR]

The FoF1-ATPase, which synthesizes ATP with a rotary motion, is highly regulated in vivo in order to function efficiently, although there remains a limited understanding of the physiological significance of this regulation. Compared with its bacterial and mitochondrial counterparts, the γ subunit of cyanobacterial F1, which makes up the central shaft of the motor enzyme, contains an additional inserted region. Although deletion of this region results in the acceleration of the rate of ATP hydrolysis, the functional significance of the region has not yet been determined. By analysis of rotation, we successfully determined that this region confers the ability to shift frequently into an ADP inhibition state; this is a highly conserved regulatory mechanism which prevents ATP synthase from carrying out the reverse reaction. We believe that the physiological significance of this increased likelihood of shifting into the ADP inhibition state allows the intracellular ATP levels to be maintained, which is especially critical for photosynthetic organisms. [ABSTRACT FROM PUBLISHER]

Determination of the role of mitochondrial (mt) ATP synthesis in plant metabolism is complicated by chloroplastic ATP synthesis. To differentiate ATP synthesis from these two organelles, we created transgenic Arabidopsis plants in which two different subunits of the mt ATP synthase, the oligomycin sensitivity-conferring protein (OSCP) (=δ) (ATP5) and the γ (ATP3) subunit, were expressed individually in antisense orientation under the control of a dexamethasone-inducible promoter. The phenotypic effects of antisense expression were identical for both atp5 and atp3. Seedling lethality resulted from induction during germination in the light, demonstrating the essentiality of both gene products. Reduced expression of either gene resulted in stunting of dark-grown (etiolated) seedlings, downward curling or wavy-edged leaf margins of light-grown plants and ball-shaped unexpanded flowers. Antisense induction reduced total ATP levels in dark-grown (etiolated) seedlings germinated on media lacking sucrose, but increased total ATP levels in induced light-grown plants and in induced dark-grown seedlings germinated on media containing sucrose. Induction reduced transcript levels for two transcription factors (TCP3 and TCP4) whose decreased expression is associated with a similar wavy-edged leaf phenotype in Arabidopsis, and increased transcript levels for dynamin-related proteins whose increased expression is associated with increased mt division. Reduced expression of these subunits of the mt ATP synthase is proposed to disturb cellular redox states, which ultimately manifest downstream as diverse and seemingly unrelated phenotypes. [ABSTRACT FROM PUBLISHER]

Abstract: Electron transport, the proton gradient and ATP synthesis were determined in thylakoids that had been briefly exposed to a low concentration of trypsin during illumination. This treatment cleaves the γ subunit of the ATP synthase into two large fragments that remain associated with the enzyme. Higher rates of electron transport are required to generate a given value of the proton gradient in the trypsin-treated membranes than in control membranes, indicating that the treated membranes are proton leaky. Since venturicidin restores electron transport and the proton gradient to control levels, the proton leak is through the ATP synthase. Remarkably, the synthesis of ATP by the trypsin-treated membranes at saturating light intensities is only slightly inhibited even though the proton gradient is significantly lower in the treated thylakoids. ATP synthesis and the proton gradient were determined as a function of light intensity in control and trypsin-treated thylakoids. The trypsin-treated membranes synthesized ATP at lower values of the proton gradient than the control membranes. Cleavage of the γ subunit abrogates inhibition of the activity of the chloroplast ATP synthase by the ε subunit. Our results suggest that overcoming inhibition by the ε subunit costs energy. [Copyright &y& Elsevier]

Abstract: In kidney, Na+, K+-ATPase is an oligomer (αβγ) with equimolar amounts of essential α and β subunits and one small hydrophobic FXYD protein (γ subunit). This report describes γ subunit as an activator of pig kidney outer medulla Na+, K+-ATPase in aqueous medium. The effects of γ subunit on Na+, K+-ATPase were dose-dependent and preincubation-dependent. Changes in αβ/γ stoichiometry did not alter K m1 for ATP, and slightly increased K m2, but V max was increased at both catalytic and regulatory sites. Hydroxylamine treatment of enzyme phosphorylated by ATP (E-P), in the presence of additional γ subunit, revealed that 52% of the E-P accumulation was not via acyl-phosphate formation. The γ subunit was phosphorylated by endogenous kinases and by commercial catalytic subunit of protein kinase A (PKA). Additionally, we demonstrated that PKA phosphorylation of γ subunit increased its capacity to stimulate ATP hydrolysis. These results suggest that γ subunit can act as an intrinsic Na+, K+-ATPase regulator in kidney. [Copyright &y& Elsevier]

