Endoplasmic Reticulum Of Plant Cells Research Articles

Protein-protein interactions of the phenylpropanoid pathway in sorghum bicolor.

Phenylalanine ammonia-lyase (PAL) is the first enzyme of the phenylpropanoid pathway followed by cinnamate 4-hydroxylase (C4H), a cytochrome P450 monooxygenase associated with the endoplasmic reticulum of plant cells. PAL and C4H are important for the plant cells due to their involvement in kickstarting the production of primary and secondary metabolites such as phenylpropanoids and lignin. C4H also interacts with cytochrome P450 reductase (CPR; Sorghum bicolor CPR in this study) to hydrolyze trans-cinnamic acid into p-coumaric acid. This study uses fluorescence correlation spectroscopy and single-protein fluorescence tracking of labeled proteins to directly measure the interactions of all three proteins in the absence and presence of their respective substrates. This study also explores specific protein-lipid interactions as a function of state (i.e. oxidation state, heterodimeric state, substrate occupancy, etc.)

MRNA Localization to the Endoplasmic Reticulum in Plant Endosperm Cells.

Subcellular mRNA localization is an evolutionarily conserved mechanism to spatially and temporally drive local translation and, in turn, protein targeting. Hence, this mechanism achieves precise control of gene expression and establishes functional and structural networks during cell growth and development as well as during stimuli response. Since its discovery in ascidian eggs, mRNA localization has been extensively studied in animal and yeast cells. Although our knowledge of subcellular mRNA localization in plant cells lags considerably behind other biological systems, mRNA localization to the endoplasmic reticulum (ER) has also been well established since its discovery in cereal endosperm cells in the early 1990s. Storage protein mRNA targeting to distinct subdomains of the ER determines efficient accumulation of the corresponding proteins in different endosomal storage sites and, in turn, underlies storage organelle biogenesis in cereal grains. The targeting process requires the presence of RNA localization elements, also called zipcodes, and specific RNA-binding proteins that recognize and bind these zipcodes and recruit other factors to mediate active transport. Here, we review the current knowledge of the mechanisms and functions of mRNA localization to the ER in plant cells and address directions for future research.

A plant receptor domain with functional analogies to animal malectin disables ER stress responses upon infection

SummaryMalectins from the oligosaccharyltransferase (OST) complex in the endoplasmic reticulum (ER) of animal cells are involved in ER quality control and contribute to the Unfolded Protein Response (UPR). Malectins are not found in plant cells, but malectin-like domains (MLDs) are constituents of many membrane-bound receptors. In Arabidopsis thaliana, the MLD-containing receptor IOS1 promotes successful infection by filamentous plant pathogens. We show that the MLD of its exodomain retains IOS1 in the ER of plant cells and attenuates the infection-induced UPR. Expression of the MLD in the ios1-1 knockout background is sufficient to complement infection-related phenotypes of the mutant, such as increased UPR and reduced disease susceptibility. IOS1 interacts with the ER membrane-associated ribophorin HAP6 from the OST complex, and hap6 mutants show decreased pathogen-responsive UPR and increased disease susceptibility. Altogether, this study revealed a previously uncharacterized role of a plant receptor domain in the regulation of ER stress during infection.

Interaction of VvbZIP60s and VvHSP83 in response to high-temperature stress in grapes.

The occurrence of frequent, extreme high temperatures affects agriculture and causes irreversible damage during the ripening period of grapes. Breeding high-temperature-tolerant varieties of grapes is the main way to deal with this challenge, thus necessitating research on the regulatory mechanism of high-temperature tolerance. Extreme high temperature causes the mismatch of proteins in the endoplasmic reticulum in plant cells and initiates the unfolded protein response (UPR). The transcription factor bZIP60 participates in the UPR process. In the present study, VvbZIP60 and VvbZIP60s (unconventional splicing of VvbZIP60) were cloned and expressed in a transgenic system to verify heat tolerance. VvbZIP60s was found to be a key gene in adapting to heat stress. VvbZIP60s/60u interacted with VvHSP83 as observed in two yeast hybrids, with bimolecular fluorescence complementation and pull-down assays. VvHSP83 is also a key gene for plants to adapt to heat stress by participating in the renaturation and degradation of denatured proteins under adversity, causing plants to resist high temperatures. This study provides a basis for analyzing the mechanism of high-temperature tolerance in grapes.

IntEResting structures: formation and applications of organized smooth endoplasmic reticulum in plant cells.

