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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

A genetic screen for dominant chloroplast reactive oxygen species signaling mutants reveals life stage-specific singlet oxygen signaling networks

Plants employ intricate molecular mechanisms to respond to abiotic stresses, which often lead to the accumulation of reactive oxygen species (ROS) within organelles such as chloroplasts. Such ROS can produce stress signals that regulate cellular response mechanisms. One ROS, singlet oxygen ( 1 O 2 ), is predominantly produced in the chloroplast during photosynthesis and can trigger chloroplast degradation, programmed cell death (PCD), and retrograde (organelle-to-nucleus) signaling. However, little is known about the molecular mechanisms involved in these signaling pathways or how many different signaling 1 O 2 pathways may exist. The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant conditionally accumulates chloroplast 1 O 2 , making fc2 a valuable genetic system for studying chloroplast 1 O 2 -initiated signaling. Here, we have used activation tagging in a new forward genetic screen to identify eight dominant fc2 activation-tagged (fas) mutations that suppress chloroplast 1 O 2 -initiated PCD. While 1 O 2 -triggered PCD is blocked in all fc2 fas mutants in the adult stage, such cellular degradation in the seedling stage is blocked in only two mutants. This differential blocking of PCD suggests that life-stage-specific 1 O 2 -response pathways exist. In addition to PCD, fas mutations generally reduce 1 O 2 -induced retrograde signals. Furthermore, fas mutants have enhanced tolerance to excess light, a natural mechanism to produce chloroplast 1 O 2 . However, general abiotic stress tolerance was only observed in one fc2 fas mutant (fc2 fas2). Together, this suggests that plants can employ general stress tolerance mechanisms to overcome 1 O 2 production but that this screen was mostly specific to 1 O 2 signaling. We also observed that salicylic acid (SA) and jasmonate (JA) stress hormone response marker genes were induced in 1 O 2 -stressed fc2 and generally reduced by fas mutations, suggesting that SA and JA signaling is correlated with active 1 O 2 signaling and PCD. Together, this work highlights the complexity of 1 O 2 signaling by demonstrating that multiple pathways may exist and introduces a suite of new 1 O 2 signaling mutants to investigate the mechanisms controlling chloroplast-initiated degradation, PCD, and retrograde signaling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Resin Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. The workflow is adaptable across many sample types including cultured cells (e.g. mammalian, prokaryotic) and whole tissues (e.g. heart, lung, muscle) which are initially lysed/homogenized with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol affinity resin which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves our understanding of the role of redox dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

RAC, TMT, thiol redox proteomics, cysteine, PTM st↗
DOE OSTI2021

Controlling selectivity of modular microbial biosynthesis of butyryl-CoA-derived designer esters

Short-chain esters have broad utility as flavors, fragrances, solvents, and biofuels. Controlling selectivity of ester microbial biosynthesis has been an outstanding metabolic engineering problem. In this study, we enabled the de novo fermentative microbial biosynthesis of butyryl-CoA-derived designer esters (e.g., butyl acetate, ethyl butyrate, butyl butyrate) in Escherichia coli with controllable selectivity. Using the modular design principles, we generated the butyryl-CoA-derived ester pathways as exchangeable production modules compatible with an engineered chassis cell for anaerobic production of designer esters. We designed these modules derived from an acyl-CoA submodule (e.g., acetyl-CoA, butyryl-CoA), an alcohol submodule (e.g., ethanol, butanol), a cofactor regeneration submodule (e.g., NADH), and an alcohol acetyltransferase (AAT) submodule (e.g., ATF1, SAAT) for rapid module construction and optimization by manipulating replication (e.g., plasmid copy number), transcription (e.g., promoters), translation (e.g., codon optimization), pathway enzymes, and pathway induction conditions. To further enhance production of designer esters with high selectivity, we systematically screened various strategies of protein solubilization using protein fusion tags and chaperones to improve the soluble expression of multiple pathway enzymes. Finally, our engineered ester-producing strains could achieve 19-fold increase in butyl acetate production (0.64 g/L, 96% selectivity), 6-fold increase in ethyl butyrate production (0.41 g/L, 86% selectivity), and 13-fold increase in butyl butyrate production (0.45 g/L, 54% selectivity) as compared to the initial strains. Altogether, this study presented a generalizable framework to engineer modular microbial platforms for anaerobic production of butyryl-CoA-derived designer esters from renewable feedstocks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Cryo-EM structure of the diapause chaperone artemin

