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Succinate dehydrogenase (Complex II; SDH) plays an important role in mitochondrial respiratory metabolism. The SDH complex consists of four core subunits and multiple cofactors, which must be assembled correctly to ensure enzyme function. To date, only an assembly factor (SDHAF2) required for FAD insertion into subunit SDH1 has been identified in plants. Here, we report the identification of Arabidopsis ( Arabidopsis thaliana ) At5g67490 as a second SDH assembly factor. Knockout of At5g67490 ( sdhaf4 ) did not cause any phenotypic variation in seedlings but resulted in a decrease in both SDH activity and the succinate-dependent respiration rate as well as increased accumulation of succinate. Mass spectrometry analyses revealed stable levels of FAD-SDH1 in sdhaf4 , together with increased levels of the FAD-SDH1 assembly factor, SDHAF2, and reduced levels of SDH2 compared with the wild type. Loss of SDHAF4 in sdhaf4 inhibited the formation of the SDH1/SDH2 intermediate, leading to the accumulation of soluble SDH1 in the mitochondrial matrix and reduced levels of SDH1 in the membrane. The increased levels of SDHAF2 suggest that the stabilization of soluble FAD-SDH1 depends on SDHAF2 availability. We conclude that SDHAF4 acts on FAD-SDH1 and promotes its assembly with SDH2, thereby stabilizing SDH2 and enabling its full assembly with SDH3/SDH4 to form the SDH complex., (© 2018 American Society of Plant Biologists. All rights reserved.)

A dual-targeted protein belonging to the mitochondrial carrier family was characterized in rice (Oryza sativa) and designated 3'-Phosphoadenosine 5'-Phosphosulfate Transporter1 (PAPST1). The papst1 mutant plants showed a defect in thylakoid development, resulting in leaf chlorosis at an early leaf developmental stage, while normal leaf development was restored 4 to 6 d after leaf emergence. OsPAPST1 is highly expressed in young leaves and roots, while the expression is reduced in mature leaves, in line with the recovery of chloroplast development seen in the older leaves of papst1 mutant plants. OsPAPST1 is located on the outer mitochondrial membrane and chloroplast envelope. Whole-genome transcriptomic analysis reveals reduced expression of genes encoding photosynthetic components (light reactions) in papst1 mutant plants. In addition, sulfur metabolism is also perturbed in papst1 plants, and it was seen that PAPST1 can act as a nucleotide transporter when expressed in Escherichia coli that can be inhibited significantly by 3'-phosphoadenosine 5'-phosphosulfate. Given these findings, together with the altered phenotype seen only when leaves are first exposed to light, it is proposed that PAPST1 may act as a 3'-phosphoadenosine 5'-phosphosulfate carrier that has been shown to act as a retrograde signal between chloroplasts and the nucleus.

Diurnal regulation of transcripts encoding proteins located in mitochondria, plastids, and peroxisomes is important for adaptation of organelle biogenesis and metabolism to meet cellular requirements. We show this regulation is related to diurnal changes in promoter activities and the presence of specific cis-acting regulatory elements in the proximal promoter region [TGGGC(C/T)], previously defined as site II elements, and leads to diurnal changes in organelle protein abundances. These site II elements can act both as activators or repressors of transcription, depending on the night/day period and on the number and arrangement of site II elements in the promoter tested. These elements bind to the TCP family of transcriptions factors in Arabidopsis thaliana, which nearly all display distinct diurnal patterns of cycling transcript abundance. TCP2, TCP3, TCP11, and TCP15 were found to interact with different components of the core circadian clock in both yeast two-hybrid and direct protein-protein interaction assays, and tcp11 and tcp15 mutant plants showed altered transcript profiles for a number of core clock components, including LATE ELONGATED HYPOCOTYL1 and PSEUDO RESPONSE REGULATOR5. Thus, site II elements in the promoter regions of genes encoding mitochondrial, plastid, and peroxisomal proteins provide a direct mechanism for the coordination of expression for genes involved in a variety of organellar functions, including energy metabolism, with the time-of-day specific needs of the organism.

The disulfide relay system of the mitochondrial intermembrane space has been extensively characterized in Saccharomyces cerevisiae. It contains two essential components, Mia40 and Erv1. The genome of Arabidopsis thaliana contains a single gene for each of these components. Although insertional inactivation of Erv1 leads to a lethal phenotype, inactivation of Mia40 results in no detectable deleterious phenotype. A. thaliana Mia40 is targeted to and accumulates in mitochondria and peroxisomes. Inactivation of Mia40 results in an alteration of several proteins in mitochondria, an absence of copper/zinc superoxide dismutase (CSD1), the chaperone for superoxide dismutase (Ccs1) that inserts copper into CSD1, and a decrease in capacity and amount of complex I. In peroxisomes the absence of Mia40 leads to an absence of CSD3 and a decrease in abnormal inflorescence meristem 1 (Aim1), a β-oxidation pathway enzyme. Inactivation of Mia40 leads to an alteration of the transcriptome of A. thaliana, with genes encoding peroxisomal proteins, redox functions, and biotic stress significantly changing in abundance. Thus, the mechanistic operation of the mitochondrial disulfide relay system is different in A. thaliana compared with other systems, and Mia40 has taken on new roles in peroxisomes and mitochondria.

