TOC Complex Research Articles

Structural insights into the chloroplast protein import in land plants

Chloroplast proteins are imported via the translocon at the outer chloroplast membrane (TOC)-translocon at the inner chloroplast membrane (TIC) supercomplex, driven by an ATPase motor. The Ycf2-FtsHi complex has been identified as the chloroplast import motor. However, its assembly and cooperation with the TIC complex during preprotein translocation remain unclear. Here, we present the structures of the Ycf2-FtsHi and TIC complexes from Arabidopsis and an ultracomplex formed between them from Pisum. The Ycf2-FtsHi structure reveals a heterohexameric AAA+ ATPase motor module with characteristic features. Four previously uncharacterized components of Ycf2-FtsHi were identified, which aid in complex assembly and anchoring of the motor module at a tilted angle relative to the membrane. When considering the structures of the TIC complex and the TIC-Ycf2-FtsHi ultracomplex together, it becomes evident that the tilted motor module of Ycf2-FtsHi enables its close contact with the TIC complex, thereby facilitating efficient preprotein translocation. Our study provides valuable structural insights into the chloroplast protein import process in land plants.

Predictors of Impairment and Self-Concept in Children and Adolescents with Persistent Tic Disorder.

This study examined predictors of, and associations between, self-concept, demographic variables, and clinical measures in fifty-eight children and adolescents with Persistent Tic Disorder (PTD; 44 males, Mage = 11.9years, SD = 2.74). Participants completed measures that assessed self-concept, tic severity, tic-related impairment, and comorbid psychological symptoms. Results showed that generalized anxiety disorder, major depressive disorder, persistent depressive disorder, total tic severity, number and complexity of tics, and total and social tic-related impairment were associated with self-concept. Tic-related social impairment mediated the relationship between tic severity and self-concept. Exploratory analyses found that total tic severity, motor tic severity, and vocal tic severity, as well as the number, intensity, and interference of tics predicted social tic-related impairment. Results suggest that treatments to reduce the number and complexity of tics, with additional focus on navigating social interactions, may serve to decrease tic severity and impairment, and in turn, improve self-concept.

Shining light on the chloroplast protein import machinery of plants

Sunny days for chloroplast protein import: Srinivasan etal. (2023) report on the isolation and provide the first structural information on the pea TOC complex that mediates protein translocation across the outer chloroplast membrane. While two cryoEM structures for algal import complexes have been published, this is the gateway to long-sought-after structures from land plants.

Contrasting features between Tourette syndrome and secondary tic disorders.

Tics are rapid, recurrent, non-rhythmic movements or emitted sounds. Tics are the hallmark of Tourette syndrome (TS); however, a number of other disorders may be associated with tics, so-called secondary tic disorders (STD). We assessed clinical history and performed blinded evaluations of video-recordings from patients with TS and STD in order to identify features that may differentiate tics associated with TS vs STD. There were 156 patients with TS and 38 with STD, 21 of whom had functional (psychogenic) tics. Patients with TS were more frequently male and had a younger age at onset. Tics in TS tend to involve muscles in the cranial-cervical area more often and have greater severity and complexity than those in patients with STD. Similar findings were observed when contrasting patients with TS with patients with functional tics only. Simple phonic tics showed the greatest diagnostic accuracy for TS, compared with STD, but marked overlap in the types of tics and comorbidities was observed between patients with TS and STD. Patients with TS were more likely males, had a younger age at onset, phonic tics and motor tics affecting predominantly the head and neck area, and had a greater complexity and severity of tics than those with STD. When these features are absent a consideration should be given to the possibility of a tic disorder other than TS.

Understanding protein import in diverse non-green plastids.

