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

News and Views into the SNARE Complexity in Arabidopsis

Secretory organelles are engaged in a continuous flux of membranes, which is believed to occur mostly via transport vesicles. Being critical in maintaining several cellular functions, transport vesicles are membrane-enclosed sacs that temporarily store and then deliver membrane lipids, protein, and polysaccharides. SNAREs have a crucial role in vesicle traffic by driving membrane fusion and conferring fidelity through the formation of specific SNARE complexes. Additionally, specific roles of SNAREs in growth and development implicate that they are versatile components for the life of a plant. Here, we summarize the recent progress on the understanding of the role of SNAREs and highlight some of the questions that are still unsolved

The Secreted Plant N-Glycoproteome and Associated Secretory Pathways

N-Glycosylation is a common form of eukaryotic protein post-translational modification, and one that is particularly prevalent in plant cell wall proteins. Large scale and detailed characterization of N-glycoproteins therefore has considerable potential in better understanding the composition and functions of the cell wall proteome, as well as those proteins that reside in other compartments of the secretory pathway. While there have been numerous studies of mammalian and yeast N-glycoproteins, less is known about the population complexity, biosynthesis, structural variation, and trafficking of their plant counterparts. However, technical developments in the analysis of glycoproteins and the structures the glycans that they bear, as well as valuable comparative analyses with non-plant systems, are providing new insights into features that are common among eukaryotes and those that are specific to plants, some of which may reflect the unique nature of the plant cell wall. In this review we present an overview of the current knowledge of plant N-glycoprotein synthesis and trafficking, with particular reference to those that are cell wall localized

A novel di-acidic motif facilitates ER export of the syntaxin SYP31

It is generally accepted that ER protein export is largely influenced by the transmembrane domain (TMD). The situation is unclear for membrane-anchored proteins such as SNAREs, which are anchored to the membrane by their TMD at the C-terminus. For example, in plants, Sec22 and SYP31 (a yeast Sed5 homologue) have a 17 aa TMD but different locations (ER/Golgi and Golgi), indicating that TMD length alone is not sufficient to explain their targeting. To establish the identity of factors that influence SNARE targeting, mutagenesis and live cell imaging experiments were performed on SYP31. It was found that deletion of the entire N-terminus domain of SYP31 blocked the protein in the ER. Several deletion mutants of different parts of this N-terminus domain indicated that a region between the SNARE helices Hb and Hc is required for Golgi targeting. In this region, replacement of the aa sequence MELAD by GAGAG or MALAG retained the protein in the ER, suggesting that MELAD may function as a di-acidic ER export motif EXXD. This suggestion was further verified by replacing the established di-acidic ER export motif DLE of a type II Golgi protein AtCASP and a membrane-anchored type I chimaera, TMcCCASP, by MELAD or GAGAG. The MELAD motif allowed the proteins to reach the Golgi, whereas the motif GAGAG was found to be insufficient to facilitate ER protein export. Our analyses indicate that we have identified a novel and transplantable di-acidic motif that facilitates ER export of SYP31 and may function for type I and type II proteins in plants

Evidence for the involvement of the Arabidopsis SEC24A in male transmission

Eukaryotic cells use COPII-coated carriers for endoplasmic reticulum (ER)-to-Golgi protein transport. Selective cargo capture into ER-derived carriers is largely driven by the SEC24 component of the COPII coat. The Arabidopsis genome encodes three AtSEC24 genes with overlapping expression profiles but it is yet to be established whether the AtSEC24 proteins have overlapping roles in plant growth and development. Taking advantage of Arabidopsis thaliana as a model plant system for studying gene function in vivo, through reciprocal crosses, pollen characterization, and complementation tests, evidence is provided for a role for AtSEC24A in the male gametophyte. It is established that an AtSEC24A loss-of-function mutation is tolerated in the female gametophyte but that it causes defects in pollen leading to failure of male transmission of the AtSEC24A mutation. These data provide a characterization of plant SEC24 family in planta showing incompletely overlapping functions of the AtSEC24 isoforms. The results also attribute a novel role to SEC24 proteins in a multicellular model system, specifically in male fertility

Dynamic remodeling of the plastid envelope membranes - a tool for chloroplast envelope in vivo localizations

Breuers FKH, Bräutigam A, Geimer S, et al. Dynamic remodeling of the plastid envelope membranes - a tool for chloroplast envelope in vivo localizations. Frontiers in Plant Science. 2012;3: 7.Two envelope membranes delimit plastids, the defining organelles of plant cells. The inner and outer envelope membranes are unique in their protein and lipid composition. Several studies have attempted to establish the proteome of these two membranes; however, differentiating between them is difficult due to their close proximity. Here, we describe a novel approach to distinguish the localization of proteins between the two membranes using a straightforward approach based on live cell imaging coupled with transient expression. We base our approach on analyses of the distribution of GFP-fusions, which were aimed to verify outer envelope membrane proteomics data. To distinguish between outer envelope and inner envelope protein localization, we used AtTOC64-GFP and AtTIC40-GFP, as respective controls. During our analyses, we observed membrane proliferations and loss of chloroplast shape in conditions of protein over-expression. The morphology of the proliferations varied in correlation with the suborganellar distribution of the over-expressed proteins. In particular, while layers of membranes built up in the inner envelope membrane, the outer envelope formed long extensions into the cytosol. Using electron microscopy, we showed that these extensions were stromules, a dynamic feature of plastids. Since the behavior of the membranes is different and is related to the protein localization, we propose that in vivo studies based on the analysis of morphological differences of the membranes can be used to distinguish between inner and outer envelope localizations of proteins. To demonstrate the applicability of this approach, we demonstrated the localization of AtLACS9 to the outer envelope membrane. We also discuss protein impact on membrane behavior and regulation of protein insertion into membranes, and provide new hypotheses on the formation of stromules

Efficient mitochondrial targeting relies on co-operation of multiple protein signals in plants

To date, the most prevalent model for transport of pre-proteins to plant mitochondria is based on the activity of an N-terminal extension serving as a targeting peptide. Whether the efficient delivery of proteins to mitochondria is based exclusively on the action of the N-terminal extension or also on that of other protein determinants has yet to be defined. A novel mechanism is reported here for the targeting of a plant protein, named MITS1, to mitochondria. It was found that MITS1 contains an N-terminal extension that is responsible for mitochondrial targeting. Functional dissection of this extension shows the existence of a cryptic signal for protein targeting to the secretory pathway. The first 11 amino acids of the N-terminal extension are necessary to overcome the activity of this signal sequence and target the protein to the mitochondria. These data suggest that co-operation of multiple determinants within the N-terminal extension of mitochondrial proteins may be necessary for efficient mitochondrial targeting. It was also established that the presence of a tryptophan residue toward the C-terminus of the protein is crucial for mitochondrial targeting, as mutation of this residue results in a redistribution of MITS1 to the endoplasmic reticulum and Golgi apparatus. These data suggest a novel targeting model whereby protein traffic to plant mitochondria is influenced by domains in the full-length protein as well as the N-terminal extension