title: Purification and initial characterization of a potential plant vacuolar targeting receptor.
abstract: Clathrin-coated vesicles are known to be involved in the transport of proteins 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 approximately 80 kDa, extracted from clathrin-coated vesicles of developing pea (Pisum sativum 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 approximately 80 kDa protein and the proaleurain targeting determinant. A peptide with a vacuolar targeting determinant from prosporamin weakly competed for binding to the approximately-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.
authors.affiliation: Department of Botany and Microbiology, University of Oklahoma, Norman 73019.
authors.forename: T
authors.initials: T
authors.lastname: Kirsch
authors.forename: N
authors.initials: N
authors.lastname: Paris
authors.forename: J M
authors.initials: JM
authors.lastname: Butler
authors.forename: L
authors.initials: L
authors.lastname: Beevers
authors.forename: J C
authors.initials: JC
authors.lastname: Rogers
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D002966:Clathrin; D003432:Cross-Linking Reagents; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D007887:Fabaceae; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010946:Plants, