Article

Dynamics of COPII vesicles and the Golgi apparatus in cultured Nicotiana tabacum BY-2 cells provides evidence for transient association of Golgi stacks with endoplasmic reticulum exit sites.

Department of Cell Biology, Heidelberg Institute for Plant Sciences, University of Heidelberg, 69120 Heidelberg, Germany.
The Plant Cell (Impact Factor: 9.58). 05/2005; 17(5):1513-31. DOI: 10.1105/tpc.104.026757
Source: PubMed

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.

0 Bookmarks
 · 
59 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Small GTPase proteins play essential roles in the regulation of vesicular trafficking systems in eukaryotic cells. Two types of small GTPases, secretion-associated Ras-related protein (Sar) and ADP-ribosylation factor (Arf), act in the biogenesis of transport vesicles. Sar/Arf GTPases function as molecular switches by cycling between active, GTP-bound and inactive, GDP-bound forms, catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins, respectively. Activated Sar/Arf GTPases undergo a conformational change, exposing the N-terminal amphipathic α-helix for insertion into membranes. The process triggers the recruitment and assembly of coat proteins to the membranes, followed by coated vesicle formation and scission. In higher plants, Sar/Arf GTPases also play pivotal roles in maintaining the dynamic identity of organelles in the secretory pathway. Sar1 protein strictly controls anterograde transport from the endoplasmic reticulum (ER) through the recruitment of plant COPII coat components onto membranes. COPII vesicle transport is responsible for the organization of highly conserved polygonal ER networks. In contrast, Arf proteins contribute to the regulation of multiple trafficking routes, including transport through the Golgi complex and endocytic transport. These transport systems have diversified in the plant kingdom independently and exhibit several plant-specific features with respect to Golgi organization, endocytic cycling, cell polarity and cytokinesis. The functional diversification of vesicular trafficking systems ensures the multicellular development of higher plants. This review focuses on the current knowledge of Sar/Arf GTPases, highlighting the molecular details of GTPase regulation in vesicle formation in yeast and advances in knowledge of the characteristics of vesicle trafficking in plants.
    Frontiers in Plant Science 08/2014; 5:411. · 3.64 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The coat protein complex II (COPII) generates transport carriers from the endoplasmic reticulum (ER) under the control of the small GTPase Sar1. Sec23 is well known as a structural component of the COPII coat and as a GTPase-activating protein (GAP) for Sar1. Here, we showed that Saccharomyces cerevisiae contains a novel Sec23 paralog, Nel1, which appears not to function as a subunit of the COPII coat. Nel1 does not associate with any of the COPII components, but it exhibits strong Sar1 GAP activity. We also demonstrated that the chromosomal deletion of NEL1 leads to a significant growth defect in the temperature-sensitive sar1D32G background, suggesting a possible functional link between these proteins. In contrast to Sec23, which is predominantly localized at ER exit sites on the ER membrane, a major proportion of Nel1 is localized throughout the cytosol. Our findings highlight a possible role of Nel1 as a novel GAP for Sar1.
    Journal of Biological Chemistry 06/2014; · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The Golgi apparatus plays essential roles in intracellular trafficking, protein and lipid modification, and polysaccharide synthesis in eukaryotic cells. It is well known for its unique stacked structure, which is conserved among most eukaryotes. However, the mechanisms of biogenesis and maintenance of the structure, which are deeply related to ER-Golgi and intra-Golgi transport systems, have long been mysterious. Now having extremely powerful microscopic technologies developed for live-cell imaging, the plant Golgi apparatus provides an ideal system to resolve the question. The plant Golgi apparatus has unique features that are not conserved in other kingdoms, which will also give new insights into the Golgi functions in plant life. In this review, we will summarize the features of the plant Golgi apparatus and transport mechanisms around it, with a focus on recent advances in Golgi biogenesis by live imaging of plants cells.
    International review of cell and molecular biology 01/2014; 310:221-87. · 4.52 Impact Factor

Full-text (2 Sources)

Download
48 Downloads
Available from
May 26, 2014