Characterization of AtSEC12 and AtSAR1 (Proteins Likely Involved in Endoplasmic Reticulum and Golgi Transport)

Abstract

Transport of cargo proteins from the endoplasmic reticulum (ER) to the cis-Golgi network is mediated by protein-coated vesicles. The coat, called COPII coat, consists of proteins that are recruited from the cytosol and interact with integral membrane proteins of the ER. In yeast, both cytosolic proteins (Sec13/31, Sec23/24, and Sar1) and ER-associated proteins (Sec12 and others) have been purified and characterized and it has been possible to demonstrate transport vesicle formation in vitro. Arabidopsis thaliana homologs of Sar1 and Sec12 have recently been identified, but little is known about the properties of the proteins or their subcellular distribution. Here we demonstrate that AtSAR1, a 22-kD protein that binds GTP, and AtSEC12, a 43-kD GTP-exchange protein, are both associated with the ER. However, about one-half of the cellular AtSAR1 is present in the cytosol. When AtSAR1 is overexpressed in transgenic plants, the additional protein is also cytosolic. When tissue-culture cells are cold-shocked (12 h at 8[deg]C), AtSAR1 levels appeared to decline and a larger proportion of the total protein was found in the cytosol. Given the known function of AtSAR1 in yeast, we propose that the amount of ER-associated AtSAR1 is an indication of the intensity of the secretory process. Thus, we expect that such a cold shock will adversely affect ER-to-Golgi transport of proteins.

Cloning and Subcellular Location of an Arabidopsis Receptor-Like Protein That Shares Common Features with Protein-Sorting Receptors of Eukaryotic Cells

“…8) markers. Characteristic of ER proteins (Lord, 1987;Bar-Peled and Raikhel, 1997), the AtSEC12 peak shifted completely from fractions 7 and 8 to a higher Sue density corresponding to fractions 11 and 15 in the presence of Mg 2+ (Fig. 6B).…”

“…This technique separates organelles on the basis of their inherent buoyant density differences and has been used to resolve the ER, mitochondria, PM, vacuole, and Golgi complex in animal, yeast, and plant cells (Walworth and Novick, 1987;Beaumelle and Hopkins, 1989;Dewitt et al, 1996;Bar-Peled and Raikhel, 1997).…”

“…Ten milliliters of Arabidopsis tkaliana ecotype Columbia cell-suspension line T87-C33, a generous gift from Dr. Michael Axelos (Institut National de Ia Recherche Agronomique, France), was subcultured in 50 mL of media (0.32 g L-' Gamborg's 8-5 with minimal organics, with the addition of 20 g L-' SUC, and 1 mg L-l 2,4-D at pH 5.7) and was grown on a rotary shaker at 50 rpm and 22°C under light conditions as described by Bar-Peled and Raikhel (1997). Seeds of A. tkaliana ecotype Columbia (approximately 15 seeds) were sterilized and placed in a 125-mL flask containing 50 mL of germination medium liquid medium (4.3 g L-' Murashige and Skoog salts mixture [GIBCO-BRL], 0.5 g L-* Mes, 10 g SUC, 0.1 g L-' myo-inositol, 100 p L L-l of 10 mg mL-' thiamine-HCI, 50 p L L-' of 10 mg mL-' pyridoxine, and 50 p L L-l of 10 mg mL-' nicotinic acid, adjusted to pH 5.7 with KOH).…”

De Novo Formation of Plant Endoplasmic Reticulum Export Sites Is Membrane Cargo Induced and Signal Mediated

“…However, proteins with analogous functions have not yet been identified in plants. Plant homologs of components of the COPI and COPII vesicle coats have been identified and some characterization studies have been carried out (d'Enfert et al, 1992;Regad et al, 1993;Bar-Peled and Raikhel, 1997;Takeuchi et al, 1998;Movafeghi et al, 1999;Contreras et al, 2000;Pimpl et al, 2000;Vernoud et al, 2003;daSilva et al, 2004;Yang et al, 2005;Stefano et al, 2006). Although this area of research is currently one of great interest and activity, knowledge of transport in the early secretory pathway of plants still lags behind that from yeast and mammalian systems.…”

Tansley Review No. 108

“…Several of these mutants have now been used in complementation screens with plant cDNA libraries. The Arabidopsis counterparts to the yeast t-SNAREs Pep12p and Vam3p (Bassham et al, 1995 ;BarPeled & Raikhel, 1997 ;Sato et al, 1997), and at least one group of Arabidopsis and Nicotiana GTPase proteins (Bednarek et al, 1994 ;Takeuchi et al, 1998) were first characterized in this manner.…”