The protein artemin acts as both an RNA and protein chaperone and constitutes over 10% of all protein in Artemia cysts during diapause. However, its mechanistic details remain elusive since no high-resolution structure of artemin exists. Here we report the full-length structure of artemin at 2.04 Å resolution. The cryo-EM map contains density for an intramolecular disulfide bond between Cys22-Cys61 and resolves the entire C-terminus extending into the core of the assembled protein cage but in a different configuration than previously hypothesized with molecular modeling. We also provide data supporting the role of C-terminal helix F towards stabilizing the dimer form that is believed to be important for its chaperoning activity. We were able to destabilize this effect by placing a tag at the C-terminus to fully pack the internal cavity and cause limited steric hindrance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

The small protein SbtC is a functional component of the CO 2 concentrating mechanism in Synechocystis sp. PCC 6803

Oxygenic phototrophs fix CO 2 via the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which shows relatively low CO 2 affinity and specificity. To circumvent low and fluctuating CO 2 concentrations in aquatic systems, cyanobacteria and algae have evolved sophisticated inorganic carbon (Ci) concentrating mechanisms (CCMs). Bicarbonate transporters such as SbtA play a crucial role in the cyanobacterial CCM and hence display multiple layers of tight regulation. Control of sbtA gene expression and corresponding transporter activity involves the PII-like protein SbtB, whose gene is frequently co-transcribed with sbtA. A previously non-annotated gene located upstream of the sbtAB operon in the model Synechocystis sp. PCC 6803 encodes the small protein SbtC, composed of 80 amino acids. Presence of SbtC was confirmed by immunoblotting of the sbtC-coding sequence fused to a Flag-tag. Similar to sbtAB , transcription of the sbtC locus is induced by low CO 2 availability; however, it is controlled independently. Mutation of the sbtC locus in a wild-type background produced only a mild phenotype, even under low CO 2 , but impaired diurnal growth resembled that of the mutant ΔsbtB . Biochemical analysis indicated a trimeric SbtABC complex in the membrane. Bicarbonate leakage from cells was strongly elevated when either sbtB or sbtC was deleted from recombinant Synechocystis strains harboring only SbtA as single Ci uptake system. Here, our results provide evidence that SbtC contributes to the formation of the SbtAB complex, thereby regulating bicarbonate exchange at the cytoplasmic membrane. Well-conserved SbtC-like proteins encoded in the neighborhood of sbtAB exist in many cyanobacterial genomes, pointing toward an important role in the cyanobacterial CCM.

Walke, Peter [Univ. of Rostock (Germany)] (ORCID:0↗
DOE OSTI2021

Engineering Forisome Scaffolds: Elucidating Spatial Self-Assembly Patterning of Bio-inorganic Complexes

Organisms can synthesize biomaterials incorporating an array of naturally occurring elements while overcoming challenges and insults. Although, it is known that most cellular biomaterials are synthesized in specialized cellular compartments, there are knowledge gaps about how organic/inorganic biomaterial synthesis is orchestrated inside cells. In addition, there is great potential in understanding how individual monomers can self-assembly into organized patterns to form responsive biomaterials. Forisomes are a natural responsive biomaterial found in legume plants that serve as a plug sieve element in the plant phloem that undergo anisotropic conformational changes by rapid (<1 s) ATP-independent from condensed spindle to plug-like form, triggered by the influx of Ca 2+ . Addressing principles of forisome synthesis and assembly will determine how biomaterials containing inorganic elements self-assemble and conduct chemical modification to produce biomaterials or undergo biomineralization. We employ transcription and translation (TXTL) using cell-free expression systems for forisome monomer expression, self-assembly, and pattern probing. We conducted experiments to precisely control forisome proteins synthesis of various monomers SEO1, SEO2, SEO3, and SEO4 to explore self- assembly. We demonstrate forisome self-assembly of the SEO monomers is possible and indicate unique monomer fluorescent labeling patterns that require additional analysis. We investigated locations and linkers for adding tetracysteine tag fluorophore probes to determine impacts of self-assembly and anisotropic conformational changes.

