Subunit Assembly Research Articles

The ϕPA3 phage nucleus is enclosed by a self-assembling 2D crystalline lattice

To protect themselves from host attack, numerous jumbo bacteriophages establish a phage nucleus—a micron-scale, proteinaceous structure encompassing the replicating phage DNA. Bacteriophage and host proteins associated with replication and transcription are concentrated inside the phage nucleus while other phage and host proteins are excluded, including CRISPR-Cas and restriction endonuclease host defense systems. Here, we show that nucleus fragments isolated from ϕPA3 infected Pseudomonas aeruginosa form a 2-dimensional lattice, having p2 or p4 symmetry. We further demonstrate that recombinantly purified primary Phage Nuclear Enclosure (PhuN) protein spontaneously assembles into similar 2D sheets with p2 and p4 symmetry. We resolve the dominant p2 symmetric state to 3.9 Å by cryo-EM. Our structure reveals a two-domain core, organized into quasi-symmetric tetramers. Flexible loops and termini mediate adaptable inter-tetramer contacts that drive subunit assembly into a lattice and enable the adoption of different symmetric states. While the interfaces between subunits are mostly well packed, two are open, forming channels that likely have functional implications for the transport of proteins, mRNA, and small molecules.

BRM Complex in Arabidopsis Adopts ncBAF-like Composition and Requires BRD Subunits for Assembly and Stability.

ATP-dependent SWI/SNF chromatin remodelling complexes are conserved multi-subunit assemblies that control genome activity. Functions of SWI/SNF complexes in plant development and growth have been well established, but the architecture of particular assemblies is unclear. In this study, we elucidate the organization of Arabidopsis SWI/SNF complexes formed around a BRM catalytic subunit, and define the requirement of bromodomain-containing proteins BRD1/2/13 for the formation and stability of the entire complex. Using affinity purification followed by mass spectrometry, we identify a set of BRM-associated subunits and demonstrate that the BRM complexes strongly resemble mammalian non-canonical BAF complexes. Furthermore, we identify BDH1 and 2 proteins as components of the BRM complex and, using mutant analyses, show that BDH1/2 are important for vegetative and generative development, as well as hormonal responses. We further show that BRD1/2/13 represent unique subunits of the BRM complexes, and their depletion severely affects the integrity of the complex, resulting in the formation of residual assemblies. Finally, analyses of BRM complexes after proteasome inhibition revealed the existence of a module consisting of the ATPase, ARP, and BDH proteins, assembled with other subunits in a BRD-dependent manner. Together, our results suggest modular organization of plant SWI/SNF complexes and provide a biochemical explanation for mutant phenotypes.

A molecular switch modulates assembly and host factor binding of the HIV-1 capsid

The HIV-1 capsid is a fullerene cone made of quasi-equivalent hexamers and pentamers of the viral CA protein. Typically, quasi-equivalent assembly of viral capsid subunits is controlled by a molecular switch. Here, we identify a Thr-Val-Gly-Gly motif that modulates CA hexamer/pentamer switching by folding into a 310 helix in the pentamer and random coil in the hexamer. Manipulating the coil/helix configuration of the motif allowed us to control pentamer and hexamer formation in a predictable manner, thus proving its function as a molecular switch. Importantly, the switch also remodels the common binding site for host factors that are critical for viral replication and the new ultra-potent HIV-1 inhibitor lenacapavir. This study reveals that a critical assembly element also modulates the post-assembly and viral replication functions of the HIV-1 capsid and provides new insights on capsid function and inhibition.

IQM-22110, a new selective inhibitor of KChIP3 and its electrophysiological effects on Kv4.3, Kv4.3/KChIP3 and Kv4.3/KChIP2 currents.

