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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 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

ER-associated VAP27-1 and VAP27-3 proteins functionally link the lipid-binding ORP2A at the ER-chloroplast contact sites

Abstract The plant endoplasmic reticulum (ER) contacts heterotypic membranes at membrane contact sites (MCSs) through largely undefined mechanisms. For instance, despite the well-established and essential role of the plant ER-chloroplast interactions for lipid biosynthesis, and the reported existence of physical contacts between these organelles, almost nothing is known about the ER-chloroplast MCS identity. Here we show that the Arabidopsis ER membrane-associated VAP27 proteins and the lipid-binding protein ORP2A define a functional complex at the ER-chloroplast MCSs. Specifically, through in vivo and in vitro association assays, we found that VAP27 proteins interact with the outer envelope membrane (OEM) of chloroplasts, where they bind to ORP2A. Through lipidomic analyses, we established that VAP27 proteins and ORP2A directly interact with the chloroplast OEM monogalactosyldiacylglycerol (MGDG), and we demonstrated that the loss of the VAP27-ORP2A complex is accompanied by subtle changes in the acyl composition of MGDG and PG. We also found that ORP2A interacts with phytosterols and established that the loss of the VAP27-ORP2A complex alters sterol levels in chloroplasts. We propose that, by interacting directly with OEM lipids, the VAP27-ORP2A complex defines plant-unique MCSs that bridge ER and chloroplasts and are involved in chloroplast lipid homeostasis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Transcriptional competition shapes proteotoxic ER stress resolution

Through dynamic activities of conserved master transcription factors (mTFs), the unfolded protein response (UPR) relieves proteostasis imbalance of the endoplasmic reticulum (ER), a condition known as ER stress. Because dysregulated UPR is lethal, the competence for fate changes of the UPR mTFs must be tightly controlled. However, the molecular mechanisms underlying regulatory dynamics of mTFs remain largely elusive. Here, we identified the abscisic acid-related regulator G-class bZIP TF2 (GBF2) and the cis-regulatory element G-box as regulatory components of the plant UPR led by the mTFs, bZIP28 and bZIP60. We demonstrate that, by competing with the mTFs at G-box, GBF2 represses UPR gene expression. Conversely, a gbf2 null mutation enhances UPR gene expression and suppresses the lethality of a bzip28 bzip60 mutant in unresolved ER stress. Here, by demonstrating that GBF2 functions as a transcriptional repressor of the UPR, we address the long-standing challenge of identifying shared signalling components for a better understanding of the dynamic nature and complexity of stress biology. Furthermore, our results identify a new layer of UPR gene regulation hinged upon an antagonistic mTFs-GFB2 competition for proteostasis and cell fate determination.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Dynamics of ER stress-induced gene regulation in plants

Endoplasmic reticulum (ER) stress is a potentially lethal condition that is induced by the abnormal accumulation of unfolded or misfolded secretory proteins in the ER. In eukaryotes, ER stress is managed by the unfolded protein response (UPR) through a tightly regulated, yet highly dynamic, reprogramming of gene transcription. Although the core principles of the UPR are similar across eukaryotes, unique features of the plant UPR reflect the adaptability of plants to their ever-changing environments and the need to balance the demands of growth and development with the response to environmental stressors. Here, the past decades have seen notable progress in understanding the mechanisms underlying ER stress sensing and signaling transduction pathways, implicating the UPR in the effects of physiological and induced ER stress on plant growth and crop yield. Facilitated by sequencing technologies and advances in genetic and genomic resources, recent efforts have driven the discovery of transcriptional regulators and elucidated the mechanisms that mediate the dynamic and precise gene regulation in response to ER stress at the systems level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Complete biosynthesis of QS-21 in engineered yeast

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host’s native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families—a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases—from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure–activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Complete biosynthesis of salicylic acid from phenylalanine in plants

