Brefeldin A (BFA) causes a block in the secretory system of eukaryotic cells by inhibiting vesicle formation at the Golgi apparatus. Although this toxin has been used in many studies, its effects on plant cells are still shrouded in controversy. We have reinvestigated the early responses of plant cells to BFA with novel tools, namely, tobacco Bright Yellow 2 (BY-2) suspension-cultured cells expressing an in vivo green fluorescent protein-Golgi marker, electron microscopy of high-pressure frozen/freeze-substituted cells, and antisera against At ␥ -COP, a component of COPI coats, and AtArf1, the GTPase necessary for COPI coat assembly. The first effect of 10 g/mL BFA on BY-2 cells was to induce in Ͻ 5 min the complete loss of vesicle-forming At ␥ -COP from Golgi cisternae. During the subsequent 15 to 20 min, this block in Golgi-based vesicle formation led to a series of sequential changes in Golgi architecture, the loss of distinct Golgi stacks, and the formation of an endoplasmic reticulum (ER)-Golgi hybrid compartment with stacked domains. These secondary effects appear to depend in part on stabilizing intercisternal filaments and include the continued maturation of cis -and medial cisternae into trans -Golgi cisternae, as predicted by the cisternal progression model, the shedding of trans -Golgi network cisternae, the fusion of individual Golgi cisternae with the ER, and the formation of large ER-Golgi hybrid stacks. Prolonged exposure of the BY-2 cells to BFA led to the transformation of the ER-Golgi hybrid compartment into a sponge-like structure that does not resemble normal ER. Thus, although the initial effects of BFA on plant cells are the same as those described for mammalian cells, the secondary and tertiary effects have drastically different morphological manifestations. These results indicate that, despite a number of similarities in the trafficking machinery with other eukaryotes, there are fundamental differences in the functional architecture and properties of the plant Golgi apparatus that are the cause for the unique responses of the plant secretory pathway to BFA. INTRODUCTIONThe fungal toxin brefeldin A (BFA) has been used as an experimental tool to block secretion in a wide variety of eukaryotic cells (Fujiwara et al., 1988;Satiat-Jeunemaitre et al., 1996). In plants, it has been shown to block the secretion of cell wall polysaccharides and proteins (Driouich et al., 1993;Schindler et al., 1994;Kunze et al., 1995) as well as the transport of soluble proteins to the vacuole (Holwerda et al., 1992;Gomez and Chrispeels, 1993). In animals, the BFA-induced block of secretion is accompanied by a structural disruption of the secretory system, most notably involving the Golgi apparatus, which becomes extensively tubulated and eventually fuses with the endoplasmic reticulum (ER) (Sciaky et al., 1997;Hess et al., 2000). In contrast, the responses of plant cells to BFA range from a loss of ciscisternae and a concomitant increase in trans -cisternae to the accumulation of Golgi membranes in highly vesicul...

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein (GFP) (smGFP) with the pH-sensing capability of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin (plant-solubility-modified ecliptic pHluorin) gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin (plant-solubility-modified ratiomatric pHluorin), a dual-excitation sensor, allowing for precise measurements. Compartment-specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar (pH 7.3 and 7.2), while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum (ER) to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network (TGN) and multivesicular body (MVB) is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H(+)-ATPase (V-ATPase) with concanamycin A (ConcA) caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.

The plant trans-Golgi network/early endosome (TGN/EE) is a major hub for secretory and endocytic trafficking with complex molecular mechanisms controlling sorting and transport of cargo. Vacuolar transport from the TGN/EE to multivesicular bodies/late endosomes (MVBs/LEs) is assumed to occur via clathrin-coated vesicles, although direct proof for their participation is missing. Here, we present evidence that post-TGN transport toward lytic vacuoles occurs independently of clathrin and that MVBs/LEs are derived from the TGN/EE through maturation. We show that the V-ATPase inhibitor concanamycin A significantly reduces the number of MVBs and causes TGN and MVB markers to colocalize in Arabidopsis thaliana roots. Ultrastructural analysis reveals the formation of MVBs from the TGN/EE and their fusion with the vacuole. The localization of the ESCRT components VPS28, VPS22, and VPS2 at the TGN/EE and MVBs/LEs indicates that the formation of intraluminal vesicles starts already at the TGN/EE. Accordingly, a dominant-negative mutant of VPS2 causes TGN and MVB markers to colocalize and blocks vacuolar transport. RNA interference-mediated knockdown of the annexin ANNAT3 also yields the same phenotype. Together, these data indicate that MVBs originate from the TGN/EE in a process that requires the action of ESCRT for the formation of intraluminal vesicles and annexins for the final step of releasing MVBs as a transport carrier to the vacuole.