Kv4 channels generate rapidly activating and inactivating outward currents responsible for the repolarization of both cardiac and neuronal action potentials through ITO (transient outward current) and IA (A-type K+ current), respectively. Kv4 dysfunctions have been identified in both cardiac and neuronal diseases (Brugada syndrome, atrial fibrillation, epilepsy or Alzheimer's disease). However, Kv4 channels need to assemble with regulatory subunits to fully reproduce ITO and IA currents. Among them, we will focus on KChIPs (potassium channel interacting proteins), being KChIP3 predominant in the brain and KChIP2 in both brain and heart. The assembly of these regulatory subunits not only modulates the biophysical properties of the channel, but also its pharmacology. For this reason, we have analysed the electrophysiological effects of IQM-22110 (a novel KChIP3 ligand) on the currents generated by Kv4.3, Kv4.3/KChIP3 and Kv4.3/KChIP2 channels. CHO cells were transiently transfected, and the potassium currents were recorded using the whole-cell patch-clamp technique. Our results indicate that IQM-22110 exerts differential effects on these currents, being the concentration-dependence of inhibition modified, exhibiting a biphasic curve when KChIP3 is present, thus suggesting two binding sites for IQM-22110 in this complex. With molecular dynamics and site-directed mutagenesis, we have identified the binding pocket corresponding to the high affinity site. Also, we have demonstrated that IQM-22110 binds to the closed-active state of the channel. In this study: i) IQM-22110 has proven to be a selective inhibitor of Kv4.3/KChIP3 at low concentrations, and ii) a new binding pocket for selective KChIP3/Kv4.3 ligands has been identified.

Determining the role of the unique N-terminal 1b domain in the gating of heterotetrameric hERG1a/hERG1b potassium channels.

Two isoforms of hERG (human Ether-a-go-go-Related Gene) potassium channel, hERG1a and hERG1b, associate in the heart to form the rapid delayed rectifier current (IKr), which is primarily responsible for repolarizing cardiac action potentials. Inherited mutations in both hERG1a and hERG1b cause Long QT Syndrome type 2 (LQT2), marked by prolonged cardiac repolarization and sudden death arrhythmia. The assembly of hERG1a subunits with hERG1b subunits increases the number of hERG1b subunits at the plasma membrane, but the mechanism by which hERG1a enhances hERG1b is not completely understood. Recently, it was reported that when expressed in trans, the hERG1a N-terminal region markedly increased hERG1b currents and increased biotin-labelled hERG1b protein at the plasma membrane surface. A direct structural interaction between the hERG1a N-terminal residues 216-220 and the hERG1b N-terminal region was characterized and proposed to be necessary for the upregulation of hERG1b (Johnson, Crawford, and Trudeau, 2022). However, hERG1b channels with a deletion of the unique N-terminal 1b domain did not have a measurable increase in current density or in biotinylated protein when co-expressed with hERG1a N-terminal region pieces. This indicates that the 1b domain is required for the increase in hERG1b by hERG1a. Interestingly, the 1b domain-deleted hERG1b channels displayed a large leftward shift in the voltage dependence of activation when co-expressed with the full hERG1a N-terminal region. This is unexpected, as the 1b domain was not previously considered as a determinant of hERG gating. Here, we will investigate the mechanism of this large leftward shift and determine the role of the 1b domain in the gating properties of hERG1a/hERG1b heterotetrameric channels.

Mechanistic studies of endolysosomal membrane repair by the human ESCRT machinery through in vitro reconstitution.

The endosomal sorting complex required for transport (ESCRT) machinery is centrally involved in the repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale and calcium has been proposed to play a key signaling role in the process. Here, we show that the calcium-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a calcium-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to the membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, and CHMP2A subunits, along with the AAA+ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open an avenue for the mechanistic understanding of endolysosomal membrane repair.

A possible role for phase separation in heteromeric biogenesis of hERG1a/1b channels.

The human ether-a-go-go related gene (hERG) encodes a K+ channel important for repolarizing the cardiac action potential. Cardiac IKr is produced by heteromeric assemblies of hERG1a and 1b subunits, and depletion of hERG1b subunits confers proarrhythmic behavior in cellular cardiac models. hERG1a homomers can traffic to the membrane, but hERG1b homomers fail to progress efficiently out of the endoplasmic reticulum (ER). We found that hERG1b protein is sequestered and forms punctate intracellular structures when expressed heterologously in HeLa cells. When co-expressed with hERG1a, previously shown to promote hERG1b maturation, few puncta are observed and both signals are robustly present at the plasma membrane. We show that 1b puncta are primarily localized to the ER space and do not overlap with other membranous compartments, including COPII, lysosome, and autophagosome. Testing the hypothesis that hERG1b puncta are condensates arising from liquid-liquid phase separation, we used live cell imaging and observed that sequestered hERG1b puncta undergo fusion events, although rarely as might be expected from ER-delimited membrane proteins. Using truncation experiments, we found that removing the disordered C-terminus altered the size and distribution of the puncta but removing the structured cyclic nucleotide-binding homology domain (CNBhD) abolished the puncta entirely. These data suggest the puncta are formed by multivalent interactions of ordered and disordered domains of hERG1b and may sequester hERG1b in the absence of hERG1a. In this way sequestration in puncta may limit deleterious homomeric expression of hERG1b and thus promote heteromeric hERG1a/1b channel assembly.

