Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in ;70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.

To study the role of the lumenal binding protein (BiP) in the transport and secretion of proteins, we have produced plants with altered BiP levels. Transgenic plants overexpressing BiP showed dramatically increased BiP mRNA levels but only a modest increase in BiP protein levels. The presence of degradation products in BiP overproducers suggests a regulatory mechanism that increases protein turnover when BiP is abundant. Antisense inhibition of BiP synthesis was not successful, demonstrating that even a minor reduction in the basal BiP level is deleterious to cell viability. Overexpression of BiP leads to downregulation of the basal transcript levels of endogenous BiP genes and greatly reduces the unfolded protein response. The data confirm that BiP transcription is regulated via a feedback mechanism that involves monitoring of BiP protein levels. To test BiP activity in vivo, we designed a functional assay, using the secretory protein alpha-amylase and a cytosolic enzyme as a control for cell viability. During tunicamycin treatment, an overall reduction of alpha-amylase synthesis was observed when compared with the cytosolic marker. We show that the tunicamycin effect is due to the depletion of BiP in the endoplasmic reticulum because coexpressed BiP alone is able to restore efficient alpha-amylase synthesis. This is a novel assay to monitor BiP activity in promoting secretory protein synthesis in vivo.

TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.Chloroplasts perform oxygenic photosynthesis in algae and plants and have evolved by endosymbiosis from cyanobacteria. Chloroplasts have two distinct membrane systems, the double envelope surrounding the organelle and an internal membrane system named thylakoids. The envelope membrane represents the interface between the cytoplasm and chloroplast stroma, whereas the thylakoid membrane separates the stroma and the lumenal space. Altogether ϳ800 membrane proteins have been identified by proteomics in the envelope and thylakoid membranes of Arabidopsis thaliana (for reviews, see Refs. 1 and 2). As expected, the main function for the identified envelope proteins was transport of ions and metabolites, whereas photosynthesis was attributed to most of the identified thylakoid proteins. The major protein complexes in thylakoids are photosystems (PS) 4 I and II, the cytochrome b 6 f complex, and the proton-translocating ATP synthase. These photosynthetic complexes contain not only proteins but also pigments and other cofactors. Their assembly, activity, and removal require a large number of auxiliary, regulatory, and transport proteins (3, 4). Many biochemical reports pointed to the existence of transport activities in the thylakoid membrane, such as calcium transport (5), copper transport (6), anion channels (7), cation channels (8, 9), and nucleotide transport (10). Only the thylakoid copper transporter was identified at the genetic level in Arabidopsis (11). No hydrophobic proteins related to the above-mentioned transport activities were identified in the previous proteomic works on Arabidopsis thylakoid membranes (for a review, see Ref.2). Therefore, genetic strategies are required for identification and elucidation of their role in optimal function of the thylakoid.ATP is produced during the light-dependent photosynthetic reactions on the stromal side of the thylakoid membrane. Besides its utilization during CO 2 fixation in the stroma, ATP drives many energy-dependent processes in thylakoids, including protein phosphorylation, folding, import, and degradation.