The spectacular diversity of plastids in non-green organs such as flowers, fruits, roots, tubers, and senescing leaves represents a Universe of metabolic processes in higher plants that remain to be completely characterized. The endosymbiosis of the plastid and the subsequent export of the ancestral cyanobacterial genome to the nuclear genome, and adaptation of the plants to all types of environments has resulted in the emergence of diverse and a highly orchestrated metabolism across the plant kingdom that is entirely reliant on a complex protein import and translocation system. The TOC and TIC translocons, critical for importing nuclear-encoded proteins into the plastid stroma, remain poorly resolved, especially in the case of TIC. From the stroma, three core pathways (cpTat, cpSec, and cpSRP) may localize imported proteins to the thylakoid. Non-canonical routes only utilizing TOC also exist for the insertion of many inner and outer membrane proteins, or in the case of some modified proteins, a vesicular import route. Understanding this complex protein import system is further compounded by the highly heterogeneous nature of transit peptides, and the varying transit peptide specificity of plastids depending on species and the developmental and trophic stage of the plant organs. Computational tools provide an increasingly sophisticated means of predicting protein import into highly diverse non-green plastids across higher plants, which need to be validated using proteomics and metabolic approaches. The myriad plastid functions enable higher plants to interact and respond to all kinds of environments. Unraveling the diversity of non-green plastid functions across the higher plants has the potential to provide knowledge that will help in developing climate resilient crops.

Chloroplast Proteostasis: Import, Sorting, Ubiquitination, and Proteolysis.

Chloroplasts are the defining plant organelles with responsibility for photosynthesis and other vital functions. To deliver these functions, they possess a complex proteome comprising thousands of largely nucleus-encoded proteins. Composition of the proteome is controlled by diverse processes affecting protein translocation and degradation-our focus here. Most chloroplast proteins are imported from the cytosol via multiprotein translocons in the outer and inner envelope membranes (the TOC and TIC complexes, respectively), or via one of several noncanonical pathways, and then sorted by different systems to organellar subcompartments. Chloroplast proteolysis is equally complex, involving the concerted action of internal proteases of prokaryotic origin and the nucleocytosolic ubiquitin-proteasome system (UPS). The UPS degrades unimported proteins in the cytosol and chloroplast-resident proteins via chloroplast-associated protein degradation (CHLORAD). The latter targets the TOC apparatus to regulate protein import, as well as numerous internal proteins directly, to reconfigure chloroplast functions in response to developmental and environmental signals.

Architecture of chloroplast TOC-TIC translocon supercomplex.

Chloroplasts rely on the translocon complexes in the outer and inner envelope membranes (the TOC and TIC complexes, respectively) to import thousands of different nuclear-encoded proteins from the cytosol1-4. Although previous studies indicated that the TOC and TIC complexes may assemble into larger supercomplexes5-7, the overall architectures of the TOC-TIC supercomplexes and the mechanism of preprotein translocation are unclear. Here we report the cryo-electron microscopy structure of the TOC-TIC supercomplex from Chlamydomonas reinhardtii. The major subunits of the TOC complex (Toc75, Toc90 and Toc34) and TIC complex (Tic214, Tic20, Tic100 and Tic56), three chloroplast translocon-associated proteins (Ctap3, Ctap4 and Ctap5) and three newly identified small inner-membrane proteins (Simp1-3) have been located in the supercomplex. As the largest protein, Tic214 traverses the inner membrane, the intermembrane space and the outer membrane, connecting the TOC complex with the TIC proteins. An inositol hexaphosphate molecule is located at the Tic214-Toc90 interface and stabilizes their assembly. Four lipid molecules are located within or above an inner-membrane funnel formed by Tic214, Tic20, Simp1 and Ctap5. Multiple potential pathways found in the TOC-TIC supercomplex may support translocation of different substrate preproteins into chloroplasts.

Understanding protein translocation across chloroplast membranes: Translocons and motor proteins.