The endoplasmic reticulum (ER) is an organelle with remarkable plasticity, capable of rapidly changing its structure to accommodate different functions based on intra- and extracellular cues. One of the ER structures observed in plants is known as "organized smooth endoplasmic reticulum" (OSER), consisting of symmetrically stacked ER membrane arrays. In plants, these structures were first described in certain specialized tissues, e.g. the sieve elements of the phloem, and more recently in transgenic plants overexpressing ER membrane resident proteins. To date, much of the investigation of OSER focused on yeast and animal cells but research into plant OSER has started to grow. In this update, we give a succinct overview of research into the OSER phenomenon in plant cells with case studies highlighting both native and synthetic occurrences of OSER. We also assess the primary driving forces that trigger the formation of OSER, collating evidence from the literature to compare two competing theories for the origin of OSER: that OSER formation is initiated by oligomerizing protein accumulation in the ER membrane or that OSER is the result of ER membrane proliferation. This has long been a source of controversy in the field and here we suggest a way to integrate arguments from both sides into a single unifying theory. Finally, we discuss the potential biotechnological uses of OSER as a tool for the nascent plant synthetic biology field with possible applications as a synthetic microdomain for metabolic engineering and as an extensive membrane surface for synthetic chemistry or protein accumulation.

Structure and Function of the Cytochrome P450 Monooxygenase Cinnamate 4-hydroxylase from Sorghum bicolor.

Cinnamate 4-hydroxylase (C4H; CYP73A) is a cytochrome P450 monooxygenase associated externally with the endoplasmic reticulum of plant cells. The enzyme uses NADPH-cytochrome P450 reductase as a donor of electrons and hydroxylates cinnamic acid to form 4-coumaric acid in phenylpropanoid metabolism. In order to better understand the structure and function of this unique class of plant P450 enzymes, we have characterized the enzyme C4H1 from lignifying tissues of sorghum (Sorghum bicolor), encoded by Sobic.002G126600 Here we report the 1.7 Å resolution crystal structure of CYP73A33. The obtained structural information, along with the results of the steady-state kinetic analysis and the absorption spectroscopy titration, displays a high degree of similarity of the structural and functional features of C4H to those of other P450 proteins. Our data also suggest the presence of a putative allosteric substrate-binding site in a hydrophobic pocket on the enzyme surface. In addition, comparing the newly resolved structure with those of well-investigated cytochromes P450 from mammals and bacteria enabled us to identify those residues of critical functional importance and revealed a unique sequence signature that is potentially responsible for substrate specificity and catalytic selectivity of C4H.

3D reconstruction of endoplasmic reticulum in a hydrocarbon-secreting green alga, Botryococcus braunii (Race B).

Based on 3D sections through cells of Botryococcus braunii, the structure of three domains of endoplasmic reticulum, and their spatial and functional relationships to other organelles are clarified. Oil production by photosynthetic microalgae has attracted attention since these oils can be converted into renewable, carbon-neutral fuels. The green alga B. braunii accumulates large amounts of hydrocarbons, 30-50% of cell dry weight, in extracellular spaces rather than its cytoplasm. To advance the knowledge of hydrocarbon biosynthesis and transport pathways in this alga, we utilized transmission EM combined with rapid freezing and image reconstruction. We constructed detailed 3D maps distinguishing three ER domains: rdER with ribosomes on both sides, rsER with ribosomes on one side, and sER without ribosomes. The rsER and sER domains were especially prominent during the oil body formation and oil secretion stages. The ER contacted the chloroplasts, oil bodies, or plasma membrane via the rsER domains, oriented with the ribosome-free surface facing the organelles. We discuss the following transport pathway for hydrocarbons and their precursors in the cytoplasm: chloroplast→endoplasmic reticulum (ER)→oil bodies→ER→plasma membrane→secretion. This study represents the first 3D study of the three-domain classification (rdER, rsER and sER) of the ER network among eukaryotic cells. Finally, we propose the novel features of the ERs in plant cells that are distinct from the latest proposed model for the ERs in mammalian cells.