We have studied the possible mechanisms of endoplasmic reticulum (ER) export and retention by using natural residents of the plant ER. Under normal physiological conditions, calreticulin and the lumenal binding protein (BiP) are efficiently retained in the ER. When the ER retention signal is removed, truncated calreticulin is much more rapidly secreted than truncated BiP. Calreticulin carries two glycans of the typical ER high-mannose form. Both glycans are competent for Golgi-based modifications, as determined from treatment with brefeldin A or based on the deletion of the ER retention motif. In contrast to BiP, calreticulin accumulation is strongly dependent on its retention signal, thereby allowing us to test whether saturation of the retention mechanism is possible. Overexpression of calreticulin led to 100-fold higher levels in dilated globular ER cisternae as well as dilated nuclear envelopes and partial secretion of both BiP and calreticulin. This result shows that both molecules are competent for ER export and supports the concept that proteins are secreted by default. This result also supports previous data suggesting that truncated BiP devoid of its retention motif can be retained in the ER by association with calreticulin. Moreover, even under these saturating conditions, cellular calreticulin did not carry significant amounts of complex glycans, in contrast to secreted calreticulin. This result shows that calreticulin is rapidly secreted once complex glycans have been synthesized in the medial/ trans Golgi apparatus and that the modified protein does not appear to recycle back to the ER. INTRODUCTIONResident chaperones and folding enzymes, termed reticuloplasmins, constitute the majority of proteins in the lumen of the endoplasmic reticulum (ER) in most cell types. In contrast, newly synthesized secretory and vacuolar proteins usually are in the minority. The mechanism by which the cell ensures efficient export of secretory proteins to the Golgi apparatus while maintaining high levels of soluble reticuloplasmins in the ER lumen has been the subject of intense investigation during the past 15 years (Pelham, 1995; Hong, 1998).The model by which soluble proteins devoid of sorting information exit the cell by default (Denecke et al., 1990; Hunt and Chrispeels, 1991; reviewed in Vitale and Denecke, 1999) implies that all molecules can diffuse freely into and out of nascent anterograde transport vesicles formed by the ER. Therefore, it has been assumed that ER resident proteins, such as the binding protein (BiP), can escape from the ER by diffusion into anterograde transport vesicles. A conserved C-terminal motif (mainly HDEL and KDEL in plants; reviewed in Vitale et al., 1993), which is recognized in the Golgi apparatus, is responsible for the retrieval of these proteins by the transmembrane receptor ERD2 (for ER retention defective 2; Lewis and Pelham, 1992). This recycling model is required to explain how a relatively small number of receptors could mediate retention of a large number of ligands wi...

Using pulse-chase experiments combined with immunoprecipitation and N-glycan structural analysis, we showed that the retrieval mechanism of proteins from post-endoplasmic reticulum (post-ER) compartments is active in plant cells at levels similar to those described previously for animal cells. For instance, recycling from the Golgi apparatus back to the ER is sufficient to block the secretion of as much as 90% of an extracellular protein such as the cell wall invertase fused with an HDEL C-terminal tetrapeptide. Likewise, recycling can sustain fast retrograde transport of Golgi enzymes into the ER in the presence of brefeldin A. However, on the basis of our data, we propose that this retrieval mechanism in plants has little impact on the ER retention of a soluble ER protein such as calreticulin. Indeed, the latter is retained in the ER without any N-glycan-related evidence for a recycling through the Golgi apparatus. Taken together, these results indicate that calreticulin and perhaps other plant reticuloplasmins are possibly largely excluded from vesicles exported from the ER. Instead, they are probably retained in the ER by mechanisms that rely primarily on signals other than H/KDEL motifs. INTRODUCTIONIn eukaryotic cells, most proteins that reside in the endoplasmic reticulum (ER) contain information in their primary structure that determines their subcellular localization. Keeping these proteins in the ER can be achieved either by strict retention in this particular compartment or by retrieval and retrograde transport back to the ER when they leave the ER (reviewed in Gomord et al., 1999;Pagny et al., 1999; Vitale and Denecke, 1999). For soluble ER resident proteins or soluble reticuloplasmins, ER residency depends largely on the presence of the specific tetrapeptide H/KDEL-or closely related sequences-at their C termini (Munro and Pelham, 1987;Andres et al., 1990). Use of different tetrapeptide sequences fused to non-ER secretory proteins as reporter probes has shown that these signals are sufficient for retention of soluble proteins in the ER (Rose and Doms, 1988;Herman et al., 1990; Denecke et al., 1992; Wandelt et al., 1992; Boevink et al., 1996;Gomord et al., 1997). Moreover, for yeast and mammalian cells, the H/KDEL-dependent retention mechanism has been proposed to promote a continual recycling of ER resident proteins whenever they exit the ER and are secreted into a post-ER compartment (Pelham, 1988).Arguing for such a recycling of ER proteins is the observation that reporter glycoproteins fused with H/KDEL motifs undergo specific N-glycan modifications. Indeed, the latter are markers of post-ER events. For example, in animal cells, the lysosomal glycoprotein cathepsin D, when modified by the addition of a KDEL C-terminal extension, accumulates in the ER even though it carries N-glycans containing the N -acetylglucosamine 1-phosphate residues that are typical of passage through the cis Golgi compartment (Pelham, 1988). Some post-ER glycan maturations have also been found on reporter glycoproteins ...