42 ENGINEERING↗
DOE OSTI2021

Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation

Abstract Liquid–liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (i) electrostatically-mediated interactions that compact hnRNPA1 and contribute to phase separation and (ii) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Posttranslational Methylation of Arginine in Methyl Coenzyme M Reductase Has a Profound Impact on both Methanogenesis and Growth of Methanococcus maripaludis

Catalyzing the key step for anaerobic production and/or oxidation of methane and likely other short-chain alkanes, methyl coenzyme M reductase (Mcr) and its homologs play a key role in the global carbon cycle. The McrA subunit possesses up to five conserved posttranslational modifications (PTMs) at its active site. It was previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To systematically examine its physiological role, in this work, mmpX (locus tag MMP1554), the gene encoding Mmp10 in Methanococcus maripaludis, was deleted with a new genetic tool, resulting in the complete loss of the 5-C-(S)-methylarginine PTM of residue 275 in the McrA subunit. When the ΔmmpX mutant was complemented with the wild-type gene expressed by either a strong or a weak promoter, methylation was fully restored. Compared to the parental strain, maximal rates of methane formation by whole cells were reduced by 40 to 60% in the ΔmmpX mutant. The reduction in activity was fully reversed by the complement with the strong promoter. Site-directed mutagenesis of mmpX resulted in a differential loss of arginine methylation among the mutants in vivo, suggesting that activities of Mmp10 directly modulated methylation. R 275 was present in a highly conserved PXRR 275 (A/S)R(G/A) signature sequence in McrAs. The only other protein in M. maripaludis containing a similar sequence was not methylated, suggesting that Mmp10 is specific for McrA. In conclusion, Mmp10 modulates the methyl-Arg PTM on McrA in a highly specific manner, which has a profound impact on Mcr activity.

03 NATURAL GAS↗
DOE OSTI2020

Enzyme-Directed Functionalization of Designed, Two-Dimensional Protein Lattices

The design and construction of crystalline protein arrays to selectively assemble ordered nanoscale materials has potential applications in sensing, catalysis and medicine. Whereas numerous designs have been implemented for the bottom-up construction of novel protein assemblies, the generation of artificial functional materials has been relatively unexplored. Enzyme-directed post-translational modifications are responsible for the functional diversity of the proteome and thus, could be harnessed to selectively modify artificial protein assemblies. In this study, we describe the use of phosphopantetheinyl transferases (PPTases), a class of enzymes that covalently modify proteins using coenzyme A (CoA), to site-selectively tailor the surface of designed, two-dimensional (2D) protein crystals. We demonstrate that a short peptide (ybbR) or a molecular tag (CoA) can be covalently tethered to 2D arrays to enable enzymatic functionalization using Sfp PPTase. Here, the site-specific modification of two different protein array platforms is facilitated by PPTases to afford both small-molecule- and protein-functionalized surfaces with no loss in crystalline order. This work highlights the potential for chemoenzymatic modification of large protein surfaces towards the generation of sophisticated protein platforms reminiscent of the complex landscape of cell surfaces.

36 MATERIALS SCIENCE↗
DOE OSTI2021

TMTpro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level

Multiplexed proteomics is a powerful tool to assay cell states in health and disease, but accurate quantification of relative protein changes is impaired by interference from co-isolated peptides. Most widely, this issue is alleviated by MS3-based quantification, reducing sensitivity and requiring specialized instrumentation. An alternative approach is quantification by complementary ions, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with the most widely distributed instruments. However, complementary ions of the popular TMT tag form inefficiently and plexing is limited to five channels. In this work, we evaluate and optimize complementary ion quantification for the recently released TMTPro tag, which increases plexing capacity to 8 channels (TMTProC). We find that the beneficial fragmentation properties increase quantification signal five-fold compared to TMT. This increased sensitivity results in ~50% more proteins quantified compared to TMTPro-MS3 and even slightly outperforms TMTPro-MS2. Furthermore, TMTProC quantification is more accurate than TMTPro-MS2 and even superior to TMTPro-MS3. To demonstrate the power of TMTProC, we analyzed a human and yeast interference sample and were able to quantify 13,290 proteins in 24 fractions. Thus, TMTProC advances multiplexed proteomics data quality and widens access to accurate multiplexed proteomics beyond laboratories with MS3-capable instrumentation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Identification of triacylglycerol remodeling mechanism to synthesize unusual fatty acid containing oils