Salicylic acid (SA) is a pivotal phytohormone for plant responses to biotic and abiotic stresses. Plants have evolved two pathways to produce SA: the isochorismate synthase and phenylalanine ammonia lyase (PAL) pathways. Whereas the isochorismate synthase pathway has been fully identified, the PAL pathway remains incomplete. Here we report the full characterization of the PAL pathway for SA biosynthesis via functional analysis of rice (Oryza sativa) SA-DEFICIENT GENE 1 (OSD1) to OSD4. The cinnamoyl-coenzyme A (CoA) ligase OSD1 catalyses the conversion of trans-cinnamic acid to cinnamoyl-CoA, which is subsequently transformed to benzoyl-CoA via the β-oxidative pathway in peroxisomes. The resulting benzoyl-CoA is further converted to benzyl benzoate by the peroxisomal benzoyltransferase OSD2. Benzyl benzoate is subsequently hydroxylated to benzyl salicylate by the endoplasmic reticulum membrane-resident cytochrome P450 OSD3, which is ultimately hydrolysed to salicylic acid by the cytoplasmic carboxylesterase OSD4. Evolutionary analyses reveal that the PAL pathway was first assembled before the divergence of gymnosperms and has been conserved in most seed plants. Activation of the PAL pathway in rice significantly enhances salicylic acid levels and plant immunity. Completion of the PAL pathway provides critical insights into the primary salicylic acid biosynthetic pathway across plant species and offers a precise target for modulating crop immunity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Single-molecule live-cell RNA imaging with CRISPR–Csm

Abstract Understanding the diverse dynamic behaviors of individual RNA molecules in single cells requires visualizing them at high resolution in real time. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved in a generalizable manner. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR–Csm complex with multiplexed guide RNAs for direct and efficient visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we track individual nativeNOTCH2andMAP1Btranscripts in living cells and identify two distinct localization mechanisms including the cotranslational translocation ofNOTCH2mRNA at the endoplasmic reticulum and directional transport ofMAP1BmRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.

Biotechnology & Applied Microbiology↗
DOE OSTI2019

Direct visualization of degradation microcompartments at the ER membrane

To promote the biochemical reactions of life, cells can compartmentalize molecular interaction partners together within separated non–membrane-bound regions. It is unknown whether this strategy is used to facilitate protein degradation at specific locations within the cell. Leveraging in situ cryo-electron tomography to image the native molecular landscape of the unicellular alga Chlamydomonas reinhardtii , we discovered that the cytosolic protein degradation machinery is concentrated within ∼200-nm foci that contact specialized patches of endoplasmic reticulum (ER) membrane away from the ER–Golgi interface. These non–membrane-bound microcompartments exclude ribosomes and consist of a core of densely clustered 26S proteasomes surrounded by a loose cloud of Cdc48. Active proteasomes in the microcompartments directly engage with putative substrate at the ER membrane, a function canonically assigned to Cdc48. Live-cell fluorescence microscopy revealed that the proteasome clusters are dynamic, with frequent assembly and fusion events. We propose that the microcompartments perform ER-associated degradation, colocalizing the degradation machinery at specific ER hot spots to enable efficient protein quality control.

Science & Technology - Other Topics↗
DOE OSTI2020

Lipid sponge droplets as programmable synthetic organelles

Living cells segregate molecules and reactions in various subcellular compartments known as organelles. Spatial organization is likely essential for expanding the biochemical functions of synthetic reaction systems, including artificial cells. Many studies have attempted to mimic organelle functions using lamellar membrane-bound vesicles. However, vesicles typically suffer from highly limited transport across the membranes and an inability to mimic the dense membrane networks typically found in organelles such as the endoplasmic reticulum. Here, we describe programmable synthetic organelles based on highly stable nonlamellar sponge phase droplets that spontaneously assemble from a single-chain galactolipid and nonionic detergents. Due to their nanoporous structure, lipid sponge droplets readily exchange materials with the surrounding environment. In addition, the sponge phase contains a dense network of lipid bilayers and nanometric aqueous channels, which allows different classes of molecules to partition based on their size, polarity, and specific binding motifs. The sequestration of biologically relevant macromolecules can be programmed by the addition of suitably functionalized amphiphiles to the droplets. We demonstrate that droplets can harbor functional soluble and transmembrane proteins, allowing for the colocalization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled. Our results show that lipid sponge droplets permit the facile integration of membrane-rich environments and self-assembling spatial organization with biochemical reaction systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structural elucidation of the cis -prenyltransferase NgBR/DHDDS complex reveals insights in regulation of protein glycosylation