Cajal–Retzius cells are implicated in regulating neuronal migration and lamination during corticogenesis. In rodents, Cajal–Retzius cells are transient, being prevalent in the marginal zone of the embryonic neocortex and declining over the first two postnatal weeks. While studies have examined in postnatal neocortex the properties of GABAA receptors in Cajal–Retzius cells, less is known about their disposition at embryonic stages. Here, we combined patch-clamp electrophysiology and single-cell mRNA profiling to probe the expression of GABAA receptors in Cajal–Retzius cells. In embryonic neocortical slices, GABA elicited GABAA receptor-mediated current responses that were diazepam-insensitive and inhibited by Zn2+, a pharmacological profile consistent with expression of γ-less GABAA receptor isoforms. Non-Cajal–Retzius cells in the same embryonic slices, on the other hand, were robustly potentiated by diazepam and were insensitive to Zn2+, typical of γ-containing GABAA receptor isoforms, as were Cajal–Retzius cells in the postnatal neocortex. Single-cell mRNA profiling and immunohistochemistry confirmed expression of GABAA receptor γ subunit transcript and protein, respectively, in individual reelin-expressing cells in the postnatal cortex but not in their embryonic counterparts. We conclude that Cajal–Retzius cells express γ-less GABAA receptors at embryonic stages and switch to expressing γ-containing GABAA receptor isoforms during postnatal neocortical development. [ABSTRACT FROM AUTHOR]

Abstract: A prenylated peptide specific to the C terminal tail of a G protein γ subunit type, γ5, inhibits activation of a G protein by the M2 muscarinic receptor. The γ5 peptide was tested for direct effects on the M2 receptor’s properties. The wild type γ5 peptide reduced the affinity of M2 for the agonist, carbachol, more than 5-fold in an antagonist displacement assay. The peptide was inactive when its amino acid sequence was scrambled or when it was unprenylated. Although the wild type peptide reduced the affinity of M2 for the antagonist QNB, it had no effect on the antagonists NMS or atropine. These results suggest that in the presence of the peptide the M2 receptor adopts a novel conformational state that affects the ligand binding surface. The results also suggest that the G protein γ5 subunit tail interacts with a receptor. [Copyright &y& Elsevier]

The FXYD proteins are a family of seven homologous single transmembrane segment proteins (FXYD1-7), expressed in a tissue-specific fashion. The FXYD proteins modulate the function of Na,K-ATPase, thus adapting kinetic properties of active Na+ and K+ transport to the specific needs of different cells. Six FXYD proteins (1-5, 7) are known to interact with Na,K-ATPase and affect its kinetic properties in specific ways. Although effects of FXYD proteins on parameters such as K1/2Na+, K1/2K+, KmATP, and Vmax are modest, usually twofold, these effects may have important long-term consequences for homeostasis of cation balance. In this review we summarize basic features of FXYD proteins and present recent evidence for functional effects, structure-function relations and structural interactions with Na,K-ATPase. We then discuss possible physiological roles, based on in vitro observations and newly available knockout mice models. Finally, we also consider evidence that FXYD proteins affect functioning of other ion transport systems. [ABSTRACT FROM AUTHOR]

Before implantation into the uterine wall, the mammalian embryo undergoes a period of cell division, cell shape change, and cell differentiation leading to the formation of an outer epithelium, the trophectoderm. The trophectoderm is the part of the embryo that initiates uterine contact and, after transformation to become the trophoblast, uterine invasion. Similar to the kidney nephron, the trophectoderm is a transporting epithelium with distinct apical and basolateral membrane domains; its function is to facilitate transepithelial Na+ and fluid transport for blastocoel formation. That transport is driven by Na,K-adenosine triphosphatase (ATPase) localized in basolateral membranes of the trophectoderm. Preimplantation embryos express multiple α and β subunit isoforms of Na,K-ATPase, potentially constituting multiple isozymes, but the basolaterally located α1β1 isozyme appears to function uniquely to drive fluid transport. Embryos unable to express α1 subunits because of targeted deletion of the gene are able to form a blastocoel, but they fail to maintain their integrity and expire during the peri-implantation period. Preimplantation embryos also express the γ subunit, a modulator of Na,K-ATPase activity, but targeted deletion of that gene did not reveal an essential developmental role. The preimplantation embryo offers a unique model for understanding the roles of Na,K-ATPase subunit isoforms in epithelial development and transepithelial transport. [ABSTRACT FROM AUTHOR]