Molecular dynamics simulations of neuronal nicotinic acetylcholine receptor allosteric modulation.

The neuronal nicotinic acetylcholine receptor is a key experimental system for studying allosteric modulation, including by clinically useful drugs. Diverse physiological responses manifest from differential assembly of nicotinic receptor subunits (α2−α10 and β2−β4) into homo- or heteropentamers. An extensive body of electrophysiology data has demonstrated that the anthelmintic compounds morantel and oxantel display limited partial-agonist activity at mammalian nicotinic receptors, but are positive allosteric modulators of α3β2 receptors, while oxantel is a negative modulator of α4β2 receptors. Largely through mutation analyses, we have identified amino acid determinants of specificity for the compounds, as well as elements of the receptor extracellular domain that crosstalk between functional sites. Here, we initiated a systematic comparison of subtype specificity, based on molecular modeling and simulations of published cryo-electron microscopy structures and homology models. Preliminary results indicate differential movements in the C loop of orthosteric versus allosteric sites, involvement of key residues predicted to influence specificity in interactions with ligands, and inter- and intra-subunit functional connectivity assessed by network analysis. These studies promise to elucidate allosteric modulation mechanisms that govern site-to-site and ligand-to-gate communication in nicotinic and related receptors.

Tetratricopeptide-containing SMALL KERNEL 11 is essential for the assembly of cytochrome c oxidase in maize mitochondria.

Assembly of the functional complexes of the mitochondrial respiratory chain requires sophisticated and efficient regulatory mechanisms. In plants, the subunit composition and assembly factors involved in the biogenesis of cytochrome c oxidase (complex IV) are substantially less defined than in mammals and yeast. In this study, we cloned maize (Zea mays) Small kernel 11 (Smk11) via map-based cloning. Smk11 encodes a mitochondria-localized tetratricopeptide repeat protein. Disruption of Smk11 severely affected the assembly and activity of mitochondrial complex IV, leading to delayed plant growth and seed development. Protein interactions studies revealed that SMK11 might interact with four putative complex IV assembly factors, Inner membrane peptidase 1A (ZmIMP1A), MYB domain protein 3R3 (ZmMYB3R-3), cytochrome c oxidase 23 (ZmCOX23), and mitochondrial ferredoxin 1 (ZmMFDX1), among which ZmMFDX1 might interact with subunits ZmCOX6a and ZmCOX-X1; ZmMYB3R-3 might also interact with ZmCOX6a. The mutation of SMK11 perturbed the normal assembly of these subunits, leading to the inactivation of complex IV. The results of this study revealed that SMK11 serves as an accessory assembly factor required for the normal assembly of subunits into complex IV, which will accelerate the elucidation of the assembly of complex IV in plant mitochondria.

Principles of mitoribosomal small subunit assembly in eukaryotes.

Mitochondrial ribosomes (mitoribosomes) synthesize proteins encoded within the mitochondrial genome that are assembled into complexes involved in oxidative phosphorylation. Thus, mitoribosome biogenesis is essential for ATP production and cellular metabolism. In this study, we used cryo-electron microscopy to determine 9 high-resolution structures of native yeast and human mitoribosomal small subunit assembly intermediates, illuminating the mechanistic basis for how GTPases are employed to control early steps of decoding center formation, how initial rRNA folding and processing events are mediated, and how mitoribosomal proteins play active roles during assembly. This series of intermediates from two species with divergent mitoribosomal architecture uncovers both conserved principles and species-specific adaptations that govern the maturation of mitoribosomal small subunits in eukaryotes. By revealing the dynamic interplay between assembly factors, mitoribosomal proteins, and rRNA required to generate functional subunits, our structural analysis provides a vignette for how molecular complexity and diversity can evolve in large ribonucleoprotein assemblies.