Cis-prenyltransferase (cis-PTase) catalyzes the rate-limiting step in the synthesis of glycosyl carrier lipids required for protein glycosylation in the lumen of endoplasmic reticulum. Here, we report the crystal structure of the human NgBR/DHDDS complex, which represents an atomic resolution structure for any heterodimeric cis-PTase. The crystal structure sheds light on how NgBR stabilizes DHDDS through dimerization, participates in the enzyme’s active site through its C-terminal -RXG- motif, and how phospholipids markedly stimulate cis-PTase activity. Comparison of NgBR/DHDDS with homodimeric cis-PTase structures leads to a model where the elongating isoprene chain extends beyond the enzyme’s active site tunnel, and an insert within the α3 helix helps to stabilize this energetically unfavorable state to enable long-chain synthesis to occur. These data provide unique insights into how heterodimeric cis-PTases have evolved from their ancestral, homodimeric forms to fulfill their function in long-chain polyprenol synthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

EVAP: A two-photon imaging tool to study conformational changes in endogenous Kv2 channels in live tissues

A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. We term this tool EVAP (Endogenous Voltage-sensor Activity Probe). GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane–endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in fluorescence intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to degree of labeling. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. We found puncta of GxTX-594 on hippocampal CA1 neurons that responded to voltage stimulation and retain a voltage response roughly similar to heterologously expressed Kv2.1 protein. Our findings show that EVAP imaging methods enable the identification of conformational changes of endogenous Kv2 voltage sensors in tissue.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Continuity of Mitochondrial Budding: Insights from BS-C-1 Cells by In Situ Cryo-electron Tomography

Mitochondrial division is a fundamental biological process essensial for cellular functionality and vitality. The prevailing hypothesis that dynamin related protein 1 (Drp1) provides principal control in mitochondrial division, in which it also involves the endoplasmic reticulum (ER) and the cytoskeleton, does not account for all the observations. Therefore. the hypothesis may be incomplete. Our previous study in HeLa cells led to a new hypothesis of mitochondrial division by budding. To follow-up our previous study, we employed in situ cryo-electron tomography to visualize mitochondrial budding in the intact healthy monkey kidney cells (BS-C-1 cells). Our findings reaffirm single and multiple mitochondrial budding, consistent with our observations in HeLa cells. Notably, the budding regions vary significantly in diameter and length, which may represent different stages of budding. More interestingly, neither rings nor ring-like structures, nor the wrapping of ER tubes was observed in the budding regions, suggesting mitochondrial budding is independent from Drp1 and ER. Meanwhile, we uncovered direct interactions between mitochondria and large vesicles that are distinct from small mitochondrial-derived vesicles and extracellular mitovesicles. In conclusion, we propose that these interacting vesicles may have mitochondrial origins.

(Cryo-EM)↗
DOE OSTI2024

The lowdown on breakdown: Open questions in plant proteolysis

Abstract Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and postharvest regulation), plant responses to environmental signals (endoplasmic-reticulum-associated degradation, chloroplast-associated degradation, drought tolerance, and the growth-defense trade-off), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

Eckardt, Nancy A. (ORCID:0000000316581412)↗
DOE OSTI2025

Powdery mildew induces chloroplast storage lipid formation at the expense of host thylakoids to promote spore production