Abstract Typical plant membranes and storage lipids are comprised of five common fatty acids yet over 450 unusual fatty acids accumulate in seed oils of various plant species. Plant oils are important human and animal nutrients, while some unusual fatty acids such as hydroxylated fatty acids (HFA) are used in the chemical industry (lubricants, paints, polymers, cosmetics, etc.). Most unusual fatty acids are extracted from non-agronomic crops leading to high production costs. Attempts to engineer HFA into crops are unsuccessful due to bottlenecks in the overlapping pathways of oil and membrane lipid synthesis where HFA are not compatible. Physaria fendleri naturally overcomes these bottlenecks through a triacylglycerol (TAG) remodeling mechanism where HFA are incorporated into TAG after initial synthesis. TAG remodeling involves a unique TAG lipase and two diacylglycerol acyltransferases (DGAT) that are selective for different stereochemical and acyl-containing species of diacylglycerol within a synthesis, partial degradation, and resynthesis cycle. The TAG lipase interacts with DGAT1, localizes to the endoplasmic reticulum (with the DGATs) and to puncta around the lipid droplet, likely forming a TAG remodeling metabolon near the lipid droplet-ER junction. Each characterized DGAT and TAG lipase can increase HFA accumulation in engineered seed oils.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Finding new friends and revisiting old ones – how plant lipid droplets connect with other subcellular structures

Summary The number of described contact sites between different subcellular compartments and structures in eukaryotic cells has increased dramatically in recent years and, as such, has substantially reinforced the well‐known premise that these kinds of connections are essential for overall cellular organization and the proper functioning of cellular metabolic and signaling pathways. Here, we discuss contact sites involving plant lipid droplets (LDs), including LD‐endoplasmic reticulum (ER) connections that mediate the biogenesis of new LDs at the ER, LD‐peroxisome connections, that facilitate the degradation of LD‐stored triacylglycerols (TAGs), and the more recently discovered LD‐plasma membrane connections, which involve at least three novel proteins, but have a yet unknown physiological function(s).

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Reconstitution of the heliobacterial photochemical reaction center and cytochrome c 553 into a proteoliposome system

The heliobacterial reaction center (HbRC) is the simplest understood photochemical reaction center, in terms of its polypeptide composition. In the heliobacterial cells, its electron donor is a cytochrome (cyt) c 553 attached to the membrane via a covalent linkage with a diacylglycerol. We have reconstituted purified HbRC into liposomes mimicking the phospholipid composition of heliobacterial membranes. We also incorporated a lipid with a headgroup containing Ni(II):nitrilotriacetate (NTA) to provide a binding site for the soluble version of the heliobacterial cyt c 553 in which the N-terminal membrane attachment site is replaced by a hexahistidine tag. The HbRC was inserted into the liposomes with the donor side preferentially exposed to the exterior; this bias increased to nearly 100% with higher concentrations (≥ 10 mol%) of the Ni(II)-NTA lipid in the membrane, and is most likely due to the net negative charge of the surface of the membrane. The HbRC in proteoliposomes without the Ni(II)-NTA lipid exhibited normal charge separation and subsequent charge recombination of the P + 800 F - X state in 15 ms; however, the oxidized primary donor (P + 800 ) was not significantly reduced by added H 6 -cyt c 553 . In contrast, with proteoliposomes containing the Ni(II)-NTA lipid, addition of H 6 -cyt c 553 resulted in a new kinetic component resulting from fast reduction (2–5 ms) of P + 800 by H 6 -cyt c 553 . The contribution of this kinetic component varied with the concentration of added H 6 -cyt c 553 and could represent 80% or more of the total P + 800 decay. As a result, the HbRC and its interaction with its native electron donor have been reconstituted into an artificial membrane system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Lytic archaeal viruses infect abundant primary producers in Earth’s crust