Calreticulin localizes to plant intra/extracellular peripheries of highly specialized cells involved in pollen-pistil interactions

Calcium (Ca2+) plays essential roles in generative reproduction of angiosperms, but the sites and mechanisms of Ca2+ storage and mobilization during pollen-pistil interactions have not been fully defined. Both external and internal Ca2+ stores are likely important during male gametophyte communication with the sporophytic and gametophytic cells within the pistil. Given that calreticulin (CRT), a Ca2+-buffering protein, is able to bind Ca2+ reversibly, it can serve as a mobile store of easily releasable Ca2+ (so called an exchangeable Ca2+) in eukaryotic cells. CRT has typical endoplasmic reticulum (ER) targeting and retention signals and resides primarily in the ER. However, localization of this protein outside the ER has also been revealed in both animal and plant cells, including Golgi/dictyosomes, nucleus, plasma membrane/cell surface, plasmodesmata, and even extracellular matrix. These findings indicate that CRT may function in a variety of different cell compartments and specialized structures. We have recently shown that CRT is highly expressed and accumulated in the ER of plant cells involved in pollen-pistil interactions in Petunia, and we proposed an essential role for CRT in intracellular Ca2+ storage and mobilization during the key reproductive events. Here, we demonstrate that both CRT and exchangeable Ca2+ are localized in the intra/extracellular peripheries of highly specialized plant cells, such as the pistil transmitting tract cells, pollen tubes, nucellus cells surrounding the embryo sac, and synergids. Based on our present results, we propose that extracellularly located CRT is also involved in Ca2+ storage and mobilization during sexual reproduction of angiosperms.

Identification of rice cornichon as a possible cargo receptor for the Golgi-localized sodium transporter OsHKT1;3.

Membrane proteins are synthesized and folded in the endoplasmic reticulum (ER), and continue their path to their site of residence along the secretory pathway. The COPII system has been identified as a key player for selecting and directing the fate of membrane and secretory cargo proteins. Selection of cargo proteins within the COPII vesicles is achieved by cargo receptors. The cornichon cargo receptor belongs to a conserved protein family found in eukaryotes that has been demonstrated to participate in the selection of integral membrane proteins as cargo for their correct targeting. Here it is demonstrated at the cellular level that rice cornichon OsCNIH1 interacts with OsHKT1;3 and, in yeast cells, enables the expression of the sodium transporter to the Golgi apparatus. Physical and functional HKT-cornichon interactions are confirmed by the mating-based split ubiquitin system, bimolecular fluorescence complementation, and Xenopus oocyte and yeast expression systems. The interaction between the two proteins occurs in the ER of plant cells and their co-expression in oocytes leads to the sequestration of the transporter in the ER. In the yeast cornichon mutant erv14, OsHKT1;3 is mistargeted, preventing the toxic effects of sodium transport in the cell observed in wild-type cells or in the erv14 mutant that co-expressed OsHKT1;3 with either OsCNIH1 or Erv14p. Identification and characterization of rice cornichon as a possible cargo receptor opens up the opportunity to improve our knowledge on membrane protein targeting in plant cells.

Analyses of Ca2+ accumulation and dynamics in the endoplasmic reticulum of Arabidopsis root cells using a genetically encoded Cameleon sensor.

In planta, very limited information is available about how the endoplasmic reticulum (ER) contributes to cellular Ca(2+) dynamics and homeostasis. Here, we report the generation of an ER-targeted Cameleon reporter protein suitable for analysis of Ca(2+) accumulation and dynamics in the lumen of the ER in plant cells. Using stably transformed Arabidopsis (Arabidopsis thaliana) plants expressing this reporter protein, we observed a transiently enhanced accumulation of Ca(2+) in the ER in response to stimuli inducing cytosolic Ca(2+) rises in root tip cells. In all experimental conditions, ER Ca(2+) dynamics were substantially different from those monitored in the cytosol. A pharmacological approach enabled us to evaluate the contribution of the different ER-resident Ca(2+)-ATPase classes in the regulation of the ER Ca(2+) homeostasis. Taken together, our results do not provide evidence for a role of the ER as a major source that releases Ca(2+) for stimulus-induced increases in cytosolic Ca(2+) concentration. Instead, our results show that the luminal ER Ca(2+) elevations typically follow cytosolic ones, but with distinct dynamics. These findings suggest fundamental differences for the function of the ER in cellular Ca(2+) homeostasis in plants and animals.

