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...

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; ...

Developing pea cotyledons contain functionally different vacuoles, a protein storage vacuole and a lytic vacuole. Lumenal as well as membrane proteins of the protein storage vacuole exit the Golgi apparatus in dense vesicles rather than in clathrin-coated vesicles (CCVs). Although the sorting receptor for vacuolar hydrolases BP-80 is present in CCVs, it is not detectable in dense vesicles. To localize these different vacuolar sorting events in the Golgi, we have compared the distribution of vacuolar storage proteins and of α-TIP, a membrane protein of the protein storage vacuole, with the distribution of the vacuolar sorting receptor BP-80 across the Golgi stack. Analysis of immunogold labeling from cryosections and from high pressure frozen samples has revealed a steep gradient in the distribution of the storage proteins within the Golgi stack. Intense labeling for storage proteins was registered for the cis-cisternae, contrasting with very low labeling for these antigens in the trans-cisternae. The distribution of BP-80 was the reverse, showing a peak in the trans-Golgi network with very low labeling of the cis-cisternae. These results indicate a spatial separation of different vacuolar sorting events in the Golgi apparatus of developing pea cotyledons.

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.