Subcellular organelles in eukaryotes are surrounded by lipid membranes. In an endomembrane system, vesicle trafficking is the primary mechanism for the delivery of organellar proteins to specific organelles. However, organellar proteins for chloroplasts, mitochondria, the nucleus, and peroxisomes that are translated in the cytosol are directly imported into their target organelles. Chloroplasts are a plant-specific organelle with outer and inner envelope membranes, a dual-membrane structure that is similar to mitochondria. Interior chloroplast proteins translated by cytosolic ribosomes are thus translocated through TOC and TIC complexes (translocons in the outer and inner envelope of chloroplasts, respectively), with stromal ATPase motor proteins playing a critical role in pulling pre-proteins through these import channels. Over the last three decades, the identity and function of TOC/TIC components and stromal motor proteins have been actively investigated, which has shed light on the action mechanisms at a molecular level. However, there remains some disagreement over the exact composition of TIC complexes and genuine stromal motor proteins. In this review, we discuss recent findings on the mechanisms by which proteins are translocated through TOC/TIC complexes and discuss future prospects for this field of research.

Structure of a TOC-TIC supercomplex spanning two chloroplast envelope membranes

The TOC and TIC complexes are essential translocons that facilitate the import of the nuclear genome-encoded preproteins across the two envelope membranes of chloroplast, but their exact molecular identities and assembly remain unclear. Here, we report a cryoelectron microscopy structure of TOC-TIC supercomplex from Chlamydomonas, containing a total of 14 identified components. The preprotein-conducting pore of TOC is a hybrid β-barrel co-assembled by Toc120 and Toc75, while the potential translocation path of TIC is formed by transmembrane helices from Tic20 and YlmG, rather than a classic model of Tic110. A rigid intermembrane space (IMS) scaffold bridges two chloroplast membranes, and a large hydrophilic clefton the IMS scaffold connects TOC and TIC, forming a pathway for preprotein translocation. Our study provides structural insights into the TOC-TIC supercomplex composition, assembly, and preprotein translocation mechanism, and lays a foundation to interpret the evolutionary conservation and diversity of this fundamental translocon machinery.

Molecular characterization, targeting and expression analysis of chloroplast and mitochondrion protein import components in Nicotiana benthamiana.

Improved bioinformatics tools for annotating gene function are becoming increasingly available, but such information must be considered theoretical until further experimental evidence proves it. In the work reported here, the genes for the main components of the translocons of the outer membrane of chloroplasts (Toc) and mitochondria (Tom), including preprotein receptors and protein-conducting channels of N. benthamiana, were identified. Sequence identity searches and phylogenetic relationships with functionally annotated sequences such as those of A. thaliana revealed that N. benthamiana orthologs mainly exist as recently duplicated loci. Only a Toc34 ortholog was found (NbToc34), while Toc159 receptor family was composed of four orthologs but somewhat different from those of A. thaliana. Except for NbToc90, the rest (NbToc120, NbToc159A and NbToc159B) had a molecular weight of about 150 kDa and an acidic domain similar in length. Only two orthologs of the Tom20 receptors, NbTom20-1 and NbTom20-2, were found. The number of the Toc and Tom receptor isoforms in N. benthamiana was comparable to that previously reported in tomato and what we found in BLAST searches in other species in the genera Nicotiana and Solanum. After cloning, the subcellular localization of N. benthamiana orthologs was studied, resulting to be identical to that of A. thaliana receptors. Phenotype analysis after silencing together with relative expression analysis in roots, stems and leaves revealed that, except for the Toc and Tom channel-forming components (NbToc75 and NbTom40) and NbToc34, functional redundancy could be observed either among Toc159 or mitochondrial receptors. Finally, heterodimer formation between NbToc34 and the NbToc159 family receptors was confirmed by two alternative techniques indicating that different Toc complexes could be assembled. Additional work needs to be addressed to know if this results in a functional specialization of each Toc complex.

Mutations in the chloroplast inner envelope protein TIC100 impair and repair chloroplast protein import and impact retrograde signaling.