Powdery mildews are obligate biotrophic fungi that manipulate plant metabolism to supply lipids to the fungus, particularly during fungal asexual reproduction when lipid demand is high. We found levels of leaf storage lipids (triacylglycerols, TAGs) are 3.5-fold higher in whole Arabidopsis (Arabidopsis thaliana) leaves with a 15-fold increase in storage lipids at the infection site during fungal asexual reproduction. Lipid bodies, not observable in uninfected mature leaves, were found in and external to chloroplasts in mesophyll cells underlying the fungal feeding structure. Concomitantly, thylakoid disassembly occurred and thylakoid membrane lipid levels decreased. Genetic analyses showed that canonical endoplasmic reticulum TAG biosynthesis does not support powdery mildew spore production. Instead, Arabidopsis chloroplast-localized DIACYLGLYCEROL ACYLTRANSFERASE 3 (DGAT3) promoted fungal asexual reproduction. Consistent with the reported AtDGAT3 preference for 18:3 and 18:2 acyl substrates, which are dominant in thylakoid membrane lipids, dgat3 mutants exhibited a dramatic reduction in powdery mildew-induced chloroplast TAGs, attributable to decreases in TAG species largely comprised of 18:3 and 18:2 acyl substrates. This pathway for TAG biosynthesis in the chloroplast at the expense of thylakoids provides insights into obligate biotrophy and plant lipid metabolism, plasticity, and function. By understanding how photosynthetically active leaves can be converted into TAG producers, more sustainable and environmentally friendly plant oil production may be developed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Chloroplast lipid biosynthesis is fine-tuned to thylakoid membrane remodeling during light acclimation

Abstract Reprogramming metabolism, in addition to modifying the structure and function of the photosynthetic machinery, is crucial for plant acclimation to changing light conditions. One of the key acclimatory responses involves reorganization of the photosynthetic membrane system including changes in thylakoid stacking. Glycerolipids are the main structural component of thylakoids and their synthesis involves two main pathways localized in the plastid and the endoplasmic reticulum (ER); however, the role of lipid metabolism in light acclimation remains poorly understood. We found that fatty acid synthesis, membrane lipid content, the plastid lipid biosynthetic pathway activity, and the degree of thylakoid stacking were significantly higher in plants grown under low light compared with plants grown under normal light. Plants grown under high light, on the other hand, showed a lower rate of fatty acid synthesis, a higher fatty acid flux through the ER pathway, higher triacylglycerol content, and thylakoid membrane unstacking. We additionally demonstrated that changes in rates of fatty acid synthesis under different growth light conditions are due to post-translational regulation of the plastidic acetyl-CoA carboxylase activity. Furthermore, Arabidopsis mutants defective in one of the two glycerolipid biosynthetic pathways displayed altered growth patterns and a severely reduced ability to remodel thylakoid architecture, particularly under high light. Overall, this study reveals how plants fine-tune fatty acid and glycerolipid biosynthesis to cellular metabolic needs in response to long-term changes in light conditions, highlighting the importance of lipid metabolism in light acclimation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Packaging “vegetable oils”: Insights into plant lipid droplet proteins

Abstract Plant neutral lipids, also known as “vegetable oils”, are synthesized within the endoplasmic reticulum (ER) membrane and packaged into subcellular compartments called lipid droplets (LDs) for stable storage in the cytoplasm. The biogenesis, modulation, and degradation of cytoplasmic LDs in plant cells are orchestrated by a variety of proteins localized to the ER, LDs, and peroxisomes. Recent studies of these LD-related proteins have greatly advanced our understanding of LDs not only as steady oil depots in seeds but also as dynamic cell organelles involved in numerous physiological processes in different tissues and developmental stages of plants. In the past 2 decades, technology advances in proteomics, transcriptomics, genome sequencing, cellular imaging and protein structural modeling have markedly expanded the inventory of LD-related proteins, provided unprecedented structural and functional insights into the protein machinery modulating LDs in plant cells, and shed new light on the functions of LDs in nonseed plant tissues as well as in unicellular algae. Here, we review critical advances in revealing new LD proteins in various plant tissues, point out structural and mechanistic insights into key proteins in LD biogenesis and dynamic modulation, and discuss future perspectives on bridging our knowledge gaps in plant LD biology.

