Purification and initial characterization of a potential plant vacuolar targeting receptor.

Abstract

Clathrin-coated vesicles are known to be involved in the transport ofproteins from the Golgi to the vacuole in plant cells. The mechanisms by which proteins are directed into this pathway are not known. Here we identify an integral membrane protein of -80 kDa, extracted from clathrin-coated vesicles of developing pea (Pisum saivum L.) cotyledons, that bound at neutral pH to an affinity column prepared with the N-terminal targeting determinant of the vacuolar thiol protease, proaleurain, and eluted when the pH was lowered to 4. The protein was not retained on a control column prepared with the N-terminal sequence of a homologous, secreted thiol protease, endopeptidase B. The 80-kDa protein also accumulated in a membrane fraction that is less dense than clathrin-coated vesicles. In vitro studies demonstrated a binding constant of 37 nM between the =80-kDa protein and the proaleurain targeting determinant. A peptide with a vacuolar targeting determinant from prosporamin weakly competed for binding to the "80-kDa protein, while a peptide carrying a single amino acid substitution known to abolish prosporamin vacuolar targeting had no measurable binding affinity for the protein. The binding protein is a glycoprotein with a transmembrane orientation in which the C terminus is exposed to the cytoplasm. The binding domain is located in the N-terminal luminal portion of the protein. These properties of the binding protein are consistent with the function of a receptor that would select proteins in the trans-Golgi for sorting to clathrin-coated vesicles and delivery to the vacuole.Vacuoles are acidic compartments occupying up to 80% of the volume of mature plant cells. In addition to functioning in the maintenance ofturgor and as a depository for such solutes as amino acids, sugars, organic acids, and mineral salts, the vacuoles also contain hydrolytic enzymes and, at certain stages of plant development, may serve as sites of accumulation of storage proteins (1). The accumulation of hydrolytic enzymes has been used as evidence to consider that the vacuole is a lytic compartment in plants analogous to the lysosome of the mammalian cell (2). Because of the presence of hydrolytic enzymes and the accumulation of reserve proteins, the vacuole/protein body is a useful system in which to study protein targeting in plants (3, 4). As in the mammalian lysosome system, soluble vacuolar proteins are synthesized in the endoplasmic reticulum and progress through the Golgi apparatus and clathrin-coated vesicles (CCVs) (5, 6) prior to accumulating in the vacuole. Although some soluble vacuolar proteins are glycosylated, N-linked oligosaccharides have no role in sorting glycoproteins to the vacuole (3, 4, 7). There is an accumulating body of information indicating that targeting of soluble proteins to the vacuole is mediated by determinants that reside in the polypeptide (3, 4, 7). To date, N-terminal (8-11) and C-terminal targeting determinant sequences (12, 13) have been identified; internal peptide sequences also appear to be in...

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

“…Similar to AtELP, the 80-kD protein from pea was not found to localize with ER or Golgi markers but fractionated as a lessdense compartment on Suc density centrifugation experiments (Beevers, 1996;Okita and Rogers, 1996). However, when this less-dense membrane fraction from pea cotyledons was incubated and allowed to associate with clathrin components, it sedimented in SUC density gradients at a density equivalent to that of CCVs (Kirsch et al, 1994a).…”

“…Nevertheless, a number of studies have provided definitive evidence for the participation of CCVs in intracellular protein transport in plants. These include the recent cloning of the clathrin heavy-chain gene from soybean and the localization of the protein to the PM (Blackbourn and Jackson, 1996), the identification of a p-type adaptin from zucchini (Holstein et al, 1994), the cloning of a o-adaptin from Camptotkeca acuminata (Maldonado-Mendoza and Nessler, 1996), and the isolation of a putative vacuolar-targeting receptor (BP-80; Kirsch et al, 1994a) having a broadbinding specificity for plant vacuolar-targeting signals from pea CCVs (Kirsch et al, 1996).…”

Trafficking of Vacuolar Proteins: The Crucial Role of Arabidopsis Vacuolar Protein Sorting 29 in Recycling Vacuolar Sorting Receptor

“…In Arabidopsis thaliana, seven VSR isoforms have been identified (Ahmed et al, 1997;Shimada et al, 2003;Zouhar et al, 2010). They are type I membrane proteins and are highly conserved in different plant species (Kirsch et al, 1994(Kirsch et al, , 1996Ahmed et al, 1997;Paris and Neuhaus, 2002). These cargo sorting receptors travel together with cargo proteins from the TGN to the late endosome or the PVC.…”

TNO1 Is Involved in Salt Tolerance and Vacuolar Trafficking in Arabidopsis

“…From the ER, they are transported to the trans-Golgi network (TGN) via the Golgi (Törmäkangas et al, 2001). In the TGN, vacuolar proteins with sequencespecific vacuolar sorting determinants are thought to be recognized by vacuolar sorting receptors (VSRs; Kirsch et al, 1994;Ahmed et al, 2000;daSilva et al, 2005), although a recent report has suggested that recognition of cargo by VSRs may occur as early as the ER (Niemes et al, 2010). Storage proteins with C-terminal sorting sequences are also bound by VSRs or by a second putative sorting receptor, RMR1 (Jiang et al, 2000;Shimada et al, 2003a;Park et al, 2005Park et al, , 2007Hinz et al, 2007).…”

“…Generally, one v-SNARE on the vesicle and three t-SNAREs on the target membrane form a trans-SNARE complex, bringing the two membranes in contact and driving membrane fusion (McNew et al, 2000). Clathrin-coated vesicles containing VSR1 contain the v-SNARE VTI11, which forms a SNARE complex with SYP5, SYP2, and VAMP727 at the PVC during anterograde trafficking from the TGN (Kirsch et al, 1994;Sanderfoot et al, 2001a;Ebine et al, 2008). However, the mechanism of recycling of VSR1 from the PVC to the TGN is not yet fully understood.…”