Reticulons 3 and 6 interact with viral movement proteins

Funding; This research was funded by the Science and Technology Facilities Council Programme (grant no. 14230008), a British Biotechnology and Biological Sciences Research Council (grant no. BB/J004987/1 to Professor Chris Hawes), and a Vice-Chancellors Research Fellowship to V.K. Parts of this work were funded by the U.K. Biotechnology and Biomedical Sciences Research Council (BBSRC) grant BB/M007200/1 to J.T. Work in J.T.'s laboratory is supported by the Scottish Government's Rural and Environment Science and Analytical Services Division (RESAS).Plant reticulon (RTN) proteins are capable of constricting membranes and are vital for creating and maintaining tubules in the endoplasmic reticulum (ER), making them prime candidates for the formation of the desmotubule in plasmodesmata (PD). RTN3 and RTN6 have previously been detected in an Arabidopsis PD proteome and have been shown to be present in primary PD at cytokinesis. It has been suggested that RTN proteins form protein complexes with proteins in the PD plasma membrane and desmotubule to stabilize the desmotubule constriction and regulate PD aperture. Viral movement proteins (vMPs) enable the transport of viruses through PD and can be ER-integral membrane proteins or interact with the ER. Some vMPs can themselves constrict ER membranes or localize to RTN-containing tubules; RTN proteins and vMPs could be functionally linked or potentially interact. Here we show that different vMPs are capable of interacting with RTN3 and RTN6 in a membrane yeast two-hybrid assay, coimmunoprecipitation, and Förster resonance energy transfer measured by donor excited-state fluorescence lifetime imaging microscopy. Furthermore, coexpression of the vMP CMV-3a and RTN3 results in either the vMP or the RTN changing subcellular localization and reduces the ability of CMV-3a to open PD, further indicating interactions between the two proteins.Publisher PDFPeer reviewe

Defining the dance: Quantification and classification of ER dynamics

The availability of quantification methods for sub-cellular organelle dynamic analysis has increased rapidly over the last 20 years. The application of these techniques to contiguous sub-cellular structures that exhibit dynamic re-modelling over a range of scales and orientations is challenging as quantification of ‘movement’ rarely corresponds to traditional, qualitative classifications of types of organelle movement. The plant endoplasmic reticulum represents a particular challenge for dynamic quantification as it itself is an entirely contiguous organelle that is in a constant state of flux and gross remodelling, controlled by the actinomyosin cytoskeleton

The plant endoplasmic reticulum: An organized chaos of tubules and sheets with multiple functions

The endoplasmic reticulum (ER) is a fascinating organelle at the core of the secretory pathway. It is responsible for the synthesis of one third of the cellular proteome and, in plant cells, it produces receptors and transporters of hormones as well as the proteins responsible for the biosynthesis of critical components of a cellulosic cell wall. The ER structure resembles a spider-web network of interconnected tubules and cisternae that pervades the cell. The study of the dynamics and interaction of this organelles with other cellular structures such as the plasma membrane, the Golgi apparatus and the cytoskeleton, have been permitted by the implementation of fluorescent protein and advanced confocal imaging. In this review, we report on the findings that contributed toward the understanding of the ER morphology and function with the aid of fluorescent proteins, focusing on the contributions provided by pioneering work from the lab of the late Professor Chris Hawes

Labeling the ER for Light and Fluorescence Microscopy

The ER is a highly dynamic network of tubules and membrane sheets. Hence imaging this organelle in its native and mobile state is of great importance. Here we describe methods of labeling the native ER using fluorescent proteins and lipid dyes as well as methods for immunolabeling on plant tissue

Reticulons 3 and 6 interact with viral movement proteins

Plant reticulon proteins (RTN) are capable of constricting membranes and vital for creating and maintaining tubules in the endoplasmic reticulum (ER), making them prime candidates for the formation of the desmotubule in plasmodesmata (PD). RTN3 and RTN6 have previously been detected in an Arabidopsis PD proteome and have been shown to be present in primary PD at cytokinesis. It was suggested that RTN proteins form protein complexes with proteins in the PD plasma membrane and desmotubule to stabilize the desmotubule constriction and regulate PD aperture. Viral Movement Proteins (vMPs) enable the transport of viruses through PD and can be ER-integral membrane proteins or interact with the ER. Some vMPs can themselves constrict ER membranes or localise to RTN-containing tubules; RTN proteins and vMPs could be functionally linked or potentially interact. Here we show that different vMPs are capable of interacting with RTN3 and 6 in a membrane yeast-2-hybrid assay, co-immunoprecipitation and Förster resonance energy transfer measured by donor excited-state fluorescence lifetime imaging microscopy (FRET-FLIM). Furthermore, coexpression of the vMP CMV-3a and RTN3 results in either the vMP or the RTN changing subcellular localisation and reduces the ability of CMV-3a to open PD, further indicating interactions between the two proteins