Simultaneous live-imaging of peroxisomes and the ER in plant cells suggests contiguity but no luminal continuity between the two organelles

Transmission electron micrographs of peroxisomes in diverse organisms, including plants, suggest their close association and even luminal connectivity with the endoplasmic reticulum (ER). After several decades of debate de novo peroxisome biogenesis from the ER is strongly favored in yeasts and mammals. Unfortunately many of the proteins whose transit through the ER constitutes a major evidence for peroxisome biogenesis from the ER do not exhibit a similar localization in plants. Consequently, at best the ER acts as a membrane source for peroxisome in plants. However, in addition to their de novo biogenesis from the ER an increase in peroxisome numbers also occurs through fission of existing peroxisomes. In recent years live-imaging has been used to visualize peroxisomes and the ER but the precise spatio-temporal relationship between the two organelles has not been well-explored. Here we present our assessment of the peroxisome-ER relationship through imaging of living Arabidopsis thaliana plants simultaneously expressing different color combinations of fluorescent proteins targeted to both organelles. Our observations on double transgenic wild type and a drp3a/apm1 mutant Arabidopsis plants suggest strong correlations between the dynamic behavior of peroxisomes and the neighboring ER. Although peroxisomes and ER are closely aligned there appears to be no luminal continuity between the two. Similarly, differentially colored elongated peroxisomes of a drp3a mutant expressing a photoconvertible peroxisomal matrix protein are unable to fuse and share luminal protein despite considerable intermingling. Substantiation of our observations is suggested through 3D iso-surface rendering of image stacks, which shows closed ended peroxisomes enmeshed among ER tubules possibly through membrane contact sites (MCS). Our observations support the idea that increase in peroxisome numbers in a plant cell occurs mainly through the fission of existing peroxisomes in an ER aided manner.

Redox regulation of glutenin subunit assembly in the plant endoplasmic reticulum

The glutenin fraction of wheat storage proteins consists of large polymers in which high- and low-molecular-weight subunits are connected by inter-chain disulfide bonds. We found that assembly of a low-molecular-weight glutenin subunit in the endoplasmic reticulum is a rapid process that leads to accumulation of various oligomeric forms, and that this assembly is sensitive to perturbation of the cellular redox environment. In endoplasmic reticulum-derived microsomes, low-molecular-weight glutenin subunits are subjected to hyper-polymerization, indicating that cytosolic factors play an important role in limiting polymer size. Addition of physiological concentrations of reduced glutathione is sufficient to maintain the original polymerization pattern of the glutenin subunits upon cytosol dilution. Furthermore, we show that a low-molecular-weight glutenin subunit can be glutathionylated in endoplasmic reticulum-derived microsomes, and that it can be directly reduced by glutathione in vitro. These results indicate that glutenin polymerization is sensitive to changes in the redox state of the cell, and suggest that the presence of a reducing cytosolic environment plays an important role in regulating disulfide bond formation in the endoplasmic reticulum of plant cells.

KMS1 and KMS2, two plant endoplasmic reticulum proteins involved in the early secretory pathway

We have identified two endoplasmic reticulum (ER)-associated Arabidopsis proteins, KMS1 and KMS2, which are conserved among most species. Fluorescent protein fusions of KMS1 localised to the ER in plant cells, and over-expression induced the formation of a membrane structure, identified as ER whorls by electron microscopy. Hydrophobicity analysis suggested that KMS1 and KMS2 are integral membrane proteins bearing six transmembrane domains. Membrane protein topology was assessed by a redox-based topology assay (ReTA) with redox-sensitive GFP and confirmed by a protease protection assay. A major loop domain between transmembrane domains 2 and 3, plus the N- and C-termini were found on the cytosolic side of the ER. A C-terminal di(tri)-lysine motif is involved in retrieval of KMS1 and deletion led to a reduction of the GFP-KMS1 signal in the ER. Over-expression of KMS1/KMS2 truncations perturbed ER and Golgi morphology and similar effects were also seen when KMS1/KMS2 were knocked-down by RNA interference. Microscopy and biochemical experiments suggested that expression of KMS1/KMS2 truncations inhibited ER to Golgi protein transport.

A TSPO-related protein localizes to the early secretory pathway in Arabidopsis, but is targeted to mitochondria when expressed in yeast