The continental subsurface houses a major portion of life’s abundance and diversity, yet little is known about viruses infecting microbes that reside there. Here, we use a combination of metagenomics and virus-targeted direct-geneFISH (virusFISH) to show that highly abundant carbon-fixing organisms of the uncultivated genus Candidatus Altiarchaeum are frequent targets of previously unrecognized viruses in the deep subsurface. Analysis of CRISPR spacer matches display resistances of Ca. Altiarchaea against eight predicted viral clades, which show genomic relatedness across continents but little similarity to previously identified viruses. Based on metagenomic information, we tag and image a putatively viral genome rich in protospacers using fluorescence microscopy. VirusFISH reveals a lytic lifestyle of the respective virus and challenges previous predictions that lysogeny prevails as the dominant viral lifestyle in the subsurface. CRISPR development over time and imaging of 18 samples from one subsurface ecosystem suggest a sophisticated interplay of viral diversification and adapting CRISPR-mediated resistances of Ca. Altiarchaeum. We conclude that infections of primary producers with lytic viruses followed by cell lysis potentially jump-start heterotrophic carbon cycling in these subsurface ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Microfabrication of a Chamber for High-Resolution, In Situ Imaging of the Whole Root for Plant–Microbe Interactions

Fabricated ecosystems (EcoFABs) offer an innovative approach to in situ examination of microbial establishment patterns around plant roots using nondestructive, high-resolution microscopy. Previously high-resolution imaging was challenging because the roots were not constrained to a fixed distance from the objective. Here, we describe a new ‘Imaging EcoFAB’ and the use of this device to image the entire root system of growing Brachypodium distachyon at high resolutions (20×, 40×) over a 3-week period. The device is capable of investigating root–microbe interactions of multimember communities. We examined nine strains of Pseudomonas simiae with different fluorescent constructs to B. distachyon and individual cells on root hairs were visible. Succession in the rhizosphere using two different strains of P. simiae was examined, where the second addition was shown to be able to establish in the root tissue. The device was suitable for imaging with different solid media at high magnification, allowing for the imaging of fungal establishment in the rhizosphere. Overall, the Imaging EcoFAB could improve our ability to investigate the spatiotemporal dynamics of the rhizosphere, including studies of fluorescently-tagged, multimember, synthetic communities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Tandem Mass Tag Labeling Facilitates Reversed-Phase Liquid Chromatography-Mass Spectrometry Analysis of Hydrophilic Phosphopeptides

Protein phosphorylation is a critical post-translational modification (PTM). Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides with enriched regions of serines, threonines, and tyrosines that often orchestrate critical biological functions. It is known that G protein coupled receptors (GPCRs) are regulators of cellular signaling events and rely on phosphorylation of serines and threonines within the GPCR C-terminus for appropriate signaling. However, the low abundance of GPCRs, combined with the hydrophilicity of C-terminal phosphopeptides, has prevented robust RPLC-MS analysis of potentially crucial regions. To address this issue, we developed a simple, easily implemented method to introduce a commonly used tandem mass tag (TMT) to increase peptide hydrophobicity, effectively enhancing RPLC-MS analysis of hydrophilic peptides. This method was applied to study the peptide QPSSSR, a tryptic peptide located on the C-terminus of the GPCR CXCR3 not been reported, and validated by comparative RPLC-MS analyses of both a hydrophilic QPSSSR peptide library as well as common phosphopeptides before and after TMT labeling. We further confirmed the utility of this method by quantifying QPSSSR phosphorylation abundances in HEK 293 cells under different treatement conditions predicted to alter QPSSSR phosphorylation. We anticipate that this simple TMT-labeling method can be broadly used not only for decoding GPCR phosphoproteomes, but also for effective RPLC-MS analysis of other highly hydrophilic analytes.