STAT3 promotes RNA polymerase III-directed transcription by controlling the miR-106a-5p/TP73 axis.

Deregulation of Pol III products causes a range of diseases, including neural diseases and cancers. However, the factors and mechanisms that modulate Pol III-directed transcription remain to be found, although massive advances have been achieved. Here, we show that STAT3 positively regulates the activities of Pol III-dependent transcription and cancer cell growth. RNA-seq analysis revealed that STAT3 inhibits the expression of TP73, a member of the p53 family. We found that TP73 is not only required for the regulation of Pol III-directed transcription mediated by STAT3 but also independently suppresses the synthesis of Pol III products. Mechanistically, TP73 can disrupt the assembly of TFIIIB subunits and inhibit their occupancies at Pol III target loci by interacting with TFIIIB subunit TBP. MiR-106a-5p can activate Pol III-directed transcription by targeting the TP73 mRNA 3' UTR to reduce TP 73 expression. We show that STAT3 activates the expression of miR-106a-5p by binding to the miRNA promoter, indicating that the miR-106a-5p links STAT3 with TP73 to regulate Pol III-directed transcription. Collectively, these findings indicate that STAT3 functions as a positive regulator in Pol III-directed transcription by controlling the miR-106a-5p/TP73 axis.

Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

GluA3 subunits are required for appropriate assembly of AMPAR GluA2 and GluA4 subunits on cochlear afferent synapses and for presynaptic ribbon modiolar-pillar morphology.

Cochlear sound encoding depends on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), but reliance on specific pore-forming subunits is unknown. With 5-week-old male C57BL/6J Gria3-knockout mice (i.e., subunit GluA3KO) we determined cochlear function, synapse ultrastructure, and AMPAR molecular anatomy at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons. GluA3KO and wild-type (GluA3WT) mice reared in ambient sound pressure level (SPL) of 55-75 dB had similar auditory brainstem response (ABR) thresholds, wave-1 amplitudes, and latencies. Postsynaptic densities (PSDs), presynaptic ribbons, and synaptic vesicle sizes were all larger on the modiolar side of the IHCs from GluA3WT, but not GluA3KO, demonstrating GluA3 is required for modiolar-pillar synapse differentiation. Presynaptic ribbons juxtaposed with postsynaptic GluA2/4 subunits were similar in quantity, however, lone ribbons were more frequent in GluA3KO and GluA2-lacking synapses were observed only in GluA3KO. GluA2 and GluA4 immunofluorescence volumes were smaller on the pillar side than the modiolar side in GluA3KO, despite increased pillar-side PSD size. Overall, the fluorescent puncta volumes of GluA2 and GluA4 were smaller in GluA3KO than GluA3WT. However, GluA3KO contained less GluA2 and greater GluA4 immunofluorescence intensity relative to GluA3WT (threefold greater mean GluA4:GluA2 ratio). Thus, GluA3 is essential in development, as germline disruption of Gria3 caused anatomical synapse pathology before cochlear output became symptomatic by ABR. We propose the hearing loss in older male GluA3KO mice results from progressive synaptopathy evident in 5-week-old mice as decreased abundance of GluA2 subunits and an increase in GluA2-lacking, GluA4-monomeric Ca2+-permeable AMPARs.

The role of post-translational modifications in synaptic AMPA receptor activity.

AMPA-type receptors for the neurotransmitter glutamate are very dynamic entities, and changes in their synaptic abundance underlie different forms of synaptic plasticity, including long-term synaptic potentiation (LTP), long-term depression (LTD) and homeostatic scaling. The different AMPA receptor subunits (GluA1-GluA4) share a common modular structure and membrane topology, and their intracellular C-terminus tail is responsible for the interaction with intracellular proteins important in receptor trafficking. The latter sequence differs between subunits and contains most sites for post-translational modifications of the receptors, including phosphorylation, O-GlcNAcylation, ubiquitination, acetylation, palmitoylation and nitrosylation, which affect differentially the various subunits. Considering that each single subunit may undergo modifications in multiple sites, and that AMPA receptors may be formed by the assembly of different subunits, this creates multiple layers of regulation of the receptors with impact in synaptic function and plasticity. This review discusses the diversity of mechanisms involved in the post-translational modification of AMPA receptor subunits, and their impact on the subcellular distribution and synaptic activity of the receptors.

