An official website of the United States government
ATP citrate lyase (ACL) catalyzes the ATP-dependent conversion of citrate to the fatty acid precursor, acetyl-CoA. ACL presence in yeasts has been associated with their ability to accumulate lipids (i.e., oleaginous phenotype), but little is known about the regulation of this enzyme in oleaginous yeasts. In the model oleaginous yeast Yarrowia lipolytica, ACL is a heterodimer comprised of a catalytic and a regulatory subunit, encoded by the ACL1 and ACL2 genes, respectively. From the earlier studies, it was shown that the loss of ACL1 resulted in lower lipid levels and altered fatty acid profiles. However, the regulation of ACL expression and activity during lipogenesis has not been studied. To better understand the role, ACL plays during lipogenesis in Y.lipolytica, we generated antibodies against its two subunits (i.e., Acl1 and Acl2). We also constructed strains that lack Acl2 (i.e., acl2Δ) and strains that overexpress Acl1 and Acl2 either alone or in combination. Preliminary experiments showed that the overexpression of Acl1 increased the protein levels of Acl2. We are currently analyzing the effects of Acl2 overexpression and the time-dependent regulation of Acl1 and Acl2.
more »
« less
Modulation of the Arabidopsis Starch Metabolic Network by the Cytosolic Acetyl-CoA Pathway in the Context of the Diurnal Illumination Cycle
The starch metabolic network was investigated in relation to other metabolic processes by examining a mutant with altered single-gene expression of ATP citrate lyase (ACL), an enzyme responsible for generating cytosolic acetyl-CoA pool from citrate. Previous research has shown that transgenic antisense plants with reduced ACL activity accumulate abnormally enlarged starch granules. In this study, we explored the underlying molecular mechanisms linking cytosolic acetyl-CoA generation and starch metabolism under short-day photoperiods. We performed transcriptome and quantification of starch accumulation in the leaves of wild-type and antisense seedlings with reduced ACL activity. The antisense-ACLA mutant accumulated more starch than the wild type under short-day conditions. Zymogram analyses were conducted to compare the activities of starch-metabolizing enzymes with transcriptomic changes in the seedling. Differential expression between wild-type and antisense-ACLA plants was detected in genes implicated in starch and acetyl-CoA metabolism, and cell wall metabolism. These analyses revealed a strong correlation between the transcript levels of genes responsible for starch synthesis and degradation, reflecting coordinated regulation at the transcriptomic level. Furthermore, our data provide novel insights into the regulatory links between cytosolic acetyl-CoA metabolism and starch metabolic pathways.
more »
« less
- Award ID(s):
- 2238942
- PAR ID:
- 10555413
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- International Journal of Molecular Sciences
- Volume:
- 25
- Issue:
- 19
- ISSN:
- 1422-0067
- Page Range / eLocation ID:
- 10850
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Lipoxygenase (LOX) is associated with responses to plant hormones, environmental stresses, and signaling substances. Methyl jasmonate (MeJA) treatment triggers the production of LOX, polyphenol oxidase, and protease inhibitors in various plants, producing resistance to herbivory. To examine the response of MtLOX24 to MeJA, the phenotypic and physiological changes in Medicago truncatula MtLOX24 overexpression and lox mutant plants were investigated. Additionally, wild-type R108, the MtLOX24-overexpressing line L4, and the mutant lox-1 were utilized as experimental materials to characterize the differentially expressed genes (DEGs) and metabolic pathways in response to MeJA. The results indicate that after treatment with 200 µM of MeJA, the damage in the mutants lox-1 and lox-2 was more serious than in the overexpressing lines L4 and L6, with more significant leaf wilting, yellowing, and oxidative damage in lox-1 and lox-2. Exogenous application of MeJA induced H2O2 production and POD activity but reduced CAT activity in the lox mutants. Transcriptome analysis revealed 10,238 DEGs in six libraries of normal-growing groups (cR108, cL4, and clox1) and MeJA-treated groups (R108, L4, and lox1). GO and KEGG functional enrichment analysis demonstrated that under normal growth conditions, the DEGs between the cL4 vs. cR108 and the clox-1 vs. cR108 groups were primarily enriched in signaling pathways such as plant–pathogen interactions, flavonoid biosynthesis, plant hormone signal transduction, the MAPK signaling pathway, and glutathione metabolism. The DEGs of the R108 vs. cR108 and L4 vs. cL4 groups after MeJA treatment were mainly enriched in glutathione metabolism, phenylpropanoid biosynthesis, the MAPK signaling pathway, circadian rhythm, and α-linolenic acid metabolism. Among them, under normal growth conditions, genes like PTI5, PR1, HSPs, PALs, CAD, CCoAOMT, and CYPs showed significant differences between L4 and the wild type, suggesting that the expression of these genes is impacted by MtLOX24 overexpression. CDPKs, CaMCMLs, IFS, JAZ, and other genes were also significantly different between L4 and the wild type upon MeJA treatment, suggesting that they might be important genes involved in JA signaling. This study provides a reference for the study of the response mechanism of MtLOX24 under MeJA signaling.more » « less
