Giovanni Stefano

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.

Assessing the role of root plasma membrane and tonoplast Na + /H + exchangers in salinity tolerance in wheat: in planta quantification methods

This work investigates the role of cytosolic Na+ exclusion in roots as a means of salinity tolerance in wheat, and offers in planta methods for the functional assessment of major transporters contributing to this trait. An electrophysiological protocol was developed to quantify the activity of plasma membrane Na+ efflux systems in roots, using the microelectrode ion flux estimation (MIFE) technique. We show that active efflux of Na+ from wheat root epidermal cells is mediated by a SOS1-like homolog, energized by the plasma membrane H+-ATPase. SOS1-like efflux activity was highest in Kharchia 65, a salt-tolerant bread wheat cultivar. Kharchia 65 also had an enhanced ability to sequester large quantities of Na+ into the vacuoles of root cells, as revealed by confocal microscopy using Sodium Green. These findings were consistent with the highest level of expression of both SOS1 and NHX1 transcripts in plant roots in this variety. In the sensitive wheat varieties, a greater proportion of Na+ was located in the root cell cytosol. Overall, our findings suggest a critical role of cytosolic Na+ exclusion for salinity tolerance in wheat and offer convenient protocols to quantify the contribution of the major transporters conferring this trait, to screen plants for salinity tolerance.

Spatiotemporal dynamics of the electrical network activity in the root apex

The study of electrical network systems, integrated with chemical signaling networks, is becoming a common trend in contemporary biology. Classical techniques are limited to the assessment of signals from doublets or triplets of cells at a fixed temporal bin width. At present, full characteristics of the electrical network distribution and dynamics in plant cells and tissues has not been established. Here, a 60-channels multielectrode array (MEA) is applied to study spatiotemporal characteristics of the electrical network activity of the root apex. Both intense spontaneous electrical activities and stimulation-elicited bursts of locally propagating electrical signals have been observed. Propagation of the spikes indicates the existence of excitable traveling waves in plants, similar to those observed in non-nerve electrogenic tissues of animals. Obtained data reveal synchronous electric activities of root cells emerging in a specific root apex region. The dynamic electrochemical activity of root apex cells is proposed to continuously integrate internal and external signaling for developmental adaptations in a changing environment.

Transorganellar complementation redefines the biochemical continuity of endoplasmic reticulum and chloroplasts

Tocopherols are nonpolar compounds synthesized and localized in plastids but whose genetic elimination specifically impacts fatty acid desaturation in the endoplasmic reticulum (ER), suggesting a direct interaction with ER-resident enzymes. To functionally probe for such interactions, we developed transorganellar complementation, where mutated pathway activities in one organelle are experimentally tested for substrate accessibility and complementation by active enzymes retargeted to a companion organelle. Mutations disrupting three plastid-resident activities in tocopherol and carotenoid synthesis were complemented from the ER in this fashion, demonstrating transorganellar access to at least seven nonpolar, plastid envelope-localized substrates from the lumen of the ER, likely through plastid:ER membrane interaction domains. The ability of enzymes in either organelle to access shared, nonpolar plastid metabolite pools redefines our understanding of the biochemical continuity of the ER and chloroplast with profound implications for the integration and regulation of organelle-spanning pathways that synthesize nonpolar metabolites in plants.

Arabidopsis RHD3 mediates the generation of the tubular ER network and is required for Golgi distribution and motility in plant cells

In plant cells, the endoplasmic reticulum (ER) and Golgi apparatus form a unique system in which single Golgi stacks are motile and in close association with the underlying ER tubules. Arabidopsis has three RHD3 (ROOT HAIR DEFECTIVE 3) isoforms that are analogous to the mammalian atlastin GTPases involved in shaping ER tubules. We used live-cell imaging, genetic complementation, split ubiquitin assays and western blot analyses in Arabidopsis and tobacco to show that RHD3 mediates the generation of the tubular ER network and is required for the distribution and motility of Golgi stacks in root and leaf epidermal cells. We established that RHD3 forms homotypic interactions at ER punctae. In addition, the activity of RHD3 on the tubular ER is specifically correlated with the cellular distribution and motility of Golgi stacks because ER to Golgi as well as Golgi to plasma membrane transport was not affected by RHD3 mutations in the conserved GDP/GTP motifs. We found a possible partial redundancy within the RHD3 isoforms in Arabidopsis. However, yeast Sey1p, a functional atlastin homologue, and RHD3 are not interchangeable in complementing the respective loss-of-function mutants, suggesting that the molecular mechanisms controlling ER tubular morphology might not be entirely conserved among eukaryotic lineages.