AtTSPO is a TspO/MBR domain-protein potentially involved in multiple stress regulation in Arabidopsis. As in most angiosperms, AtTSPO is encoded by a single, intronless gene. Expression of AtTSPO is tightly regulated both at the transcriptional and post-translational levels. It has been shown previously that overexpression of AtTSPO in plant cell can be detrimental, and the protein was detected in the endoplasmic reticulum (ER) and Golgi stacks, contrasting with previous findings and suggesting a mitochondrial subcellular localization for this protein. To ascertain these findings, immunocytochemistry and ABA induction were used to demonstrate that, in plant cells, physiological levels of AtTSPO colocalized with AtArf1, a mainly Golgi-localized protein in plant cells. In addition, fluorescent protein-tagged AtTSPO was targeted to the secretory pathway and did not colocalize with MitoTracker-labelled mitochondria. These results suggest that the polytopic membrane protein AtTSPO is cotranslationally targeted to the ER in plant cells and accumulates in the Trans-Golgi Network. Heterologous expression of AtTSPO in Saccharomyces cerevisiae, yeast devoid of TSPO-related protein, resulted in growth defects. However, subcellular fractionation and immunoprecipitation experiments showed that AtTSPO was targeted to mitochondria where it colocalized and interacted with the outer mitochondrial membrane porin VDAC1p, reminiscent of the subcellular localization and activity of mammalian translocator protein 18 kDa TSPO. The evolutionarily divergent AtTSPO appears therefore to be switching its sorting mode in a species-dependent manner, an uncommon peculiarity for a polytopic membrane protein in eukaryotic cells. These results are discussed in relation to the recognition and organelle targeting mechanisms of polytopic membrane proteins in eukaryotic cells.

Chemical Basis of Nitrogen Recovery through the Ureide Pathway: Formation and Hydrolysis ofS-Ureidoglycine in Plants and Bacteria

While some organisms, including humans, eliminate oxidized purines to get rid of excess nitrogen, for many others the recovery of the purine ring nitrogen is vital. In the so-called ureide pathway, nitrogen is released as ammonia from allantoate through a series of reactions starting with allantoate amidohydrolase (AAH), a manganese-dependent enzyme found in plants and bacteria. We report NMR evidence that the true product of the AAH reaction is S-ureidoglycine, a nonstandard alpha-amino acid that spontaneously releases ammonia in vitro. Using gene proximity and logical genome analysis, we identified a candidate gene (ylbA) for S-ureidoglycine metabolism. The proteins encoded by Escherichia coli and Arabidopsis thaliana genes catalyze the manganese-dependent release of ammonia through hydrolysis of S-ureidoglycine. Hydrolysis then inverts the configuration and yields S-ureidoglycolate. S-Ureidoglycine aminohydrolase (UGHY) is cytosolic in bacteria, whereas in plants it is localized, like allantoate amidohydrolase, in the endoplasmic reticulum. These findings strengthen the basis for the known sensitivity of the ureide pathway to Mn availability and suggest a further rationale for the active transport of Mn in the endoplasmic reticulum of plant cells.

Anchoring Secreted Proteins in Endoplasmic Reticulum by Plant Oleosin: The Example of Vitamin B12 Cellular Sequestration by Transcobalamin

BackgroundOleosin is a plant protein localized to lipid droplets and endoplasmic reticulum of plant cells. Our idea was to use it to target functional secretory proteins of interest to the cytosolic side of the endoplasmic reticulum of mammalian cells, through expressing oleosin-containing chimeras. We have designed this approach to create cellular models deficient in vitamin B12 (cobalamin) because of the known problematics associated to the obtainment of effective vitamin B12 deficient cell models. This was achieved by the overexpression of transcobalamin inside cells through anchoring to oleosin.Methodologychimera gene constructs including transcobalamin-oleosin (TC-O), green fluorescent protein-transcobalamin-oleosin (GFP-TC-O) and oleosin-transcobalamin (O-TC) were inserted into pAcSG2 and pCDNA3 vectors for expression in sf9 insect cells, Caco2 (colon carcinoma), NIE-115 (mouse neuroblastoma), HEK (human embryonic kidney), COS-7 (Green Monkey SV40-transfected kidney fibroblasts) and CHO (Chinese hamster ovary cells). The subcellular localization, the changes in vitamin B12 binding activity and the metabolic consequences were investigated in both Caco2 and NIE-115 cells.Principal findingsvitamin B12 binding was dramatically higher in TC-O than that in O-TC and wild type (WT). The expression of GFP-TC-O was observed in all cell lines and found to be co-localized with an ER-targeted red fluorescent protein and calreticulin of the endoplasmic reticulum in Caco2 and COS-7 cells. The overexpression of TC-O led to B12 deficiency, evidenced by impaired conversion of cyano-cobalamin to ado-cobalamin and methyl-cobalamin, decreased methionine synthase activity and reduced S-adenosyl methionine to S-adenosyl homocysteine ratio, as well as increases in homocysteine and methylmalonic acid concentration.Conclusions/Significancethe heterologous expression of TC-O in mammalian cells can be used as an effective strategy for investigating the cellular consequences of vitamin B12 deficiency. More generally, expression of oleosin-anchored proteins could be an interesting tool in cell engineering for studying proteins of pharmacological interest.