Regulating Shaker Kv channel clustering by hetero-oligomerization.

Scaffold protein-mediated voltage-dependent ion channel clustering at unique membrane sites, such as nodes of Ranvier or the post-synaptic density plays an important role in determining action potential properties and information coding. Yet, the mechanism(s) by which scaffold protein-ion channel interactions lead to channel clustering and how cluster ion channel density is regulated are mostly unknown. This molecular-cellular gap in understanding channel clustering can be bridged in the case of the prototypical Shaker voltage-activated potassium channel (Kv), as the mechanism underlying the interaction of this channel with its PSD-95 scaffold protein partner is known. According to this mechanism, changes in the length of the intrinsically disordered channel C-terminal chain, brought about by alternative splicing to yield the short A and long B chain subunit variants, dictate affinity to PSD-95 and further controls cluster homo-tetrameric Kv channel density. These results raise the hypothesis that heteromeric subunit assembly serves as a means to regulate Kv channel clustering. Since both clustering variants are expressed in similar fly tissues, it is reasonable to assume that hetero-tetrameric channels carrying different numbers of high- (A) and low-affinity (B) subunits could assemble, thereby giving rise to distinct cluster Kv channel densities. Here, we tested this hypothesis using high-resolution microscopy, combined with quantitative clustering analysis. Our results reveal that the A and B clustering variants can indeed assemble to form heteromeric channels and that controlling the number of the high-affinity A subunits within the hetero-oligomer modulates cluster Kv channel density. The implications of these findings for electrical signaling are discussed.

RanBP9 controls the oligomeric state of CTLH complex assemblies

The CTLH (C-terminal to lissencephaly-1 homology motif) complex is a multisubunit RING E3 ligase with poorly defined substrate specificity and flexible subunit composition. Two key subunits, muskelin and Wdr26, specify two alternative CTLH complexes that differ in quaternary structure, thereby allowing the E3 ligase to presumably target different substrates. With the aid of different biophysical and biochemical techniques, we characterized CTLH complex assembly pathways, focusing not only on Wdr26 and muskelin but also on RanBP9, Twa1, and Armc8β subunits, which are critical to establish the scaffold of this E3 ligase. We demonstrate that the ability of muskelin to tetramerize and the assembly of Wdr26 into dimers define mutually exclusive oligomerization modules that compete with nanomolar affinity for RanBP9 binding. The remaining scaffolding subunits, Armc8β and Twa1, strongly interact with each other and with RanBP9, again with nanomolar affinity. Our data demonstrate that RanBP9 organizes subunit assembly and prevents higher order oligomerization of dimeric Wdr26 and the Armc8β-Twa1 heterodimer through its tight binding. Combined, our studies define alternative assembly pathways of the CTLH complex and elucidate the role of RanBP9 in governing differential oligomeric assemblies, thereby advancing our mechanistic understanding of CTLH complex architectures.

Dynamics in the assembly of the 30S ribosomal subunit investigated by coarse-grained simulations

The ribosome is a large biomolecular complex responsible for protein synthesis. In <i>Escherichia coli</i> (<i>E. coli</i>), a complete ribosome is composed of a 30S small subunit and a 50S large subunit. For approximately half a century, the 30S subunit has been a key model system for studying the in vitro assembly of the ribosome, and an assembly map has been proposed. However, structural details in the assembly of this protein‒RNA complex remain elusive. In this paper, we conducted a series of coarse-grained simulations following the order of the assembly map to investigate conformational dynamics during the assembly process of the 30S subunit. It has been found that the tertiary structure of naked 16S rRNA is very unstable, which is the case after binding of early-assembly proteins. The mid-assembly proteins can significantly restrict the mobility of the 16S rRNA and make the latter close to the native structure. The final binding of the late-assembly proteins would fully obtain the collective motion of the 16S rRNA. In particular, proteins S9 and S3 may have more important contributions to the assembly of the 30S subunit than other S proteins. Our strategy of coarse-grained simulations can be generally used to study assembly dynamics of large biomolecular complexes as long as the assembly map is available.