Signal transducer and activator of transcription-3 (STAT3) activation has been associated with suppressed inflammatory processes in experimental animals, murine myeloid cells and macrophage cell lines. Manipulation of STAT3 activity may therefore be a focus for pharmacological intervention of inflammatory diseases in humans. However, the ability of STAT3 to reduce the production of inflammatory mediators by activated human monocytes and macrophages has been characterized inadequately. To establish this, we used a recently optimized adenoviral approach to study the effect of overexpressed STAT3 or a transcriptionally inactive mutant STAT3 in lipopolysaccharide (LPS)-stimulated human monocytes. STAT3 activated by LPS did not directly regulate inhibitor of kappa B alpha (IkappaBalpha) activation or tumour necrosis factor (TNF)-alpha production, a process dependent on the transcriptional activity of nuclear factor kappa B (NFkappaB), although the transcriptional activity of STAT3 contributed to the mechanism by which interleukin (IL)-10 suppressed LPS-induced TNF-alpha levels. This contrasted with the efficient block in IL-10 induction of suppressor of cytokine signalling-3 (SOCS3) in monocytes infected with an adenovirus expressing mutant STAT3. These results indicate that STAT3 activation cannot directly regulate LPS-signalling in human monocytes and represents only part of the mechanism by which IL-10 suppresses TNF-alpha production by activated human monocytes. This study concludes that pharmacological manipulation of STAT3 transcriptional activity alone would be insufficient to control NFkappaB-associated inflammation in humans.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine that suppresses the production of tumour necrosis factor-alpha (TNF-alpha) by monocytes and macrophages. Suppressor of cytokine signalling-3 (SOCS3), a negative regulator of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, is induced following IL-10 exposure but recent studies in mice suggest that SOCS3 only targets gp-130-dependent signal transduction pathways. Understanding the signalling pathways responsible for IL-10-mediated effects in primary human monocytes is relevant to human inflammatory disease and necessary for the identification of potential therapeutic targets. An adenoviral transfection system was used to express different levels of SOCS3 (quantified experimentally with its tag green fluorescent protein (GFP)) with the aim of investigating the role of SOCS3 in LPS-induced and IL-10-mediated suppression of TNF-alpha production by non-transformed human monocytes. SOCS3 over-expression had no effect on TNF-alpha mRNA levels induced by LPS or LPS plus IL-10, or on IL-10 phosphorylation of STAT3, STAT1 and ERK1/2. When data from all donors were combined, adenoviral overexpression of SOCS3 significantly reversed the suppressive effects of IL-10 on LPS-induced TNF-alpha production after 2 hr. However, there was a direct correlation between mean GFP intensity (extent of viral infection) and extent of reversal of IL-10's inhibitory effects. Physiological levels of SOCS3 detected in IL-10-exposed human monocytes had no effect on LPS-induced TNF-alpha production. Although overexpression of SOCS3 to supraphysiological levels transiently antagonized the regulatory properties of IL-10 by a post-transcriptional mechanism, these findings suggest that under pathological conditions SOCS3 does not control LPS-activation or the anti-inflammatory properties of IL-10 in primary human monocytes.

The import pathways of the alternative oxidase and the F(A)d subunit of the ATP synthase from soybean were characterised. The F(A)d precursor does not require extramitochondrial ATP for import and this was shown to be a characteristic of the mature protein. The alternative oxidase and F(A)d precursors were shown to differ in their requirement for a membrane potential. The membrane potential was modified using malonate, a competitive inhibitor to complex II. The alternative oxidase could be imported at higher malonate concentrations compared to the F(A)d. This difference could not be ascribed to the number of positive charges in each presequence as would be predicted from similar studies in fungi.

Characterisation of the amount of protein import of the alternative oxidase (AOX) and the F(A)d precursor proteins (previously shown to use different import pathways) into mitochondria from developing soybean tissues indicated that they displayed different patterns. Import of the AOX declined in both cotyledon and root mitochondria with increasing age, whereas the import of the F(A)d into cotyledon mitochondria remained high throughout the same period. Using primary leaf mitochondria, it was evident that import of AOX remained high while it declined in cotyledon and root mitochondria. The amount of import of the AOX into mitochondria from different tissues closely matched the amount of the Tom 20 receptor.