Arabidopsis bax inhibitor-1

Unfolded and misfolded proteins in the endoplasmic reticulum (ER) of eukaryotic cells elicit a highly conserved unfolded protein response (UPR) that leads to an increase in the capacity of the ER to deal with protein folding by hightened expression of enzymes such as chaperone and protein disulfide isomerases. However, cells die by apoptosis if the function of the ER cannot be restored in metazoans. To what extent is this mechanism evolutionarily conserved in plant cells remains to be elucidated. Emerging data from our recent study now provide compelling evidence that a conserved cell death suppressor, BAX inhibitor-1 (BI-1), plays a pivotal role as a survival factor against endoplasmic reticulum stress-mediated programmed cell death (PCD) that likely acts in parallel to the UPR pathway. This finding suggests a clear functional correlation to the predicted ER localization of AtBI1 as well as directly implicating the ER of plant cells as an important modulator of cell death activation. Furthermore, ER stress and its associated cell death in plants can be relieved by administration of chemical chaperones which have been clinically used for treatment of many human diseases linked to neurodegenerative disorders that are triggered by the dysfunction of ER homeostasis. This opens the way for future studies to decipher the mechanisms and pathways of ER-mediated PCD, and function of this pathway in plant development and stress response.

AtUTr1, a UDP-glucose/UDP-galactose Transporter from Arabidopsis thaliana, Is Located in the Endoplasmic Reticulum and Up-regulated by the Unfolded Protein Response

The folding of glycoproteins in the endoplasmic reticulum (ER) depends on a quality control mechanism mediated by the calnexin/calreticulin cycle. During this process, continuous glucose trimming and UDP-glucose-dependent re-glucosylation of unfolded glycoproteins takes place. To ensure proper folding, increases in misfolded proteins lead to up-regulation of the components involved in quality control through a process known as the unfolded protein response (UPR). Reglucosylation is catalyzed by the ER lumenal located enzyme UDP-glucose glycoprotein glucosyltransferase, but as UDP-glucose is synthesized in the cytosol, a UDP-glucose transporter is required in the calnexin/calreticulin cycle. Even though such a transporter has been hypothesized, no protein playing this role in the ER yet has been identified. Here we provide evidence that AtUTr1, a UDP-galactose/glucose transporter from Arabidopsis thaliana, responds to stimuli that trigger the UPR increasing its expression around 9-fold. The accumulation of AtUTr1 transcript is accompanied by an increase in the level of the AtUTr1 protein. Moreover, subcellular localization studies indicate that AtUTr1 is localized in the ER of plant cells. We reasoned that an impairment in AtUTr1 expression should perturb the calnexin/calreticulin cycle leading to an increase in misfolded protein and triggering the UPR. Toward that end, we analyzed an AtUTr1 insertional mutant and found an up-regulation of the ER chaperones BiP and calnexin, suggesting that these plants may be constitutively activating the UPR. Thus, we propose that in A. thaliana, AtUTr1 is the UDP-glucose transporter involved in quality control in the ER.

In tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of ER export sites depends on active COPI machinery

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.

Diacidic Motifs Influence the Export of Transmembrane Proteins from the Endoplasmic Reticulum in Plant Cells

In yeast and mammals, amino acid motifs in the cytosolic tails of transmembrane domains play a role in protein trafficking by facilitating export from the endoplasmic reticulum (ER). However, little is known about ER export signals of membrane proteins in plants. Therefore, we investigated the role of diacidic motifs in the ER export of Golgi-localized membrane proteins. We show that diacidic motifs perform a significant function in the export of transmembrane proteins to the Golgi apparatus, as mutations of these signals impede the efficient anterograde transport of multispanning, type II, and type I proteins. Furthermore, we demonstrate that diacidic motifs instigate the export of proteins that reside in the ER due to the lengths of their transmembrane domains. However, not all of the diacidic motifs in the cytosolic tails of the proteins studied were equally important in ER export. Transport of Golgi proteins was disrupted only by mutagenesis of specific diacidic signals, suggesting that the protein environment of these signals affects their function. Our findings indicate that diacidic ER export motifs are present and functional in plant membrane proteins and that they are dominant over transmembrane domain length in determining the export of proteins from the ER in plant cells.