Coat protein complex I (COPI) and COPII are required for bidirectional membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. While these core coat machineries and other transport factors are highly conserved across species, high-resolution imaging studies indicate that the organization of the ER–Golgi interface is varied in eukaryotic cells. Regulation of COPII assembly, in some cases to manage distinct cellular cargo, is emerging as one important component in determining this structure. Comparison of the ER–Golgi interface across different systems, particularly mammalian and plant cells, reveals fundamental elements and distinct organization of this interface. A better understanding of how these interfaces are regulated to meet varying cellular secretory demands should provide key insights into the mechanisms that control efficient trafficking of proteins and lipids through the secretory pathway.

A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributions to be determined. Consequently, genuine organelle residents have been distinguished from contaminating proteins and proteins in transit through the secretory pathway.endomembrane ͉ localization of organelle proteins by isotope tagging ͉ isotope tags for relative and absolute quantitation ͉ organelle proteomics P roteins are spatially organized according to their functions within the eukaryotic cell. Therefore, protein localization is an important step toward assigning functions to the thousands of uncharacterized proteins predicted by the genome-sequencing projects. Proteomics provides powerful tools for characterizing the protein contents of organelles. Confident protein localization, however, requires that either organelle preparations are free of contaminants or that techniques are used to discriminate between genuine organelle residents and contaminating proteins (1). Although reasonably pure preparations of some organelles, such as mitochondria, can be achieved, the components of the endomembrane system so far have proved recalcitrant to purification (2, 3). The constituent organelles of the endomembrane system have similar sizes and densities, making them difficult to separate. In addition, the proteins that reside within this system are in a constant state of flux. Endomembrane proteins traffic through the system en route to their final destination; for example, plasma membrane (PM) proteins travel although the endoplasmic reticulum (ER) and the Golgi apparatus before reaching the cell surface. Proteins within the endomembrane system also cycle between compartments; for example, ER residents continuously escape to the Golgi apparatus and are retrieved in COPI vesicles (4). Consequently, it is not sufficient merely to identify the proteins within a single organelle-enriched fraction. Instead, the steady-state distributions of proteins within the whole endomembrane system must be determined if a realistic insight into the subcellular localization of endomembrane proteins is to be achieved.Localization of organelle proteins by...

In contrast with animals, plant cells contain multiple mobile Golgi stacks distributed over the entire cytoplasm. However, the distribution and dynamics of protein export sites on the plant endoplasmic reticulum (ER) surface have yet to be characterized. A widely accepted model for ER-to-Golgi transport is based on the sequential action of COPII and COPI coat complexes. The COPII complex assembles by the ordered recruitment of cytosolic components on the ER membrane. Here, we have visualized two early components of the COPII machinery, the small GTPase Sar1p and its GTP exchanging factor Sec12p in live tobacco (Nicotiana tabacum) leaf epidermal cells. By in vivo confocal laser scanning microscopy and fluorescence recovery after photobleaching experiments, we show that Sar1p cycles on mobile punctate structures that track with the Golgi bodies in close proximity but contain regions that are physically separated from the Golgi bodies. By contrast, Sec12p is uniformly distributed along the ER network and does not accumulate in these structures, consistent with the fact that Sec12p does not become part of a COPII vesicle. We propose that punctate accumulation of Sar1p represents ER export sites (ERES). The sites may represent a combination of Sar1p-coated ER membranes, nascent COPII membranes, and COPII vectors in transit, which have yet to lose their coats. ERES can be induced by overproducing Golgi membrane proteins but not soluble bulk-flow cargos. Few punctate Sar1p loci were observed that are independent of Golgi bodies, and these may be nascent ERES. The vast majority of ERES form secretory units that move along the surface of the ER together with the Golgi bodies, but movement does not influence the rate of cargo transport between these two organelles. Moreover, we could demonstrate using the drug brefeldin A that formation of ERES is strictly dependent on a functional retrograde transport route from the Golgi apparatus.

Cells operate a signaling network termed unfolded protein response (UPR) to monitor protein-folding capacity in the endoplasmic reticulum (ER). IRE1 is an ER transmembrane sensor that activates UPR to maintain ER and cellular function. While mammalian IRE1 promotes cell survive, it can initiate apoptosis via decay of anti-apoptotic microRNAs. Convergent and divergent IRE1 characteristics between plants and animals underscore its significance in cellular homeostasis. This review provides an updated scenario of IRE1 signaling model, discusses emerging IRE1 sensing mechanisms, compares IRE1 features among species, and outlines exciting future directions in UPR research.

The mechanisms that control protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus are poorly characterized in plants. Here, we examine in tobacco leaves the structural relationship between Golgi and ER membranes using electron microscopy and demonstrate that Golgi membranes contain elements that are in close association and/or in direct contact with the ER. We further visualized protein trafficking between the ER and the Golgi using Golgi marker proteins tagged with green fluorescent protein. Using photobleaching techniques, we showed that Golgi membrane markers constitutively cycle to and from the Golgi in an energy-dependent and N -ethylmaleimidesensitive manner. We found that membrane protein transport toward the Golgi occurs independently of the cytoskeleton and does not require the Golgi to be motile along the surface of the ER. Brefeldin A treatment blocked forward trafficking of Golgi proteins before their redistribution into the ER. Our results indicate that in plant cells, the Golgi apparatus is a dynamic membrane system whose components continuously traffic via membrane trafficking pathways regulated by brefeldin A-and N -ethylmaleimide-sensitive machinery.