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ER-to-Golgi transport by COPII vesicles in Arabidopsis involves a ribosome-excluding scaffold that is transferred with the vesicles to the Golgi matrix

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Abstract

Plant Golgi stacks are mobile organelles that can travel along actin filaments. How COPII (coat complex II) vesicles are transferred from endoplasmic reticulum (ER) export sites to the moving Golgi stacks is not understood. We have examined COPII vesicle transfer in high-pressure frozen/freeze-substituted plant cells by electron tomography. Formation of each COPII vesicle is accompanied by the assembly of a ribosome-excluding scaffold layer that extends approximately 40 nm beyond the COPII coat. These COPII scaffolds can attach to the cis-side of the Golgi matrix, and the COPII vesicles are then transferred to the Golgi together with their scaffolds. When Atp115-GFP, a green fluorescent protein (GFP) fusion protein of an Arabidopsis thaliana homolog of the COPII vesicle-tethering factor p115, was expressed, the GFP localized to the COPII scaffold and to the cis-side of the Golgi matrix. Time-lapse imaging of Golgi stacks in live root meristem cells demonstrated that the Golgi stacks alternate between phases of fast, linear, saltatory movements (0.9–1.25 μm/s) and slower, wiggling motions (<0.4 μm/s). In root meristem cells, approximately 70% of the Golgi stacks were connected to an ER export site via a COPII scaffold, and these stacks possessed threefold more COPII vesicles than the Golgi not associated with the ER; in columella cells, only 15% of Golgi stacks were located in the vicinity of the ER. We postulate that the COPII scaffold first binds to and then fuses with the cis-side of the Golgi matrix, transferring its enclosed COPII vesicle to the cis-Golgi.

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Abbreviations

COPII:

coat complex II

ER:

endoplasmic reticulum

GFP:

green fluorescent protein

ManI:

mannosidase I

TGN:

trans-Golgi network

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Acknowledgements

We thank for Dr. Andreas Nebenführ (Univ. of Tennessee) for providing A. thaliana lines expressing ManI-GFP. We also thank Dr. David G. Robinson (Univ. of Heidelberg) for the AtSar1 antibody. We are grateful to Dr. Sebastian Y. Bednarek and Dr. Sookhee Park (Univ. of Wisconsin) for helping generate the transgenic BY-2 cell lines, Dr. David Mastronarde for assistance in using the IMOD software, and Dr. David A. Christopher (Univ. of Hawaii) for critical reading of the manuscript. We also appreciate Ms. Alexis Bencze (University of Colorado) for her assistance in generating 3D models. This work was supported by National Institutes of Health Grant GM61306 to LAS and funds from the University of Florida, Microbiology and Cell Science Department and Interdisciplinary Center for Biotechnology Research to B-HK.

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Authors and Affiliations

  1. Department of Molecular Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309, USA

    Byung-Ho Kang & L. Andrew Staehelin

  2. Department of Microbiology and Cell Science & Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL, 32611, USA

    Byung-Ho Kang

  3. Department of Microbiology and Cell Science, Electron Microscopy and Bioimaging Lab, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL, 32611, USA

    Byung-Ho Kang

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  1. Byung-Ho Kang
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Correspondence to Byung-Ho Kang.

Electronic Supplementary Material

Below is the image is a link to a high resolution version

Fig. S1

Thin section electron micrographs of A. thaliana root meristem cells immunolabeled with an AtSar1 antibody. The COPII vesicles associated with immunogold particles (arrowheads) are located between the ER and the cis-side of Golgi stacks. in A. thaliana root meristem cells. Scale bars (a, b): 300 nm. Scale bars: 200 nm (GIF 74.8 KB)

High resolution image file (TIF 473 KB)

Fig. S2

Tomographic slice image (a) and 3D model image (b) of the cortical cytoplasm of an A. thaliana root meristem cell showing the PM, ER, and ribosomes (light-blue spheres). Ribosomes are in contact with the ER membrane but are not with the PM. Scale bar: 200 nm (GIF 87.3 KB)

High resolution image file (TIF 2.61 MB)

Fig. S3

Immunoblot of protein extracts (10 μg) from BY-2 cells (a) and A. thaliana seedling roots (b). Untransformed BY-2 cells/A. thaliana roots (lane 1), BY-2 cells/A. thaliana roots stably-transformed with the Atp115-GFP construct (lane 2, 130 kDa), and A. thaliana roots expressing DRP1A-GFP (lane 3, 96 kDa) were analyzed. Blots were probed with a GFP antibody. The AtUso1-GFP lines express a full-length GFP fusion protein without other forms of GFP. Immunoblot analyses were performed as described in Kang et al. (2001) (GIF 60.4 KB)

High resolution image file (TIF 1.21 MB)

Fig. S4

Tracking of five Golgi stacks shown in Fig. 6 and in Video S1. Two fast-moving (G1, G2) and three wiggling Golgi stacks (G3, G4, G5) were traced. The three Golgi stacks marked as #1, #2, and #3 in Fig. 6 correspond to Golgi1, Golgi2, and Golgi3, respectively. (GIF 11.2 KB)

High resolution image file (TIF 2.60 MB)

Below is the link to the electronic supplementary material

Video S1

Movie of ManI-GFP labeled Golgi stacks in an A. thaliana root meristem cell. The root was stained briefly with FM4–64 to outline the cell walls prior to viewing. Serial images from this movie are presented in Fig. 6. (MOV 3.72 MB)

A movie of ManI-GFP labeled Golgi stacks in two A. thaliana root meristem cells. The time-lapse image series was taken with the same setting as the cell shown in Fig. 6 and Video S1. Scale bar: 10 μm. (MOV 2.33 MB)

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Kang, BH., Staehelin, L.A. ER-to-Golgi transport by COPII vesicles in Arabidopsis involves a ribosome-excluding scaffold that is transferred with the vesicles to the Golgi matrix. Protoplasma 234, 51–64 (2008). https://doi.org/10.1007/s00709-008-0015-6

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