Abstract: Cyanobacteria and red algae have intricate light-harvesting systems comprised of phycobilisomes that are attached to the outer side of the thylakoid membrane. The phycobilisomes absorb light in the wavelength range of 500–650 nm and transfer energy to the chlorophyll for photosynthesis. Phycobilisomes, which biochemically consist of phycobiliproteins and linker polypeptides, are particularly wonderful subjects for the detailed analysis of structure and function due to their spectral properties and their various components affected by growth conditions. The linker polypeptides are believed to mediate both the assembly of phycobiliproteins into the highly ordered arrays in the phycobilisomes and the interactions between the phycobilisomes and the thylakoid membrane. Functionally, they have been reported to improve energy migration by regulating the spectral characteristics of colored phycobiliproteins. In this review, the progress regarding linker polypeptides research, including separation approaches, structures and interactions with phycobiliproteins, as well as their functions in the phycobilisomes, is presented. In addition, some problems with previous work on linkers are also discussed. [Copyright &y& Elsevier]

Abstract: The calcium channel gamma (γ) subunit family consists of eight members whose functions include modulation of high voltage-activated (HVA) calcium currents in skeletal muscle and neurons, and regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propanoic acid (AMPA) receptor targeting. Cardiac myocytes express at least three γ subunits, γ4, γ6 and γ7, whose function(s) in the heart are unknown. Here we compare the effects of the previously uncharacterized γ6 subunit with that of γ4 and γ7 on a low voltage-activated calcium channel (Cav3.1) that is expressed in cardiac myoctyes. Co-expression of both the long and short γ6 subunit isoforms, γ6L and γ6S, with Cav3.1 in HEK-293 cells significantly decreases current density by 49% and 69%, respectively. Two other γ subunits expressed in cardiac myocytes, γ4 and γ7, have no significant effect on Cav3.1 current. Neither γ6L, γ6S, γ4 nor γ7 significantly affect the voltage dependency of activation or inactivation or the kinetics of Cav3.1 current. Transient expression of γ6L in an immortalized atrial cell line (HL-1) significantly reduces the endogenous low voltage-activated current in these cells by 63%. Green fluorescent protein tagged γ6L is localized primarily in HEK-293 cell surface membranes where it is evenly distributed. Expression of γ6L does not affect the level of Cav3.1 mRNA or the amount of total Cav3.1 protein in transfected HEK-293 cells. These results demonstrate that the γ6 subunit has a unique ability to inhibit Cav3.1 dependent calcium current that is not shared with the γ4 and γ7 isoforms and is thus a potential regulator of cardiac low voltage-activated calcium current. [Copyright &y& Elsevier]

To understand the regulatory function of the γ and ϵ subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric FoF1 complex of thermophilic bacteria. When a part of the chloroplast F1 γ subunit was introduced into the bacterial FoF1 complex, the inverted membrane vesicles with this chimeric FoF1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal α-helices region of the ϵ subunit was substituted with the corresponding region from CF1-ϵ together with the mutation of γ, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-γ and the entire ϵ subunit from CF1 were introduced. These results provide important features for the regulation of FoF1 by these subunits: (1) the interaction between γ and ϵ is important for the redox regulation of FoF1 complex by the γ subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial FoF1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity. [Copyright &y& Elsevier]

AMP-activated protein kinase (AMPK) is a αβγ heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the α subunit and by AMP allosteric control previously thought to be mediated by both α and γ subunits. Here we present evidence that adjacent γ subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the γ1 CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast γ homolog, snf4 contains a His151Gly substitution, and when this is introduced into γ1, AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in γ1 corresponds to the site of mutation in human γ2 and pig γ3 genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the α and γ subunits and that AMP functions to derepress AMPK activity. [ABSTRACT FROM AUTHOR]