Schisanhenol Attenuates OxLDL-Induced Endothelial Dysfunction via an AMPK-Dependent Mechanism.

Atherosclerotic cardiovascular diseases, commonly known as the formation of fibrofatty lesions in the artery wall, are the leading causes of death globally. Oxidized low-density lipoprotein (oxLDL) is one of the major components of atherosclerotic plaques. It is evident that dietary supplementation containing sources of antioxidants can prevent atherogenic diseases. Schisanhenol (SAL), a dibenzocyclooctene lignin, has been shown to attenuate oxLDL-induced apoptosis and the generation of reactive oxygen species (ROS) in endothelial cells. However, the underlying molecular mechanisms are still largely unknown. In this study, human umbilical vein endothelial cells (HUVECs) were pre-treated with SAL and oxLDL. Our results showed that adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was enhanced in cells pre-treated with SAL in time-dependent and dose-dependent manners. Subsequently, oxLDL-induced AMPK dephosphorylation and protein kinase C (PKC) phosphorylation were significantly reversed in the presence of SAL. In addition, SAL treatment led to an inhibiting effect on the oxLDL-induced membrane assembly of NADPH oxidase subunits, and a similar effect was observed in ROS generation. This effect was further confirmed using knockdown AMPK with small interfering RNA (siRNA) and pharmaceutical reagents, such as the AMPK activator (AICAR), PKC inhibitor (Gö 6983), and ROS inhibitor (DPI). Furthermore, the oxLDL-induced intracellular calcium rise and the potential collapse of the mitochondrial membrane reduced the Bcl-2/Bax ratio, and released cytochrome c from the mitochondria, leading to the subsequent activation of caspase-3 in HUVECs, which were also markedly suppressed by SAL pretreatment. The results mentioned above may provide additional insights into the possible molecular mechanisms underlying the cardiovascular protective effects of SAL.

Cell dehydration of intergeneric hybrid induces subgenome-related specific responses.

The aim was to identify subgenome-related specific responses in two types of triticale, that is, of the wheat-dominated genome (WDG) and rye-dominated genome (RDG), to water stress induced in the early phase (tillering) of plant growth. Higher activity of the primary metabolism of carbohydrates is a feature of the WDG type, while the dominance of the rye genome is associated with a higher activity of the secondary metabolism of phenolic compounds in the RDG type. The study analyzed carbohydrates and key enzymes of their synthesis, free phenolic compounds and carbohydrate-related components of the cell wall, monolignols, and shikimic acid (ShA), which is a key link between the primary and secondary metabolism of phenolic compounds. Under water stress, dominance of the wheat genome in the WDG type was manifested by an increased accumulation of the large subunit of Rubisco and sucrose phosphate synthase and a higher content of raffinose and stachyose compared with the RDG type. In dehydrated RDG plants, higher activity of L-phenylalanine ammonia lyase (PAL) and L-tyrosine ammonia lyase (TAL), as well as a higher level of ShA, free and cell wall-bound p-hydroxybenzoic acid, free homovanillic acid, free sinapic acid, and cell wall-bound syringic acid can be considered biochemical indicators of the dominance of the rye genome.

The membrane domain of respiratory complex I accumulates during muscle aging in Drosophila melanogaster

The boot-shaped respiratory complex I (CI) consists of a mitochondrial matrix and membrane domain organized into N-, Q- and P-modules. The N-module is the most distal part of the matrix domain, whereas the Q-module is situated between the N-module and the membrane domain. The proton-pumping P-module is situated in the membrane domain. We explored the effect of aging on the disintegration of CI and its constituent subcomplexes and modules in Drosophila flight muscles. We find that the fully-assembled complex remains largely intact in aged flies. And while the effect of aging on the stability of many Q- and N-module subunits in subcomplexes was stochastic, NDUFS3 was consistently down-regulated in subcomplexes with age. This was associated with an accumulation of many P-module subunits in subcomplexes. The potential significance of these studies is that genetic manipulations aimed at boosting, perhaps, a few CI subunits may suffice to restore the whole CI biosynthesis pathway during muscle aging.