-
Abstract Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle ‘metabolon’ which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was present together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.more » « less
-
Magnesium (Mg2+) is an essential nutrient in all organisms. However, high levels of Mg2+ in the environment are toxic to plants. In this study, we identified the vacuolar-type H+-pyrophosphatase, AVP1, as a critical enzyme for optimal plant growth under high-Mg conditions. The Arabidopsis avp1 mutants displayed severe growth retardation, as compared to the wild-type plants upon excessive Mg2+. Unexpectedly, the avp1 mutant plants retained similar Mg content to wild-type plants under either normal or high Mg conditions, suggesting that AVP1 may not directly contribute to Mg2+ homeostasis in plant cells. Further analyses confirmed that the avp1 mutant plants contained a higher pyrophosphate (PPi) content than wild type, coupled with impaired vacuolar H+-pyrophosphatase activity. Interestingly, expression of the Saccharomyces cerevisiae cytosolic inorganic pyrophosphatase1 gene IPP1, which facilitates PPi hydrolysis but not proton translocation into vacuole, rescued the growth defects of avp1 mutants under high-Mg conditions. These results provide evidence that high-Mg sensitivity in avp1 mutants possibly resulted from elevated level of cytosolic PPi. Moreover, genetic analysis indicated that mutation of AVP1 was additive to the defects in mgt6 and cbl2 cbl3 mutants that are previously known to be impaired in Mg2+ homeostasis. Taken together, our results suggest AVP1 is required for cellular PPi homeostasis that in turn contributes to high-Mg tolerance in plant cells.more » « less
-
ABSTRACT Vibrio cholerae controls the pathogenicity of interactions with arthropod hosts via the activity of the CrbS/R two-component system. This signaling pathway regulates the consumption of acetate, which in turn alters the relative virulence of interactions with arthropods, including Drosophila melanogaster . CrbS is a histidine kinase that links a transporter-like domain to its signaling apparatus via putative STAC and PAS domains. CrbS and its cognate response regulator are required for the expression of acetyl coenzyme A (acetyl-CoA) synthetase (product of acs ), which converts acetate to acetyl-CoA. We demonstrate that the STAC domain of CrbS is required for signaling in culture; without it, acs transcription is reduced in LB medium, and V. cholerae cannot grow on acetate minimal media. However, the strain remains virulent toward Drosophila and expresses acs similarly to the wild type during infection. This suggests that there is a unique signal or environmental variable that modulates CrbS in the gastrointestinal tract of Drosophila . Second, we present evidence in support of CrbR, the response regulator that interacts with CrbS, binding directly to the acs promoter, and we identify a region of the promoter that CrbR may target. We further demonstrate that nutrient signals, together with the cAMP receptor protein (CRP)-cAMP system, control acs transcription, but regulation may occur indirectly, as CRP-cAMP activates the expression of the crbS and crbR genes. Finally, we define the role of the Pta-AckA system in V. cholerae and identify redundancy built into acetate excretion pathways in this pathogen. IMPORTANCE CrbS is a member of a unique family of sensor histidine kinases, as its structure suggests that it may link signaling to the transport of a molecule. However, mechanisms through which CrbS senses and communicates information about the outside world are unknown. In the Vibrionaceae , orthologs of CrbS regulate acetate metabolism, which can, in turn, affect interactions with host organisms. Here, we situate CrbS within a larger regulatory framework, demonstrating that crbS is regulated by nutrient-sensing systems. Furthermore, CrbS domains may play various roles in signaling during infection and growth in culture, suggesting a unique mechanism of host recognition. Finally, we define the roles of additional pathways in acetate flux, as a foundation for further studies of this metabolic nexus point.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/ijms251910850