In chloroplasts, synthesis of ATP is energetically coupled with the utilization of a proton gradient formed by photosynthetic electron transport. The involved enzyme, the chloroplast ATP synthase, can potentially hydrolyze ATP when the magnitude of the transmembrane electrochemical potential difference of protons (ΔμH+) is small, e.g. at low light intensity or in the dark. To prevent this wasteful consumption of ATP, the activity of chloroplast ATP synthase is regulated as the occasion may demand. As regulation systems ΔμH+ activation, thiol modulation, tight binding of ADP and the role of the intrinsic inhibitory subunit ϵ is well documented. In this article, we discuss recent progress in understanding of the regulation system of the chloroplast ATP synthase at the molecular level. [Copyright &y& Elsevier]

We have cloned and characterized a new member of the voltage-dependent Ca2+ channel γ subunit family, with a novel gene structure and striking properties. Unlike the genes of other potential γ subunits identified by their homology to the stargazin gene, CACNG7 is a five-, and not four-exon gene whose mRNA encodes a protein we have designated γ7. Expression of human γ7 has been localized specifically to brain. N-type current through CaV2.2 channels was almost abolished when co-expressed transiently with γ7 in either Xenopus oocytes or COS-7 cells. Furthermore, immunocytochemistry and western blots show that γ7 has this effect by causing a large reduction in expression of CaV2.2 rather than by interfering with trafficking or biophysical properties of the channel. No effect of transiently expressed γ7 was observed on pre-existing endogenous N-type calcium channels in sympathetic neurones. Low homology to the stargazin-like γ subunits, different gene structure and the unique functional properties of γ7 imply that it represents a distinct subdivision of the family of proteins identified by their structural and sequence homology to stargazin. [ABSTRACT FROM AUTHOR]

The heterotrimeric GTP binding proteins, G proteins, consist of three distinct subunits: α, β, and γ. There are 12 known mammalian γ subunit genes whose products are the smallest and most variable of the G protein subunits. Sequencing of the bovine brain γ10 protein by electrospray mass spectrometry revealed that it differs from the human protein by an Ala to Val substitution near the N-terminus. Comparison of γ isoform subunit sequences indicated that they vary substantially more at the N-terminus than at other parts of the protein. Thus, species variation of this region might reflect the lack of conservation of a functionally unimportant part of the protein. Analysis of 38 γ subunit sequences from four different species shows that the N-terminus of a given γ subunit isoform is as conserved between different species as any other part of the protein, including highly conserved regions. These data suggest that the N-terminus of γ is a functionally important part of the protein exhibiting substantial isoform-specific variation. [ABSTRACT FROM AUTHOR]

Incubation of chloroplast thylakoids with pyridoxal 5'-phosphate for a short time (5 s) modified the lysine residues of the y subunit of ATP synthase. Energization of thylakoids by illumination increased the reactivity of Lys24 by a factor of three and decreased the reactivity of Lys30 to 60%. The reactivities of these residues reached their maximum and minimum values, respectively, within 1 s alter the onset of illumination. Illumination of thylakoids increased the reactivitics of Lys222 and Lys231 in two steps by a factor of three. The first step was completed within 1 s and the second step was completed 20-30 s after the onset of illumination. In the presence of 10 mM NH4Cl, illumination of thylakoids did not change the reactivities of these lysine residues. These results suggest that the Lys24- and Lys30-containing region of the γ subunit changes its conformation rapidly in response to ΔμH+ and that thc Lys222- and Lys231-containing region of the γ subunit changes its conformation in two steps in response to ΔμH+ formation. [ABSTRACT FROM AUTHOR]

The expression plasmids for the subunit γ (γc) and the subunit ε (εc)of chloroplast coupling factor (CF1) from spinach were constructed, and the desired proteins were expressed in Escherichia coli. Both expressed subunits were obtained as inclusion bodies. When recombinant γc was mixed with recombinant α and β subunits of F1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (α3β3γc) was reconstituted and it showed significant ATP hydrolysis activity. The ATP hydrolysis activity of this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCl2, which induces formation of a disulfide bond between two cysteine residues in γc. Hence, this complex has similar modulation characteristics as CF1. The effects of recombinant εc and ε subunit from TF1 (εt) on α3β3γc, were also investigated. εc strongly inhibited the ATP hydrolysis activity of chimeric α3β3γc complex but εt did not. The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium. The addition of εc or εt to the α3β3γt complex, which is the authentic subunit complex from TF1, resulted in weak stimulation of the ATP hydrolysis activity. These results suggest that (a) the specific regulatory function of γc can be transferred to the bacterial subunit complex; (b) the interaction between the γc subunit and εc strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the α3β3 core. [ABSTRACT FROM AUTHOR]