Chloroplast biogenesis requires synthesis of proteins in the nucleocytoplasm and the chloroplast itself. Nucleus-encoded chloroplast proteins are imported via multiprotein translocons in the organelle’s envelope membranes. Controversy exists around whether a 1-MDa complex comprising TIC20, TIC100, and other proteins constitutes the inner membrane TIC translocon. The Arabidopsis thaliana cue8 virescent mutant is broadly defective in plastid development. We identify CUE8 as TIC100. The tic100cue8 mutant accumulates reduced levels of 1-MDa complex components and exhibits reduced import of two nucleus-encoded chloroplast proteins of different import profiles. A search for suppressors of tic100cue8 identified a second mutation within the same gene, tic100soh1, which rescues the visible, 1 MDa complex-subunit abundance, and chloroplast protein import phenotypes. tic100soh1 retains but rapidly exits virescence and rescues the synthetic lethality of tic100cue8 when retrograde signaling is impaired by a mutation in the GENOMES UNCOUPLED 1 gene. Alongside the strong virescence, changes in RNA editing and the presence of unimported precursor proteins show that a strong signaling response is triggered when TIC100 function is altered. Our results are consistent with a role for TIC100, and by extension the 1-MDa complex, in the chloroplast import of photosynthetic and nonphotosynthetic proteins, a process which initiates retrograde signaling.

Chloroplast protein import machinery and quality control.

Most chloroplast proteins are nucleus-encoded, translated on cytoplasmic ribosomes as precursor proteins, and imported into chloroplasts through TOC and TIC, the translocons of the outer and inner chloroplast envelope membranes. While the composition of the TOC complex is well established, there is still some controversy about the importance of a recently identified TIC complex consisting of Tic20, Tic214, Tic100, and Tic56. TOC and TIC form a supercomplex with a protein channel at the junction of the outer and inner envelope membranes through which preproteins are pulled into the stroma by the ATP-powered Ycf2 complex consisting of several FtsH-like ATPases and/or by chloroplast Hsp proteins. Several components of the TOC/TIC system are moonlighting proteins with additional roles in chloroplast gene expression and metabolism. Chaperones and co-chaperones, associated with TOC and TIC on the cytoplasmic and stromal side of the chloroplast envelope, participate in the unfolding and folding of the precursor proteins and act together with the ubiquitin-proteasome system in protein quality control. Chloroplast protein import is also intimately linked with retrograde signaling, revealing altogether an unsuspected complexity in the regulation of this process.

Sex differences in patients with Tourette syndrome.

Tourette syndrome (TS) is a neurodevelopmental disorder characterized by the presence of motor and phonic tics. It is at least three times more common in males compared with females; however, the clinical phenomenology between sexes has not been fully examined. We aimed to contrast the clinical features between males and females with TS and chronic tic disorder. We studied 201 consecutive patients fulfilling the diagnostic criteria for TS, persistent (or chronic) motor and vocal tic disorder and provisional tic disorder that were considered within the TS spectrum disorder. We performed blinded evaluations of video-recordings and retrospectively reviewed the clinical charts of all patients. Age ranges between 4 and 65years. Males represented 77.6% of patients in the cohort. Overall, no differences were observed in the frequency, distribution and complexity of tics between sexes, except for a higher frequency of attention-deficit/hyperactivity disorder (ADHD) (P=.003) among males. Patients younger than 18-years old, in addition to a higher frequency of ADHD (P=.026), males had a statistically higher frequency of complex motor tics (P=.049) and earlier age at onset (P=.072) than females in the multivariate regression analysis. However, these differences were lost in patients older than 18years, due to increased complexity of tics in females with aging. A sexual dimorphism was observed between patients with TS mainly before age of 18years, suggesting an earlier onset of some types of tics and ADHD in males compared to females.

Crosstalk between the chloroplast protein import and SUMO systems revealed through genetic and molecular investigation in Arabidopsis.

