Abstract
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 COPII-coat 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.
Highlights
The endoplasmic reticulum (ER) is a highly versatile membrane compartment that extends throughout the cytoplasm of eukaryotic cells
Membrane-bound Sec13/31 should represent fully assembled ER exit sites (ERES) and is the reason why the bulk of our observations have been made with Sec13 antibodies or with a transiently expressed LeSec13:green fluorescent protein (GFP) construct
The expression of Sec13:GFP has no effect on the secretion index of protoplasts, whereas the expression of Sar1 mutants prevent export out of the ER and in the case of the Sar1-GDP mutant lead to an altered pattern of LeSec13:GFP fluorescence
Summary
The endoplasmic reticulum (ER) is a highly versatile membrane compartment that extends throughout the cytoplasm of eukaryotic cells. It is universally accepted that ER-to-Golgi protein transport in mammalian cells is mediated by the sequential action of COPII- and COPI-coat protein complexes (Duden, 2003; Lee et al, 2004). This is because a pleiomorphic structure known alternatively as the ER–Golgi intermediate compartment (ERGIC) and vesicular tubular clusters (VTCs) transits along microtubules. There is general agreement that only the COPII machinery is responsible for the actual transport of cargo out of the ER (Barlowe, 1998, 2003)
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