Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex

<p>Abstract</p> <p>Background</p> <p><it>Carnation Italian ringspot virus </it>(CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36), are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined.</p> <p>Results</p> <p>Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs) and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC) that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM), but not with the sorting and assembly machinery (SAM).</p> <p>Conclusion</p> <p>Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer membrane also sheds light on the mechanisms involved in sorting of host-cell membrane proteins to mitochondria, a process that has been largely unexplored in plants.</p

New Insights Into Sunflower (Helianthus annuus L.) FatA and FatB Thioesterases, Their Regulation, Structure and Distribution

Sunflower seeds (Helianthus annuus L.) accumulate large quantities of triacylglycerols (TAG) between 12 and 28 days after flowering (DAF). This is the period of maximal acyl-acyl carrier protein (acyl-ACP) thioesterase activity in vitro, the enzymes that terminate the process of de novo fatty acid synthesis by catalyzing the hydrolysis of the acyl-ACPs synthesized by fatty acid synthase. Fatty acid thioesterases can be classified into two families with distinct substrate specificities, namely FatA and FatB. Here, some new aspects of these enzymes have been studied, assessing how both enzymes contribute to the acyl composition of sunflower oil, not least through the changes in their expression during the process of seed filling. Moreover, the binding pockets of these enzymes were modeled based on new data from plant thioesterases, revealing important differences in their volume and geometry. Finally, the subcellular location of the two enzymes was evaluated and while both possess an N-terminal plastid transit peptide, only in FatB contains a hydrophobic sequence that could potentially serve as a transmembrane domain. Indeed, using in vivo imaging and organelle fractionation, H. annuus thioesterases, HaFatA and HaFatB, appear to be differentially localized in the plastid stroma and membrane envelope, respectively. The divergent roles fulfilled by HaFatA and HaFatB in oil biosynthesis are discussed in the light of our data.España MINECO y FEDER Projects AGL2014- 53537-R y AGL2017-83449-

Protein–Protein Interaction Network and Subcellular Localization of the Arabidopsis Thaliana ESCRT Machinery

The endosomal sorting complex required for transport (ESCRT) consists of several multi-protein subcomplexes which assemble sequentially at the endosomal surface and function in multivesicular body (MVB) biogenesis. While ESCRT has been relatively well characterized in yeasts and mammals, comparably little is known about ESCRT in plants. Here we explored the yeast two-hybrid protein interaction network and subcellular localization of the Arabidopsis thaliana ESCRT machinery. We show that the Arabidopsis ESCRT interactome possesses a number of protein–protein interactions that are either conserved in yeasts and mammals or distinct to plants. We show also that most of the Arabidopsis ESCRT proteins examined at least partially localize to MVBs in plant cells when ectopically expressed on their own or co-expressed with other interacting ESCRT proteins, and some also induce abnormal MVB phenotypes, consistent with their proposed functional role(s) as part of the ESCRT machinery in Arabidopsis. Overall, our results help define the plant ESCRT machinery by highlighting both conserved and unique features when compared to ESCRT in other evolutionarily diverse organisms, providing a foundation for further exploration of ESCRT in plants

Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

Background: Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope. Methodology/Principal Findings: Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein. Conclusions/Significance: Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA proteins to their proper intracellular locations in general

Identification of a New Class of Lipid Droplet-Associated Proteins in Plants

Article on the identification of a new class of lipid droplet-associated proteins in plants

Genome-wide analysis of Homo sapiens, Arabidopsis thaliana, and Saccharomyces cerevisiae reveals novel attributes of tail-anchored membrane proteins

Abstract Background Tail-anchored membrane proteins (TAMPs) differ from other integral membrane proteins, because they contain a single transmembrane domain at the extreme carboxyl-terminus and are therefore obliged to target to membranes post-translationally. Although 3–5% of all transmembrane proteins are predicted to be TAMPs only a small number are well characterized. Results To identify novel putative TAMPs across different species, we used TAMPfinder software to identify 859, 657 and 119 putative TAMPs in human (Homo sapiens), plant (Arabidopsis thaliana), and yeast (Saccharomyces cerevisiae), respectively. Bioinformatics analyses of these putative TAMP sequences suggest that the list is highly enriched for authentic TAMPs. To experimentally validate the software predictions several human and plant proteins identified by TAMPfinder that were previously uncharacterized were expressed in cells and visualized at subcellular membranes by fluorescence microscopy and further analyzed by carbonate extraction or by bimolecular fluorescence complementation. With the exception of the pro-apoptotic protein harakiri, which is, peripherally bound to the membrane this subset of novel proteins behave like genuine TAMPs. Comprehensive bioinformatics analysis of the generated TAMP datasets revealed previously unappreciated common and species-specific features such as the unusual size distribution of and the propensity of TAMP proteins to be part of larger complexes. Additionally, novel features of the amino acid sequences that anchor TAMPs to membranes were also revealed. Conclusions The findings in this study more than double the number of predicted annotated TAMPs and provide new insights into the common and species-specific features of TAMPs. Furthermore, the list of TAMPs and annotations provide a resource for further investigation

Engineering the production of conjugated fatty acids in Arabidopsis thaliana leaves

This article discusses whether the fatty acid composition of leaf oil could be engineered to accumulate unusual fatty acids