Identification and characterization of AtCASP, a plant transmembrane Golgi matrix protein

Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin. Similar to its homologues, the plant CASP contains a long N-terminal coiled-coil region protruding into the cytosol and a C-terminal transmembrane domain with amino acid residues which are highly conserved across species. Through fluorescent protein tagging experiments, we show that plant CASP localizes at the plant Golgi apparatus and that the C-terminus of this protein is sufficient for its localization, as has been shown for its mammalian counterpart. In addition, we demonstrate that the plant CASP is able to localize at the mammalian Golgi apparatus. However, mutagenesis of a conserved tyrosine in the transmembrane domain revealed that it is necessary for ER export and Golgi localization of the Arabidopsis CASP in mammalian cells, but is not required for its correct localization in plant cells. These data suggest that mammalian and plant cells have different mechanisms for concentrating CASP in the Golgi apparatus.

Crossing the Divide – Transport Between the Endoplasmic Reticulum and Golgi Apparatus in Plants

The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI‐ and COPII‐mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented. We summarize current knowledge of these protein transport routes, as well as highlighting key differences between those of plant systems and those of mammals and yeast. These differences have necessitated the study of plant‐specific aspects of protein transport in the early secretory pathway, and this review discusses recent developments in this area. Advances in live‐cell‐imaging technology have allowed the observation of protein movement in vivo, giving a new insight into many of the processes involved in vesicle formation and protein trafficking. The use of these new technologies has been combined with more traditional methods, such as protein biochemistry and electron microscopy, to increase our understanding of the transport routes in the cell.

Multiple Roles of ADP-Ribosylation Factor 1 in Plant Cells Include Spatially Regulated Recruitment of Coatomer and Elements of the Golgi Matrix

Recent evidence indicates that ADP-ribosylation factor 1 (ARF1) carries out multiple roles in plant cells that may be independent from the established effector complex COPI. To investigate potential COPI-independent functions, we have followed the dynamics of ARF1 and a novel putative effector, the plant golgin GRIP-related ARF-binding domain-containing Arabidopsis (Arabidopsis thaliana) protein 1 (GDAP1) in living plant cells. We present data that ascribe a new role to ARF1 in plant cell membrane traffic by showing that the GTPase functions to recruit GDAP1 to membranes. In addition, although ARF1 appears to be central to the recruitment of both COPI components and the golgin, we have established a different subcellular distribution of these ARF1 effectors. Live cell imaging demonstrates that GDAP1 and COPI are distributed on Golgi membranes. However, GDAP1 is also found on ARF1-labeled structures that lack coatomer, suggesting that the membrane environment, rather than ARF1 alone, influences the differential recruitment of ARF1 effectors. In support of this hypothesis, fluorescence recovery after photobleaching analyses demonstrated that GDAP1 and COPI have different kinetics on membranes during the cycle of activation and inactivation of ARF1. Therefore, our data support a model where modulation of the cellular functions of ARF1 in plant cells encompasses not only the intrinsic activities of the effectors, but also differential recruitment onto membranes that is spatially regulated.

In Arabidopsis, the spatial and dynamic organization of the endoplasmic reticulum and Golgi apparatus is influenced by the integrity of the C‐terminal domain of RHD3, a non‐essential GTPase

The mechanisms underlying the organization and dynamics of plant endomembranes are largely unknown. Arabidopsis RHD3, a distant member of the dynamin superfamily, has recently been implicated in plant ER morphology and Golgi movement through analyses of dominant-negative mutants of the putative GTPase domain in a heterologous system. Whether RHD3 is indispensable for ER architecture and what role regions other than the putative GTPase domain play in RHD3 function are unanswered questions. Here we characterized an EMS mutant, gom8, with disrupted Golgi movement and positioning and compromised ER shape and dynamics. gom8 mapped to a missense mutation in the RHD3 hairpin loop domain, causing accumulation of the mutant protein into large structures, a markedly different distribution compared with wild-type RHD3 over the ER network. Despite the GOM8 distribution, tubules fused in the peripheral ER of the gom8 mutant. These data imply that integrity of the hairpin region is important for the subcellular distribution of RHD3, and that reduced availability of RHD3 over the ER can cause ER morphology defects, but does not prevent peripheral fusion between tubules. This was confirmed by evidence that gom8 was phenocopied in an RHD3 null background. Furthermore, we established that the region encompassing the RHD3 hairpin domain and the C-terminal cytosolic domain is necessary for RHD3 function. We conclude that RHD3 is important in ER morphology, but is dispensable for peripheral ER tubulation in an endogenous context, and that its activity relies on the C-terminal region in addition to the GTPase domain.

COPII-mediated traffic in plants

The secretory pathway encloses functionally interlinked organelles for the synthesis and deposition of most of the building blocks of eukaryotic cells, such as lipids, proteins and sugars. The coat protein complex II (COPII) is a specialized protein complex for the transport between secretory organelles, specifically from the endoplasmic reticulum (ER) to the Golgi apparatus. This review focuses on the developments on COPII research in the plant system. Here, we address the most recent advances in the distribution and regulation of ER-to-Golgi protein transport intermediates and functional analyses of COPII isoforms. New studies support that such isoforms might not be functionally redundant and that they might have unanticipated roles in maintaining the integrity of the ER.