Cai, Yingqi (ORCID:0000000203575809)↗
DOE OSTI2026

Arabidopsis cytochrome b 5 proteins support fatty acid ω-3 but not ω-6 desaturation

Fatty acids are primary components of lipids, which serve as major energy sources in cells and play essential roles in membrane structure, signaling, and metabolic regulation (Shanklin and Cahoon 1998). The degree of fatty acid unsaturation critically influences lipid physicochemical properties, thereby affecting membrane fluidity and biological function (Nguyen et al. 2019). In Arabidopsis thaliana, fatty acid desaturation occurs via 2 parallel pathways: the “prokaryotic pathway” in plastids, involving glycosylglycerides, such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and phospholipid phosphatidylglycerol (PG); and the “eukaryotic pathway” in the endoplasmic reticulum (ER), involving phosphatidylcholine (PC) (Lou et al. 2014) (Supplementary Figure S1). Seven fatty acid desaturases (FADs) in Arabidopsis differentially desaturate each glycerolipid class in the plastid and ER (Nguyen et al. 2019). FAD2, an ER-resident ω-6 fatty acid desaturase, catalyzes the conversion of oleic acid (18:1) to linoleic acid (18:2), which can be further desaturated to α-linolenic acid (18:3) by FAD3, an ER-resident ω-3 fatty acid desaturase. In plastids, FAD6 catalyzes the desaturation of 18:1/16:1 to produce 18:2/16:2, while FAD7 and FAD8 redundantly convert 18:2/16:2 to 18:3/16:3 (Li-Beisson et al. 2013; Nguyen et al. 2019). Additionally, fatty acids synthesized in the ER can also be reimported into plastids to their site of de novo synthesis (Xu et al. 2010). All FADs require reducing power, in the form of 2 electrons, for catalysis, but the sources of the electrons vary between their subcellular localizations. In the ER, FAD2 and FAD3 receive electrons from a cytochrome b 5 (CB5)-based electron transfer chain comprising cytochrome b 5 reductase (CBR) and CB5. In contrast, ferredoxin serves as the electron donor for plastid-localized FAD6, FAD7, and FAD8 (Ohlrogge and Browse 1995; Andreu et al. 2007). While the relative contributions of the 2 pathways to total cellular desaturation products vary across tissues and species, most polyunsaturated FA biosynthesis in seeds occurs via ER-resident FAD2 and FAD3 (Miquel and Browse 1992; Ohlrogge and Browse 1995).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Arabidopsis lipins mediate lipid droplet biogenesis to protect cells from lipotoxicity

Lipin proteins, a family of phosphatidic acid phosphatases (PAHs), are key regulators of lipid metabolism, storage, and homeostasis across eukaryotes. While Arabidopsis (Arabidopsis thaliana) lipins function in lipid biosynthesis and gene regulation, their roles in lipid droplet (LD) biogenesis and lipid homeostasis remain largely unknown. Here, we show that double knockout of two PAH genes (PAH1/2) results in impaired LD biogenesis, accelerated triacylglycerol (TAG) hydrolysis, and lipid imbalance. pah1/2 mutant leaves exhibited a marked reduction in TAG levels and a significant decrease in LD size, while the rates of TAG and diacylglycerol (DAG) synthesis remained largely unchanged. In seeds, PAH1/2 disruption minimally affected TAG content but significantly reduced LD size. Fatty acid feeding experiments demonstrated impaired LD formation and increased lipotoxicity in pah1/2 leaves and seedlings. Furthermore, knockout of PAH1/2 in mutants with enhanced fatty acid flux through phosphatidylcholine (PC) led to severe reductions in leaf TAG levels, despite increases in TAG synthesis rates, indicating accelerated TAG turnover. Phosphatidic acid, free fatty acids, and PC accumulated, leading to massive proliferation of endoplasmic reticulum membranes and severe growth and developmental defects. These findings demonstrate evolutionarily conserved roles for PAH1/2 in LD biogenesis, membrane lipid homeostasis, and cellular protection against lipotoxicity, particularly under conditions of elevated fatty acid flux.

59 BASIC BIOLOGICAL SCIENCES↗