A dominant negative mutant of Sar1 GTPase inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus in tobacco and <i>Arabidopsis</i> cultured cells

Takeuchi, Masaki; Ueda, Takashi; Sato, Ken; Abe, Hiroshi; Nagata, Toshiyuki; Nakano, Akihiko

doi:10.1046/j.1365-313x.2000.00823.x

Cited by 180 publications

(211 citation statements)

References 48 publications

(78 reference statements)

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“…Sar-1 is a COPII-recruiting guanosine triphosphatehydrolyzing protein (GTPase; Takeuchi et al, 2000) found at the ER exit site (ERES) but also over the ER network and mediating vesicle trafficking from the ER to the Golgi (Bassham et al, 2008;Chung et al, 2016). To determine whether the tonoplast targeting of PAT10 relies on COPII, we expressed a DN-Sar-1b specifically in N cells using Pro CPC .…”

Section: Ap-3-dependent Vacuolar Trafficking Involves a Subpopulationmentioning

confidence: 99%

Adaptor Protein-3-Dependent Vacuolar Trafficking Involves a Subpopulation of COPII and HOPS Tethering Proteins

Feng

Song

et al. 2017

Plant Physiol.

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ORCID IDs: 0000-0001-6345-6855 (J.-G.W.); 0000-0002-3501-5857 (Y.Z.).Plant vacuoles are versatile organelles critical for plant growth and responses to environment. Vacuolar proteins are transported from the endoplasmic reticulum via multiple routes in plants. Two classic routes bear great similarity to other phyla with major regulators known, such as COPII and Rab5 GTPases. By contrast, vacuolar trafficking mediated by adaptor protein-3 (AP-3) or that independent of the Golgi has few recognized cargos and none of the regulators. In search of novel regulators for vacuolar trafficking routes and by using a fluorescence-based forward genetic screen, we demonstrated that the multispan transmembrane protein, Arabidopsis (Arabidopsis thaliana) PROTEIN S-ACYL TRANSFERASE10 (PAT10), is an AP-3-mediated vacuolar cargo. We show that the tonoplast targeting of PAT10 is mediated by the AP-3 complex but independent of the Rab5-mediated post-Golgi trafficking route. We also report that AP-3-mediated vacuolar trafficking involves a subpopulation of COPII and requires the vacuolar tethering complex HOPS. In addition, we have identified two novel mutant alleles of AP-3d, whose point mutations interfered with the formation of the AP-3 complex as well as its membrane targeting. The results presented here shed new light on the vacuolar trafficking route mediated by AP-3 in plant cells.

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Section: Ap-3-dependent Vacuolar Trafficking Involves a Subpopulationmentioning

confidence: 99%

Adaptor Protein-3-Dependent Vacuolar Trafficking Involves a Subpopulation of COPII and HOPS Tethering Proteins

Feng

Song

et al. 2017

Plant Physiol.

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“…For example, changes in the distribution of membrane GFP-Golgi markers have been followed in live cells to test the effect of overexpression of Sec12, the activating protein of the small GTPase Sar1, and the negative dominant mutants of regulatory small GTPases, such as Rab1, ARF1, and Sar1, on membrane transport from the Golgi and on Golgi integrity (Batoko et al, 2000;Takeuchi et al, 2000Takeuchi et al, , 2002Lee et al, 2002;daSilva et al, 2004;Stefano et al, 2006). Similarly, soluble secretory forms of GFP have been instrumental in analyzing protein traffic from the ER.…”

Section: Fluorescent Proteins As Tools To Assay Traffic Within and Onmentioning

confidence: 99%

Advances in Fluorescent Protein-Based Imaging for the Analysis of Plant Endomembranes

2008

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An exciting new era for live cell imaging of plant endomembranes was ushered in just over 10 years ago when Jim Haseloff and colleagues reported on the removal of a cryptic intron in the sequence of wildtype GFP for successful expression in plant cells (Haseloff et al., 1997). Haseloff's success was quickly welcomed by labs around the globe; by targeting GFP to secretory organelles, scientists could now bring to light the secret life of plant endomembranes. The plant endomembranes comprise several organelles functionally interlinked for the production and transport of secretory compounds, such as proteins, lipids, and sugars. Most of the secretory compounds are synthesized in the endoplasmic reticulum (ER) and then transported to the Golgi apparatus to be sorted for transport to distal compartments such as the plasma membrane and vacuoles. A forward transport of secretory molecules from the ER is counterbalanced by a retrograde flow from distal compartments that allows membrane homeostasis and communication with the cell's surroundings. Fluorescent protein technology applied to live cell imaging of plant endomembranes has aided immensely in providing subcellular markers for the study of the complex spatial and temporal relationships among secretory organelles. Live cell imaging is now moving forward to novel challenges. With the integration of genomic, transcriptomic, and proteomic techniques, we begin to collect data on the putative functions of plant genes; we need to understand the intricate relationships among thousands of proteins. To complete the puzzle, we must understand how the pieces fit together; we must define the functions of gene products. Important questions include: When are our proteins synthesized? Where are they targeted? With what elements do they interact? Advances in the development of optical imaging at the cellular level now offer the exciting opportunity to answer these questions.In this Update, we discuss several state-of-the-art applications of GFP imaging and how these techniques have been used to answer critical biological questions, with a focus on plant endomembrane trafficking.

