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

Abstract

Many receptors involved in clathrin-mediated protein transport through the endocytic and secretory pathways of yeast and animal cells share common features. They are all type I integral membrane proteins containing cysteine-rich lumenal domains and cytoplasmic tails with tyrosine-containing sorting signals. The cysteine-rich domains are thought to be involved in ligand binding, whereas the cytoplasmic tyrosine motifs interact with clathrin-associated adaptor proteins during protein sorting along these pathways. In addition, tyrosine-containing signals are required for the retention and recycling of some of these membrane proteins to the trans-Golgi network. Here we report the characterization of an approximately 80-kD epidermal growth factor receptor-like type I integral membrane protein containing all of these functional motifs from Arabidopsis thaliana (called AtELP for A. thaliana Epidermal growth factor receptor-Like Protein). Biochemical analysis indicates that AtELP is a membrane protein found at high levels in the roots of both monocots and dicots. Subcellular fractionation studies indicate that the AtELP protein is present in two membrane fractions corresponding to a novel, undefined compartment and a fraction enriched in vesicles containing clathrin and its associated adaptor proteins. AtELP may therefore serve as a marker for compartments involved in intracellular protein trafficking in the plant cell.

Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis

“…However, little is known about the trafficking of VSRs through the SYP61 compartment. VSR1 is thought to direct ntVSS cargo to the PVC in a clathrin-dependent pathway [43], although recent studies have expanded this view by showing that ctVSS cargo follows the same route [41,44,45]. Our proteomic analysis revealed the presence of several VSRs in SYP61 vesicles, including VSR4 (At2g14720), VSR3 (At2g14740) and VSR7 (At4g20110; Supplementary information, Tables S1 and S2), with localization studies showing clearly that VSR4-YFP was colocalized with SYP61-CFP in stably transformed plants ( Figure 2D-2F).…”

An Arabidopsis VPS45p Homolog Implicated in Protein Transport to the Vacuole1

“…Most of the protein was associated with the P150 fraction, which contained microsomal membranes. A small amount of AtVPS45p was found in the P8 fraction, which contains most of the tonoplast and plasma membrane (Ahmed et al, 1997). Upon longer exposures, a small amount of AtVPS45p could also be detected in the soluble fraction (data not shown).…”

“…protein), a potential vacuolar protein sorting receptor, is found in clathrin-coated vesicles and in an additional, uncharacterized compartment (Ahmed et al, 1997). On the Suc gradient, AtVPS45p co-fractionated with AtELP, with a major peak observed at 23% Suc (1.08 g cm Ϫ3 ) and a minor peak at 35% Suc (1.15 g cm Ϫ3 ) for both proteins.…”

“…Protein in each fraction was precipitated using TCA, and was analyzed by SDS-PAGE and immunoblotting. Blots were probed using AtVPS45p antibodies, AtPEP12p antibodies (Conceição et al, 1997), or AtELP antibodies (Ahmed et al, 1997), followed by secondary antibodies conjugated to alkaline phosphatase or horseradish peroxidase. The blots were digitized on a flat-bed scanner, and protein amounts were quantitated by densitometry using NIH Image software (National Institutes of Health, Bethesda, MD).…”

“…AtVPS45 is able to complement the yeast vps45⌬ mutant, and the majority of the protein in Arabidopsis roots is membrane associated. AtVPS45p does not completely cofractionate with AtPEP12p on Suc gradients, but instead cofractionates with AtELP, a receptor-like protein potentially involved in vacuolar protein sorting (Ahmed et al, 1997).…”