The chloroplast proteome contains thousands of different proteins that are encoded by the nuclear genome. These proteins are imported into the chloroplast via the action of the TOC translocase and associated downstream systems. Our recent work has revealed that the stability of the TOC complex is dynamically regulated by the ubiquitin-dependent chloroplast-associated protein degradation pathway. Here, we demonstrate that the TOC complex is also regulated by the small ubiquitin-like modifier (SUMO) system. Arabidopsis mutants representing almost the entire SUMO conjugation pathway can partially suppress the phenotype of ppi1, a pale-yellow mutant lacking the Toc33 protein. This suppression is linked to increased abundance of TOC proteins and improvements in chloroplast development. Moreover, data from molecular and biochemical experiments support a model in which the SUMO system directly regulates TOC protein stability. Thus, we have identified a regulatory link between the SUMO system and the chloroplast protein import machinery.

Metabolic activities and molecular investigations of the ameliorative impact of some growth biostimulators on chilling-stressed coriander (Coriandrum sativum L.) plant

BackgroundPriming of seed prior chilling is regarded as one of the methods to promote seeds germination, whole plant growth, and yield components. The application of biostimulants was reported as beneficial for protecting many plants from biotic or abiotic stresses. Their value was as important to be involved in improving the growth parameters of plants. Also, they were practiced in the regulation of various metabolic pathways to enhance acclimation and tolerance in coriander against chilling stress. To our knowledge, little is deciphered about the molecular mechanisms underpinning the ameliorative impact of biostimulants in the context of understanding the link and overlap between improved morphological characters, induced metabolic processes, and upregulated gene expression. In this study, the ameliorative effect(s) of potassium silicate, HA, and gamma radiation on acclimation of coriander to tolerate chilling stress was evaluated by integrating the data of growth, yield, physiological and molecular aspects.ResultsPlant growth, yield components, and metabolic activities were generally diminished in chilling-stressed coriander plants. On the other hand, levels of ABA and soluble sugars were increased. Alleviation treatment by humic acid, followed by silicate and gamma irradiation, has notably promoted plant growth parameters and yield components in chilling-stressed coriander plants. This improvement was concomitant with a significant increase in phytohormones, photosynthetic pigments, carbohydrate contents, antioxidants defense system, and induction of large subunit of RuBisCO enzyme production. The assembly of Toc complex subunits was maintained, and even their expression was stimulated (especially Toc75 and Toc 34) upon alleviation of the chilling stress by applied biostimulators. Collectively, humic acid was the best the element to alleviate the adverse effects of chilling stress on growth and productivity of coriander.ConclusionsIt could be suggested that the inducing effect of the pretreatments on hormonal balance triggered an increase in IAA + GA3/ABA hormonal ratio. This ratio could be linked and engaged with the protection of cellular metabolic activities from chilling injury against the whole plant life cycle. Therefore, it was speculated that seed priming in humic acid is a powerful technique that can benefit the chilled along with non-chilled plants and sustain the economic importance of coriander plant productivity.

Art as relaxation for tic disorders: a pilot randomised control study

ABSTRACT Background Despite the known benefits of art therapy, there are a limited number of studies on art therapy for tic disorders. This pilot randomised controlled study investigated effects of art as a relaxation technique for tic disorders. Methods Twenty-two children aged 7–9 years were randomly allocated to art intervention (n= 11) and control (n= 11) groups. Pre– and post-test measurements included the Yale Global Tic Severity Scale (YGTSS), Heart Rate Variability (HRV), and Hassles Scale for Children (HSC). Results Art as relaxation significantly decreased the YGTSS total score, motor tic frequency, motor tic intensity, motor tic complexity, vocal tic complexity, and total daily stressors compared to the control group. The intervention group showed significantly greater physiological relaxation, as indicated by increases in HRV parameters. Conclusions Art appears to be an effective relaxation technique for tic disorders. Extensive research is necessary for rigorous examination of its effectiveness.