Tsai, Chia-Feng↗
DOE OSTI2025

Cryogenic Vibrational Spectroscopy of the Deprotonated Dimer of Phosphoric Acid

Phosphate-containing molecules are ubiquitous in nature, where they play crucial roles in biochemical processes. Further, they are of technical importance, for example, in certain batteries and in fuel cells, where a unique property of phosphoric acid is exploited its exceptionally high proton conductivity. Proton transport in phosphoric acid is known to involve proton shuttling; however, the elementary steps involved are not clear. To elucidate the hydrogen bonding preferences of phosphoric acid, we investigate the dihydrogen phosphate anion as well as the deprotonated dimer of phosphoric acid (H 3 PO 4 ·H 2 PO 4 − ) in the gas phase using infrared action spectroscopy in helium nanodroplets and infrared D 2 -tagging photodissociation spectroscopy, and the experimental spectra are compared to theoretical ones. Theory finds for H 3 PO 4 ·H 2 PO 4 − two different structures that are predicted to be nearly isoenergetic. The comparison to the experimental spectra, however, allows for a clear assignment and structure identification. The resulting structure has an interesting binding motif, which might be of relevance to interactions of phosphoric acid in the condensed phase and which can serve as a benchmark for quantum chemical calculations.

Chemical structure↗
DOE OSTI2023

Transforming our understanding of chloroplast-associated genes through comprehensive characterization of protein localizations and protein-protein interactions

Bioenergy crops are a renewable source of fuels and are a critical base for building a carbon-neutral economy. Rational engineering of bioenergy crops has the potential to enhance the yields. However, our ability to engineer plants is limited because the functions of most genes remain unknown. Systematic characterization of gene function in plants thus has the potential to greatly accelerate bioenergy research. Here, we focus on the chloroplast, an underexplored energy-producing organelle that is a hallmark of plants. The chloroplast is one of the promising targets of biofuel crop engineering efforts because of its central role in photosynthesis, metabolism, and intracellular signaling. However, the protein composition of the chloroplast and the functions of most of its proteins remain poorly characterized. At the core of this project, we sought to comprehensively determine the localization of chloroplast-associated proteins and generate a spatially defined protein-protein interaction network for chloroplast. For this purpose, we used the leading model alga Chlamydomonas reinhardtii, which greatly increased experimental speed and throughput. We illustrated the value of our findings to land plants by determining the localization of Arabidopsis thaliana land plant homologs of the Chlamydomonas proteins. Altogether, we were successful in determining the localization of 1,034 chloroplast-associated proteins in Chlamydomonas. The localizations provide numerous insights into the spatial organization of chloroplasts and how they function to support photosynthesis. The localization patterns of distinct proteins revealed new chloroplast structures and revealed new spatial organization inside the chloroplast. We also identified new components of known chloroplast structures, such as the chloroplast envelope, nucleoid, plastoglobuli, and pyrenoid. We identified these new components by investigating the interacting partners of known proteins. Many proteins localized in both the chloroplast and other cellular structures, thereby hinting at new functions and communication between cellular structures. We also applied machine learning on the atlas to generate predictions for the location of all of the proteins in Chlamydomonas. This enabled us to assign putative functions to many uncharacterized proteins based on their cellular location. Altogether, this research establishes a rich resource that opens new avenues of investigation and guides future work in deciphering and manipulating chloroplast function. Next, we developed an extensive protein-protein interaction network for the chloroplast by performing affinity purification-mass spectrometry on ~1,150 tagged chloroplast-associated proteins, the first such large-scale study in any photosynthetic organism. This dataset reveals 4,694 high-confidence protein-protein interactions, offering insights into the functions of thousands of conserved poorly-characterized chloroplast proteins. This systematic identification of protein-protein interactions in the chloroplast also provides multiple exciting new research directions and a detailed blueprint of the chloroplast's operation. This research lays the groundwork to decipher the inner workings of the chloroplast, the cell structure at the heart of photosynthesis. The spatial atlas and protein-protein interactions reveal chloroplast organizational features that would not have been accessible with traditional approaches. The localization mapping, insights into the function, and research materials generated further provide a rich resource for the research community to advance the understanding of how the chloroplast is organized to enable engineering of enhanced photosynthetic organisms.

59 BASIC BIOLOGICAL SCIENCES↗