The expression plasmids for the subunit γ (γc) and the subunit ε (εc)of chloroplast coupling factor (CF1) from spinach were constructed, and the desired proteins were expressed in Escherichia coli. Both expressed subunits were obtained as inclusion bodies. When recombinant γc was mixed with recombinant α and β subunits of F1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (α3β3γc) was reconstituted and it showed significant ATP hydrolysis activity. The ATP hydrolysis activity of this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCl2, which induces formation of a disulfide bond between two cysteine residues in γc. Hence, this complex has similar modulation characteristics as CF1. The effects of recombinant εc and ε subunit from TF1 (εt) on α3β3γc, were also investigated. εc strongly inhibited the ATP hydrolysis activity of chimeric α3β3γc complex but εt did not. The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium. The addition of εc or εt to the α3β3γt complex, which is the authentic subunit complex from TF1, resulted in weak stimulation of the ATP hydrolysis activity. These results suggest that (a) the specific regulatory function of γc can be transferred to the bacterial subunit complex; (b) the interaction between the γc subunit and εc strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the α3β3 core. [ABSTRACT FROM AUTHOR]

A positive selection technique was used to identify novel auxiliary calcium channel subunits that are similar to the skeletal muscle γ subunit. A new rat γ subunit cDNA was found, which was highly expressed in skeletal muscle tissue and was detected by RT-PCR in cardiac tissue. The 223-amino-acid-protein shares 84% and 79% identity, respectively, with the human and rabbit skeletal muscle subunits. Northern blot analysis revealed a single transcript of 1.5 kb in rat skeletal muscle, but not in cardiac tissue. Transient coexpression with the cardiac calcium channel complex demonstrated that the γ subunit shifted the inactivation curve to negative potentials and accelerated current inactivation without changing other voltage-dependent properties of the channel. [ABSTRACT FROM AUTHOR]

The F o F 1 synthase produces ATP from ADP and inorganic phosphate. The γ subunit of F o F 1 ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic β-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole F o F 1 complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire F o F 1 ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the β-hairpin structure of F o F 1 ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the β-hairpin structure, which provided novel insights into the regulatory mechanisms of F o F 1 ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The activity of the molecular motor enzyme, chloroplast ATP synthase, is regulated in a redox-dependent manner. The γ subunit, CF 1 -γ, is the central shaft of this enzyme complex and possesses the redox-active cysteine pair, which is reduced by thioredoxin (Trx). In light conditions, Trx transfers the reducing equivalent obtained from the photosynthetic electron transfer system to the CF 1 -γ. Previous studies showed that the light-dependent reduction of CF 1 -γ is more rapid than those of other Trx target proteins in the stroma. Although there are multiple Trx isoforms in chloroplasts, it is not well understood as to which chloroplast Trx isoform primarily contributes to the reduction of CF 1 -γ, especially under physiological conditions. We therefore performed direct assessment of the CF 1 -γ reduction capacity of each of the Trx isoforms. The kinetic analysis of the reduction process showed no significant difference in the reduction efficiency between two major chloroplast Trxs, namely Trx- f and Trx- m. Based on the thorough analyses of the CF 1 -γ redox dynamics in Arabidopsis thaliana Trx mutant plants, we found that lack of Trx- f or Trx- m had no significant impact on the in vivo light-dependent reduction of CF 1 -γ. The results showed that CF 1 -γ can accept the reducing power from both Trx- f and Trx- m in chloroplasts. • The light-dependent rapid reduction on CF 1 -γ is the unique molecular mechanism of this regulation. • The CF 1 -γ reduction capacity of each of the Trx isoforms was directly examined in vivo. • CF 1 -γ shows the modest specificity on thioredoxin isoforms in chloroplasts. [ABSTRACT FROM AUTHOR]

Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a γ subunit of the 2.6 megadalton α 6 -β 6 S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions. The γ subunit spans the length of the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The γ subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS. • Discovery of a regulatory γ subunit of the yeast fatty acid synthase (FAS) • The γ subunit regulates FAS reductase activities by kinetic hysteresis • Establishes structural basis for γ subunit-mediated FAS activity regulation • The γ subunit defines a higher efficiency FAS functional compartment Structural analysis identifies a new regulator of yeast fatty acid synthase. [ABSTRACT FROM AUTHOR]