We have studied the transport of soluble cargo molecules by inhibiting specific transport steps to and from the Golgi apparatus. Inhibition of export from the Golgi via coexpression of a dominant-negative GTP-restricted ARF1 mutant (Q71L) inhibits the secretion of ␣ -amylase and simultaneously induces the secretion of the vacuolar protein phytepsin to the culture medium. By contrast, specific inhibition of endoplasmic reticulum export via overexpression of Sec12p or coexpression of a GTP-restricted form of Sar1p inhibits the anterograde transport of either cargo molecule in a similar manner. Increased secretion of the vacuolar protein was not observed after incubation with the drug brefeldin A or after coexpression of the GDP-restricted mutant of ARF1 (T31N). Therefore, the differential effect of inducing the secretion of one cargo molecule while inhibiting the secretion of another is dependent on the GTP hydrolysis by ARF1p and is not caused by a general inhibition of Golgi-derived COPI vesicle traffic. Moreover, we demonstrate that GTP-restricted ARF1-stimulated secretion is observed only for cargo molecules that are expected to be sorted in a BP80-dependent manner, exhibiting sequence-specific, context-independent, vacuolar sorting signals. Induced secretion of proteins carrying C-terminal vacuolar sorting signals was not observed. This finding suggests that ARF1p influences the BP80-mediated transport route to the vacuole in addition to transport steps of the default secretory pathway to the cell surface.

COPII-coated vesicles, first identified in yeast and later characterized in mammalian cells, mediate protein export from the endoplasmic reticulum (ER) to the Golgi apparatus within the secretory pathway. In these organisms, the mechanism of vesicle formation is well understood, but the process of soluble cargo sorting has yet to be resolved. In plants, functional complements of the COPII-dependent protein traffic machinery were identified almost a decade ago, but the selectivity of the ER export process has been subject to considerable debate. To study the selectivity of COPII-dependent protein traffic in plants, we have developed an in vivo assay in which COPII vesicle transport is disrupted at two distinct steps in the pathway. First, overexpression of the Sar1p-specific guanosine nucleotide exchange factor Sec12p was shown to result in the titration of the GTPase Sar1p, which is essential for COPII-coated vesicle formation. A second method to disrupt COPII transport at a later step in the pathway was based on coexpression of a dominant negative mutant of Sar1p (H74L), which is thought to interfere with the uncoating and subsequent membrane fusion of the vesicles because of the lack of GTPase activity. A quantitative assay to measure ER export under these conditions was achieved using the natural secretory protein barley ␣ -amylase and a modified version carrying an ER retention motif. Most importantly, the manipulation of COPII transport in vivo using either of the two approaches allowed us to demonstrate that export of the ER resident protein calreticulin or the bulk flow marker phosphinothricin acetyl transferase is COPII dependent and occurs at a much higher rate than estimated previously. We also show that the instability of these proteins in post-ER compartments prevents the detection of the true rate of bulk flow using a standard secretion assay. The differences between the data on COPII transport obtained from these in vivo experiments and in vitro experiments conducted previously using yeast components are discussed. INTRODUCTIONIn yeast and mammalian cells, the export of proteins from the endoplasmic reticulum (ER) occurs in COPII-coated vesicles. The process of COPII vesicle formation is well understood and can be reconstituted using purified yeast components (Barlowe et al., 1994). Vesicle formation in vitro depends solely on the ER donor membrane, the GTPase Sar1p, the Sar1p-specific guanosine nucleotide exchange factor Sec12p, the cytosolic COPII coat components, and GTP (Barlowe et al., 1994). Considerably less is known about the sorting of soluble cargo molecules during ER export in eukaryotes (Klumperman, 2000), and conflicting reports from the plant field have contributed to the ongoing discussions (Crofts et al., 1999;Gomord and Faye, 2000;Pagny et al., 2000;.To support protein synthesis and folding, the ER lumen maintains high levels of soluble residents such as the lumenal binding protein (BiP), protein disulfide isomerase, or calreticulin. The concentration of nonresidents in the ER lume...

Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells. Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach. Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus. COPI-coated buds primarily reside on the cis -face of the Golgi stack. In addition, COPI and Arf1p show predominant labeling of the cis -Golgi stack, gradually diminishing toward the trans -Golgi stack. In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/ Golgi membranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism. In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley ␣ -amylase-HDEL yielded a COPI-coated vesicle fraction that contained ␣ -amylase as well as calreticulin. INTRODUCTIONProtein trafficking in the secretory and endocytotic pathways is facilitated by coated vesicles (Rothman and Wieland, 1996;Robinson et al., 1998). Research on mammalian and yeast cells has established that two different types of non-clathrin-coated vesicles are responsible for transport events occurring between the endoplasmic reticulum (ER) and the Golgi apparatus and for intra-Golgi transport. Whereas investigators generally agree that COPIIcoated vesicles bud from the ER and transport proteins in the anterograde direction (Barlowe et al., 1994;Bannykh and Balch, 1998), the function of COPI-coated vesicles remains controversial. Strong evidence suggests that COPIcoated vesicles are responsible for the recycling of escaped ER-resident proteins from post-ER compartments (Letourneur et al., 1994), but data have also been presented supporting a role in anterograde transport of proteins, especially within the Golgi stack (Orci et al., 1997;Lippincott-Schwartz et al., 1998).The formation of both types of COP-coated vesicles involves the recruitment of a coat protein complex (coatomer for COPI-coated vesicles, Sec23/24 and Sec13/31 dimers for COPII-coated vesicles) by a membrane-associated GTP binding protein (Arf1p and Sar1p for COPI and COPII, respectively; Schekman and Orci, 1996). Both Arf1p and Sar1p exist in the cytosol as GDP-bound forms and become converted to the GTP form through the action of a membrane-associated exchange factor, guanine nucleotide exchange factor (Helms and Rothman, 1992). For Sar1p, this is Sec12p, a transmembrane protein of the ER ; for Arf1p, the most likely candidates are Gea1/2p and ARNO (ARF nucleotide binding site opener; ...

SUMMARYReceptor-mediated sorting processes in the secretory pathway of eukaryotic cells rely on mechanisms to recycle the receptors after completion of transport. Based on this principle, plant vacuolar sorting receptors (VSRs) are thought to recycle after dissociating of receptor-ligand complexes in a pre-vacuolar compartment. This recycling is mediated by retromer, a cytosolic coat complex that comprises sorting nexins and a large heterotrimeric subunit. To analyse retromer-mediated VSR recycling, we have used a combination of immunoelectron and fluorescence microscopy to localize the retromer components sorting nexin 1 (SNX1) and sorting nexin 2a (SNX2a) and the vacuolar sorting protein VPS29p. All retromer components localize to the trans-Golgi network (TGN), which is considered to represent the early endosome of plants. In addition, we show that inhibition of retromer function in vivo by expression of SNX1 or SNX2a mutants as well as transient RNAi knockdown of all sorting nexins led to accumulation of the VSR BP80 at the TGN. Quantitative protein transport studies and live-cell imaging using fluorescent vacuolar cargo molecules revealed that arrival of these VSR ligands at the vacuole is not affected under these conditions. Based on these findings, we propose that the TGN is the location of retromer-mediated recycling of VSRs, and that transport towards the lytic vacuole downstream of the TGN is receptor-independent and occurs via maturation, similar to transition of the early endosome into the late endosome in mammalian cells.

COPII-coated vesicles, first identified in yeast and later characterized in mammalian cells, mediate protein export from the endoplasmic reticulum (ER) to the Golgi apparatus within the secretory pathway. In these organisms, the mechanism of vesicle formation is well understood, but the process of soluble cargo sorting has yet to be resolved. In plants, functional complements of the COPII-dependent protein traffic machinery were identified almost a decade ago, but the selectivity of the ER export process has been subject to considerable debate. To study the selectivity of COPII-dependent protein traffic in plants, we have developed an in vivo assay in which COPII vesicle transport is disrupted at two distinct steps in the pathway. First, overexpression of the Sar1p-specific guanosine nucleotide exchange factor Sec12p was shown to result in the titration of the GTPase Sar1p, which is essential for COPII-coated vesicle formation. A second method to disrupt COPII transport at a later step in the pathway was based on coexpression of a dominant negative mutant of Sar1p (H74L), which is thought to interfere with the uncoating and subsequent membrane fusion of the vesicles because of the lack of GTPase activity. A quantitative assay to measure ER export under these conditions was achieved using the natural secretory protein barley ␣-amylase and a modified version carrying an ER retention motif. Most importantly, the manipulation of COPII transport in vivo using either of the two approaches allowed us to demonstrate that export of the ER resident protein calreticulin or the bulk flow marker phosphinothricin acetyl transferase is COPII dependent and occurs at a much higher rate than estimated previously. We also show that the instability of these proteins in postER compartments prevents the detection of the true rate of bulk flow using a standard secretion assay. The differences between the data on COPII transport obtained from these in vivo experiments and in vitro experiments conducted previously using yeast components are discussed.