Secretory bulk flow of soluble proteins is efficient and COPII dependent.
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 alpha-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.
Phillipson
B A
BA
Centre for Plant Sciences, Leeds Institute for Plant Biotechnology and Agriculture, School of Biology, The University of Leeds, Leeds LS2 9JT, United Kingdom.
Pimpl
P
P
daSilva
L L
LL
Crofts
A J
AJ
Taylor
J P
JP
Movafeghi
A
A
Robinson
D G
DG
Denecke
J
J
D022181:COP-Coated Vesicles / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D037282:Calreticulin; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006056:Golgi Apparatus / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature; D014026:Nicotiana / Q000166:cytology* / Q000378:metabolism*; D033921:Vesicular Transport Proteins; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; C072614:HDEL receptor; D008024:Ligands; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011506:Proteins; D018000:Receptors, Peptide; D012261:Ribonucleoproteins; C071239:SEC12 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C077480:histidyl-aspartyl-glutamyl-leucine; D000516:alpha-Amylases; D020559:Monomeric GTP-Binding Proteins; C077395:SAR1 protein, S cerevisiae