Characterization of Cop I Coat Proteins in Plant Cells

Contreras, Inmaculada; Ortiz-Zapater, Elena; Castilho, Luis Miguel; Aniento, Fernando

doi:10.1006/bbrc.2000.2918

Cited by 29 publications

(24 citation statements)

References 46 publications

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“…Despite these clear morphological differences in the early secretory pathway, COPI/COPII vesiculating machineries appear to be quite conserved amongst the various eukaryotic cell types. Thus, COPI homologs can be found in the Arabidopsis database (Andreeva et al, 1998), and some have been identified in plant extracts (Movafeghi et al, 1999;Contreras et al, 2000Contreras et al, , 2004. The presence of COPI proteins at the surface of vesicles budding from the periphery of Golgi cisternae has also been demonstrated by immunolabeling at both light (Ritzenthaler et al, 2002a) and electron microscopy levels (Pimpl et al, 2000).…”

Section: Introductionmentioning

confidence: 98%

Dynamics of COPII Vesicles and the Golgi Apparatus in CulturedNicotiana tabacumBY-2 Cells Provides Evidence for Transient Association of Golgi Stacks with Endoplasmic Reticulum Exit Sites

et al. 2005

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Despite the ubiquitous presence of the COPI, COPII, and clathrin vesicle budding machineries in all eukaryotes, the organization of the secretory pathway in plants differs significantly from that in yeast and mammalian cells. Mobile Golgi stacks and the lack of both transitional endoplasmic reticulum (ER) and a distinct ER-to-Golgi intermediate compartment are the most prominent distinguishing morphological features of the early secretory pathway in plants. Although the formation of COPI vesicles at periphery of Golgi cisternae has been demonstrated in plants, exit from the ER has been difficult to visualize, and the spatial relationship of this event is now a matter of controversy. Using tobacco (Nicotiana tabacum) BY-2 cells, which represent a highly active secretory system, we have used two approaches to investigate the location and dynamics of COPII binding to the ER and the relationship of these ER exit sites (ERES) to the Golgi apparatus. On the one hand, we have identified endogenous COPII using affinity purified antisera generated against selected COPIIcoat proteins (Sar1, Sec13, and Sec23); on the other hand, we have prepared a BY-2 cell line expressing Sec13:green fluorescent protein (GFP) to perform live cell imaging with red fluorescent protein-labeled ER or Golgi stacks. COPII binding to the ER in BY-2 cells is visualized as fluorescent punctate structures uniformly distributed over the surface of the ER, both after antibody staining as well as by Sec13:GFP expression. These structures are smaller and greatly outnumber the Golgi stacks. They are stationary, but have an extremely short half-life (<10 s). Without correlative imaging data on the export of membrane or lumenal ER cargo it was not possible to equate unequivocally these COPII binding loci with ERES. When a GDP-fixed Sar1 mutant is expressed, ER export is blocked and the visualization of COPII binding is perturbed. On the other hand, when secretion is inhibited by brefeldin A, COPII binding sites on the ER remain visible even after the Golgi apparatus has been lost. Live cell imaging in a confocal laser scanning microscope equipped with spinning disk optics allowed us to investigate the relationship between mobile Golgi stacks and COPII binding sites. As they move, Golgi stacks temporarily associated with COPII binding sites at their rims. Golgi stacks were visualized with their peripheries partially or fully occupied with COPII. In the latter case, Golgi stacks had the appearance of a COPII halo. Slow moving Golgi stacks tended to have more peripheral COPII than faster moving ones. However, some stationary Golgi stacks entirely lacking COPII were also observed. Our results indicate that, in a cell type with highly mobile Golgi stacks like tobacco BY-2, the Golgi apparatus is not continually linked to a single ERES. By contrast, Golgi stacks associate intermittently and sometimes concurrently with several ERES as they move.

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Section: Introductionmentioning

confidence: 98%

Dynamics of COPII Vesicles and the Golgi Apparatus in CulturedNicotiana tabacumBY-2 Cells Provides Evidence for Transient Association of Golgi Stacks with Endoplasmic Reticulum Exit Sites

et al. 2005

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“…Evidence for the existence of COPI in plants was demonstrated using cell fractionation, biochemical reconstitution assays, and electron microscopy (Movafeghi et al, 1999;Contreras et al, 2000;Pimpl et al, 2000). The GTPase ARF1p was shown to influence ER-to-Golgi transport as well as Golgi-derived transport to the plasma membrane, dependent on the cargo molecule (Lee et al, 2002;Takeuchi et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

The GTPase ARF1p Controls the Sequence-Specific Vacuolar Sorting Route to the Lytic Vacuole

et al. 2003

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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.

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“…However, proteins with analogous functions have not yet been identified in plants. Plant homologs of components of the COPI and COPII vesicle coats have been identified and some characterization studies have been carried out (d'Enfert et al, 1992;Regad et al, 1993;Bar-Peled and Raikhel, 1997;Takeuchi et al, 1998;Movafeghi et al, 1999;Contreras et al, 2000;Pimpl et al, 2000;Vernoud et al, 2003;daSilva et al, 2004;Yang et al, 2005;Stefano et al, 2006). Although this area of research is currently one of great interest and activity, knowledge of transport in the early secretory pathway of plants still lags behind that from yeast and mammalian systems.…”

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confidence: 99%

De Novo Formation of Plant Endoplasmic Reticulum Export Sites Is Membrane Cargo Induced and Signal Mediated

et al. 2007

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The plant endoplasmic reticulum (ER) contains functionally distinct subdomains at which cargo molecules are packed into transport carriers. To study these ER export sites (ERES), we used tobacco (Nicotiana tabacum) leaf epidermis as a model system and tested whether increased cargo dosage leads to their de novo formation. We have followed the subcellular distribution of the known ERES marker based on a yellow fluorescent protein (YFP) fusion of the Sec24 COPII coat component (YFP-Sec24), which, differently from the previously described ERES marker, tobacco Sar1-YFP, is visibly recruited at ERES in both the presence and absence of overexpressed membrane cargo. This allowed us to quantify variation in the ERES number and in the recruitment of Sec24 to ERES upon expression of cargo. We show that increased synthesis of membrane cargo leads to an increase in the number of ERES and induces the recruitment of Sec24 to these ER subdomains. Soluble proteins that are passively secreted were found to leave the ER with no apparent up-regulation of either the ERES number or the COPII marker, showing that bulk flow transport has spare capacity in vivo. However, de novo ERES formation, as well as increased recruitment of Sec24 to ERES, was found to be dependent on the presence of the diacidic ER export motif in the cytosolic domain of the membrane cargo. Our data suggest that the plant ER can adapt to a sudden increase in membrane cargo-stimulated secretory activity by signal-mediated recruitment of COPII machinery onto existing ERES, accompanied by de novo generation of new ERES.

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Characterization of Cop I Coat Proteins in Plant Cells

Cited by 29 publications

References 46 publications

Dynamics of COPII Vesicles and the Golgi Apparatus in CulturedNicotiana tabacumBY-2 Cells Provides Evidence for Transient Association of Golgi Stacks with Endoplasmic Reticulum Exit Sites

Dynamics of COPII Vesicles and the Golgi Apparatus in CulturedNicotiana tabacumBY-2 Cells Provides Evidence for Transient Association of Golgi Stacks with Endoplasmic Reticulum Exit Sites

The GTPase ARF1p Controls the Sequence-Specific Vacuolar Sorting Route to the Lytic Vacuole

De Novo Formation of Plant Endoplasmic Reticulum Export Sites Is Membrane Cargo Induced and Signal Mediated

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