Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Apr;143(4):1640-50.
doi: 10.1104/pp.106.094110. Epub 2007 Feb 23.

De novo formation of plant endoplasmic reticulum export sites is membrane cargo induced and signal mediated

Sally L Hanton  1 , Laurent Chatre, Luciana Renna, Loren A Matheson, Federica Brandizzi
Affiliations

De novo formation of plant endoplasmic reticulum export sites is membrane cargo induced and signal mediated

Sally L Hanton et al. Plant Physiol. 2007 Apr.

Abstract

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.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
YFP-Sec24 overexpression does not disrupt constitutive ER export. Tobacco leaf protoplasts were transfected with a constant amount of α-amylase DNA, with an increasing concentration of DNA encoding either Sec12-GFP (A) or YFP-Sec24 (B). The α-amylase activities in the extracellular medium and cellular fraction were measured and the secretion index, which represents the ratio between the intracellular and extracellular activities, was calculated as previously described (Phillipson et al., 2001). Error bars represent se for three independent experiments.
Figure 2.
Figure 2.
Overexpression of Sec12-YFP inhibits transport of ERD2-GFP to the Golgi apparatus. A, Confocal image of a cell expressing ERD2-GFP, which labels the Golgi apparatus (arrows) and ER (arrowhead). B to D, On coexpression of Sec12-YFP (C), ERD2-GFP is partially redistributed to the ER (B and D). Bars = 5 μm; circles represent the size of the areas used to measure fluorescence intensity in the ER and Golgi. E, Quantification of the ER-localized ERD2-GFP fluorescence relative to that in the Golgi apparatus in the absence (−Sec12) or presence (+Sec12) of Sec12-YFP. A significant increase in the ratio was verified when Sec12-YFP was expressed in comparison with the control. Sample size: 240 Golgi bodies and 240 ER measurements were analyzed for each sample. Error bars represent se of the mean.
Figure 3.
Figure 3.
Coexpression of ERD2-GFP with YFP-Sec24 increases the fluorescence and number of ERES. A to C, YFP-Sec24 labels the cytosol (arrowhead) and punctate structures (ERES, arrow) when expressed alone. Note that there is no signal in the GFP channel, excluding the possibility that colocalization of the signals is due to imaging cross talk (Brandizzi et al., 2002b). Coexpression of ERD2-GFP (D) with YFP-Sec24 (E) results in an apparent increase in the fluorescence intensity of the YFP-Sec24 at ERES (E). F, Merged image of D and E. Coexpression of the soluble marker secGFP (G) with YFP-Sec24 (H) does not appear to affect the number or fluorescence of ERES (I, merge). Bars = 5 μm.
Figure 4.
Figure 4.
Quantification of the ERD2-mediated increase in fluorescence intensity and number of ERES. A, Quantification of the YFP-Sec24 fluorescence intensity at ERES relative to that in the cytosol, shown as a ratio, indicates that in the presence of ERD2-GFP, the fluorescence intensity at ERES increases in comparison to that in cells expressing YFP-Sec24 alone. However, no increase in fluorescence intensity is observed on coexpression of secGFP, a soluble cargo protein. Fluorescence was measured for at least 10 ERES and cytosol areas per cell in 150 cells, giving at least 1,500 ERES and 1,500 cytosol measurements for each combination of markers. B, Number of ERES per 100 μm3 in cells expressing YFP-Sec24 in the presence of ERD2-GFP increases in comparison to cells expressing YFP-Sec24 alone. However, coexpression of the soluble marker secGFP has no effect on the number of ERES per 100 μm3. Error bars represent se of the mean.
Figure 5.
Figure 5.
Mutation of a cytosolic diacidic motif prevents recruitment of YFP-Sec24 to ERES. A to C, TMcCCASP (A) predominantly labels the Golgi apparatus. Coexpression with YFP-Sec24 (B) leads to an apparent increase in both YFP fluorescence intensity at ERES and ERES number. C, Merged image of A and B. D to F, Mutation of a diacidic motif in TMcCCASP results in redistribution of the marker to the ER membranes (D, arrow). Coexpression of TMcCCASPDXE1 with YFP-Sec24 (E) does not noticeably affect YFP fluorescence intensity or ERES number. F, Merged image of D and E. Bars = 5 μm. G, Quantification of the YFP fluorescence intensity at ERES relative to that in the cytosol, shown as a ratio, indicates that in the presence of TMcCCASP (TMc), the fluorescence intensity at ERES increases relative to that of YFP-Sec24 alone. In comparison, no obvious increase in fluorescence intensity is observed on coexpression of TMcCCASPDXE1 (TMcDXE1). Fluorescence was measured for at least 10 ERES and cytosol areas per cell, giving an average of 1,500 ERES for each combination of markers. H, Number of ERES per 100 μm3 (sample size = 150 cells) expressing YFP-Sec24 in the presence of TMcCCASP increases in comparison to cells expressing YFP-Sec24 alone. However, coexpression of the export-incompetent TMcCCASPDXE1 slightly reduces the number of ERES per 100 μm3. Error bars represent se of the mean.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Antonny B, Madden D, Hamamoto S, Orci L, Schekman R (2001) Dynamics of the COPII coat with GTP and stable analogues. Nat Cell Biol 3 531–537 - PubMed
    1. Aridor M, Fish KN, Bannykh S, Weissman J, Roberts TH, Lippincott-Schwartz J, Balch WE (2001) The Sar1 GTPase coordinates biosynthetic cargo selection with endoplasmic reticulum export site assembly. J Cell Biol 152 213–229 - PMC - PubMed
    1. Aridor M, Weissman J, Bannykh S, Nuoffer C, Balch WE (1998) Cargo selection by the COPII budding machinery during export from the ER. J Cell Biol 141 61–70 - PMC - PubMed
    1. Barlowe C (2003) Signals for COPII-dependent export from the ER: what's the ticket out? Trends Cell Biol 13 295–300 - PubMed
    1. Barlowe C, Orci L, Yeung T, Hosobuchi M, Hamamoto S, Salama N, Rexach MF, Ravazzola M, Amherdt M, Schekman R (1994) COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77 895–907 - PubMed

Publication types

MeSH terms

Associated data

LinkOut - more resources