УПРАВЛЕНИЕ ЭКСПОРТНЫМ ПРОДОВОЛЬСТВЕННЫМ ПОТЕНЦИАЛОМ КАК ИМПУЛЬС РАЗВИТИЯ АГРОПРЕДПРИЯТИЙ

The current state of the domes- tic agro-industrial complex requires the search for new forms and methods to increase the effectiveness of impact of the economic mechanisms operating in the export of food products system, which can affect not only the results of production, but also the volume of the export of food products and agricultural raw materials. The article substantiates the urgency of the modifying the management of export food potential and its demand from the state, the agro-industrial complex, and economic entities represented by individual commodity producers. To solve this problem, the paper proposes the creation of an export potential managing architecture using a risk- based approach. External and internal macro- economic risks reduce the expected income of many participants of the food production process. The position of commodity producers loses stability under the influence of direct or indirect threats, as well as risks that cause additional, and in some cases, critical magnitude material and financial losses. The risk-oriented architecture of the export food potential management of the Russian Federation through the functioning of an effective organizational and economic management mechanism will not only protect the domestic food production market from fluctuations resulting from risky events and macroeconomic instability, but also form a scientifically based system mechanism to meet domestic and external demand. The development of export potential based on minimizing the associated risks and threats will provide agricultural enterprises with access to external markets, give an impulse to the development of competitive production and the agricultural economy as a whole.

Coexpressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts

In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons-termed TOC and TIC-located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.

Abundance of metalloprotease FtsH12 modulates chloroplast development in Arabidopsis thaliana.

The ATP-dependent metalloprotease FtsH12 (filamentation temperature sensitive protein H 12) has been suggested to participate in a heteromeric motor complex, driving protein translocation into the chloroplast. FtsH12 was immuno-detected in proplastids, seedlings, leaves, and roots. Expression of Myc-tagged FtsH12 under its native promotor allowed identification of FtsHi1, 2, 4, and 5, and plastidic NAD-malate dehydrogenase, five of the six interaction partners in the suggested import motor complex. Arabidopsis thaliana mutant seedlings with reduced FTSH12 abundance exhibited pale cotyledons and small, deformed chloroplasts with altered thylakoid structure. Mature plants retained these chloroplast defects, resulting in slightly variegated leaves and lower chlorophyll content. Label-free proteomics revealed strong changes in the proteome composition of FTSH12 knock-down seedlings, reflecting impaired plastid development. The composition of the translocon on the inner chloroplast membrane (TIC) protein import complex was altered, with coordinated reduction of the FtsH12-FtsHi complex subunits and accumulation of the 1 MDa TIC complex subunits TIC56, TIC214 and TIC22-III. FTSH12 overexpressor lines showed no obvious phenotype, but still displayed distinct differences in their proteome. N-terminome analyses further demonstrated normal proteolytic maturation of plastid-imported proteins irrespective of FTSH12 abundance. Together, our data suggest that FtsH12 has highest impact during seedling development; its abundance alters the plastid import machinery and impairs chloroplast development.

Arabidopsis ORANGE protein regulates plastid pre-protein import through interacting with Tic proteins.

Chloroplast-targeted proteins are actively imported into chloroplasts via the machinery spanning the double-layered membranes of chloroplasts. While the key translocons at the outer (TOC) and inner (TIC) membranes of chloroplasts are defined, proteins that interact with the core components to facilitate pre-protein import are continuously being discovered. A DnaJ-like chaperone ORANGE (OR) protein is known to regulate carotenoid biosynthesis as well as plastid biogenesis and development. In this study, we found that OR physically interacts with several Tic proteins including Tic20, Tic40, and Tic110 in the classic TIC core complex of the chloroplast import machinery. Knocking out or and its homolog or-like greatly affects the import efficiency of some photosynthetic and non-photosynthetic pre-proteins. Consistent with the direct interactions of OR with Tic proteins, the binding efficiency assay revealed that the effect of OR occurs at translocation at the inner envelope membrane (i.e. at the TIC complex). OR is able to reduce the Tic40 protein turnover rate through its chaperone activity. Moreover, OR was found to interfere with the interaction between Tic40 and Tic110, and reduces the binding of pre-proteins to Tic110 in aiding their release for translocation and processing. Our findings suggest that OR plays a new and regulatory role in stabilizing key translocons and in facilitating the late stage of plastid pre-protein translocation to regulate plastid pre-protein import.