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“…To establish an alternative method to manipulate COPII transport in our system, we took advantage of the known trans-dominant negative effect on COPII vesicle transport by a GTP-trapped mutant of Sar1p Takeuchi et al, 1998Takeuchi et al, , 2000. This mutant is less sensitive to the GTPase-activating activity of Sec23, and it traps vesicles in a coated configuration so that they are unable to fuse with the target membrane.…”

Section: Inhibition Of Copii Transport Via Coexpression Of Mutant Gtpmentioning

confidence: 99%

“…In addition to this, the ␣-amylase transport assays can now be used for the functional analysis of Sec12p using the inhibition assay or for the analysis of Sar1p in the rescue assay. This should prove valuable as an additional tool in combination with the use of GTPase mutants that exhibit a trans dominant negative effect on secretion (Batoko et al, 2000;Takeuchi et al, 2000) (Figure 5). …”

Section: Sec12p Overexpression Inhibits Copii Transport Through the Dmentioning

confidence: 99%

“…From the data on in vitro vesicle budding (Barlowe et al, 1994), it can be predicted that incorporation into COPII vesicles of a mutant Sar1p that is unable to respond to the GTPase-activating action of Sec23 Takeuchi et al, 1998Takeuchi et al, , 2000 would prevent vesicle uncoating and fusion with the target membrane. Perhaps this leads to an as yet undiscovered mechanism for the disposal of such defective vesicles, which would explain the loss of enzyme yield.…”

Section: Sec12p Overproduction and Mutant Sar1p Inhibit Copii Transpomentioning

confidence: 99%

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Secretory Bulk Flow of Soluble Proteins Is Efficient and COPII Dependent

Phillipson¹,

Pimpl²,

daSilva³

et al. 2001

The Plant Cell

159

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COPII-coated vesicles, first identified in yeast and later characterized in mammalian cells, mediate protein export from the endoplasmic reticulum (ER) to the Golgi apparatus within the secretory pathway. In these organisms, the mechanism of vesicle formation is well understood, but the process of soluble cargo sorting has yet to be resolved. In plants, functional complements of the COPII-dependent protein traffic machinery were identified almost a decade ago, but the selectivity of the ER export process has been subject to considerable debate. To study the selectivity of COPII-dependent protein traffic in plants, we have developed an in vivo assay in which COPII vesicle transport is disrupted at two distinct steps in the pathway. First, overexpression of the Sar1p-specific guanosine nucleotide exchange factor Sec12p was shown to result in the titration of the GTPase Sar1p, which is essential for COPII-coated vesicle formation. A second method to disrupt COPII transport at a later step in the pathway was based on coexpression of a dominant negative mutant of Sar1p (H74L), which is thought to interfere with the uncoating and subsequent membrane fusion of the vesicles because of the lack of GTPase activity. A quantitative assay to measure ER export under these conditions was achieved using the natural secretory protein barley ␣ -amylase and a modified version carrying an ER retention motif. Most importantly, the manipulation of COPII transport in vivo using either of the two approaches allowed us to demonstrate that export of the ER resident protein calreticulin or the bulk flow marker phosphinothricin acetyl transferase is COPII dependent and occurs at a much higher rate than estimated previously. We also show that the instability of these proteins in post-ER compartments prevents the detection of the true rate of bulk flow using a standard secretion assay. The differences between the data on COPII transport obtained from these in vivo experiments and in vitro experiments conducted previously using yeast components are discussed. INTRODUCTIONIn yeast and mammalian cells, the export of proteins from the endoplasmic reticulum (ER) occurs in COPII-coated vesicles. The process of COPII vesicle formation is well understood and can be reconstituted using purified yeast components (Barlowe et al., 1994). Vesicle formation in vitro depends solely on the ER donor membrane, the GTPase Sar1p, the Sar1p-specific guanosine nucleotide exchange factor Sec12p, the cytosolic COPII coat components, and GTP (Barlowe et al., 1994). Considerably less is known about the sorting of soluble cargo molecules during ER export in eukaryotes (Klumperman, 2000), and conflicting reports from the plant field have contributed to the ongoing discussions (Crofts et al., 1999;Gomord and Faye, 2000;Pagny et al., 2000;.To support protein synthesis and folding, the ER lumen maintains high levels of soluble residents such as the lumenal binding protein (BiP), protein disulfide isomerase, or calreticulin. The concentration of nonresidents in the ER lume...

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A dominant negative mutant of Sar1 GTPase inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus in tobacco and Arabidopsis cultured cells

Cited by 180 publications

References 48 publications

Adaptor Protein-3-Dependent Vacuolar Trafficking Involves a Subpopulation of COPII and HOPS Tethering Proteins

Adaptor Protein-3-Dependent Vacuolar Trafficking Involves a Subpopulation of COPII and HOPS Tethering Proteins

Advances in Fluorescent Protein-Based Imaging for the Analysis of Plant Endomembranes

Secretory Bulk Flow of Soluble Proteins Is Efficient and COPII Dependent

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