Using pulse-chase experiments combined with immunoprecipitation and N-glycan structural analysis, we showed that the retrieval mechanism of proteins from post-endoplasmic reticulum (post-ER) compartments is active in plant cells at levels similar to those described previously for animal cells. For instance, recycling from the Golgi apparatus back to the ER is sufficient to block the secretion of as much as 90% of an extracellular protein such as the cell wall invertase fused with an HDEL C-terminal tetrapeptide. Likewise, recycling can sustain fast retrograde transport of Golgi enzymes into the ER in the presence of brefeldin A. However, on the basis of our data, we propose that this retrieval mechanism in plants has little impact on the ER retention of a soluble ER protein such as calreticulin. Indeed, the latter is retained in the ER without any N-glycan-related evidence for a recycling through the Golgi apparatus. Taken together, these results indicate that calreticulin and perhaps other plant reticuloplasmins are possibly largely excluded from vesicles exported from the ER. Instead, they are probably retained in the ER by mechanisms that rely primarily on signals other than H/KDEL motifs.

To study the role of the lumenal binding protein (BiP) in the transport and secretion of proteins, we have produced plants with altered BiP levels. Transgenic plants overexpressing BiP showed dramatically increased BiP mRNA levels but only a modest increase in BiP protein levels. The presence of degradation products in BiP overproducers suggests a regulatory mechanism that increases protein turnover when BiP is abundant. Antisense inhibition of BiP synthesis was not successful, demonstrating that even a minor reduction in the basal BiP level is deleterious to cell viability. Overexpression of BiP leads to downregulation of the basal transcript levels of endogenous BiP genes and greatly reduces the unfolded protein response. The data confirm that BiP transcription is regulated via a feedback mechanism that involves monitoring of BiP protein levels. To test BiP activity in vivo, we designed a functional assay, using the secretory protein alpha-amylase and a cytosolic enzyme as a control for cell viability. During tunicamycin treatment, an overall reduction of alpha-amylase synthesis was observed when compared with the cytosolic marker. We show that the tunicamycin effect is due to the depletion of BiP in the endoplasmic reticulum because coexpressed BiP alone is able to restore efficient alpha-amylase synthesis. This is a novel assay to monitor BiP activity in promoting secretory protein synthesis in vivo.

The Arabidopsis thaliana Tonoplast Intrinsic Protein 1;1 (AtTIP1;1) is a member of the tonoplast aquaporin family. The tissue-specific expression pattern and intracellular localization of AtTIP1;1 were characterized using GUS and GFP fusion genes. Results indicate that AtTIP1;1 is expressed in almost all cell types with the notable exception of meristematic cells. The highest level of AtTIP1;1 expression was detected in vessel-flanking cells in vascular bundles. AtTIP1;1-GFP fusion protein labelled the tonoplast of the central vacuole and other smaller peripheral vacuoles. The fusion protein was not found evenly distributed along the tonoplast continuum but concentrated in contact zones of tonoplasts from adjacent vacuoles and in invaginations of the central vacuole. Such invaginations may result from partially engulfed small vacuoles. A knockout mutant was isolated and characterized to gain insight into AtTIP1;1 function. No phenotypic alteration was found under optimal growth conditions indicating that AtTIP1;1 function is not essential to the plant and that some members of the TIP family may act redundantly to facilitate water flow across the tonoplast. However, a conditional root phenotype was observed when mutant plants were grown on a glycerol-containing medium.