Bioinformatics Analysis of Phylogeny and Transcription of TAA/YUC Auxin Biosynthetic Genes

Auxin is a main plant growth hormone crucial in a multitude of developmental processes in plants. Auxin biosynthesis via the tryptophan aminotransferase of arabidopsis (TAA)/YUCCA (YUC) route involving tryptophan aminotransferases and YUC flavin-dependent monooxygenases that produce the auxin indole-3-acetic acid (IAA) from tryptophan is currently the most researched auxin biosynthetic pathway. Previous data showed that, in maize and arabidopsis, TAA/YUC-dependent auxin biosynthesis can be detected in endoplasmic reticulum (ER) microsomal fractions, and a subset of auxin biosynthetic proteins are localized to the ER, mainly due to transmembrane domains (TMD). The phylogeny presented here for TAA/TAR (tryptophan aminotransferase related) and YUC proteins analyses phylogenetic groups as well as transmembrane domains for ER-membrane localisation. In addition, RNAseq datasets are analysed for transcript abundance of YUC and TAA/TAR proteins in Arabidopsis thaliana. We show that ER membrane localisation for TAA/YUC proteins involved in auxin biosynthesis is already present early on in the evolution of mosses and club mosses. ER membrane anchored YUC proteins can mainly be found in roots, while cytosolic proteins are more abundant in the shoot. The distribution between the different phylogenetic classes in root and shoot may well originate from gene duplications, and the phylogenetic groups detected also overlap with the biological function

Auxin biosynthesis: Spatial regulation and adaptation to stress

The plant hormone auxin is essential for plant growth and development, controlling both organ development and overall plant architecture. Auxin homeostasis is regulated by coordination of biosynthesis, transport, conjugation, sequestration/storage, and catabolism to optimize concentration-dependent growth responses and adaptive responses to temperature, water stress, herbivory and pathogens. At present, the best defined pathway of auxin biosynthesis is the TAA/YUC route, in which the tryptophan aminotransferases TAA and TAR and YUCCA flavin-dependent monooxygenases produce the auxin indole-3-acetic acid from tryptophan. This review highlights recent advances in our knowledge of TAA/YUC-dependent auxin biosynthesis focussing on membrane localisation of auxin biosynthetic enzymes, differential regulation in root and shoot tissue, and auxin biosynthesis during abiotic stress

An interplay between mitochondrial and ER targeting of a bacterial signal peptide in plants

Protein targeting is essential in eukaryotic cells to maintain cell function and organelle identity. Signal peptides are a major type of targeting sequences containing a tripartite structure, which is conserved across all domains in life. They are frequently included in recombinant protein design in plants to increase yields by directing them to the endoplasmic reticulum (ER) or apoplast. The processing of bacterial signal peptides by plant cells is not well understood but could aid in the design of efficient heterologous expression systems. Here we analysed the signal peptide of the enzyme PmoB from methanotrophic bacteria. In plant cells, the PmoB signal peptide targeted proteins to both mitochondria and the ER. This dual localisation was still observed in a mutated version of the signal peptide sequence with enhanced mitochondrial targeting efficiency. Mitochondrial targeting was shown to be dependent on a hydrophobic region involved in transport to the ER. We, therefore, suggest that the dual localisation could be due to an ER-SURF pathway recently characterised in yeast. This work thus sheds light on the processing of bacterial signal peptides by plant cells and proposes a novel pathway for mitochondrial targeting in plants

Localization and interactions between Arabidopsis auxin biosynthetic enzymes in the TAA/YUC-dependent pathway

The growth regulator auxin is involved in all key developmental processes in plants. A complex network of a multiplicity of potential auxin biosynthetic pathways as well as transport, signalling plus conjugation and deconjugation lead to a complicated system of auxin function. This raises the question how such a complex and multifaceted system producing such a powerful and important molecule as auxin can be effectively organised and controlled. Here we report that a subset of auxin biosynthetic enzymes in the TAA/YUC route of auxin biosynthesis is localised to the endoplasmic reticulum (ER). ER microsomal fractions also contain a significant percentage of auxin biosynthetic activity. This could point toward a model of auxin function using ER membrane location and subcellular compartmentation for supplementary layers of regulation. Additionally we show specific protein-protein interactions between some of the enzymes in the TAA/YUC route of auxin biosynthesis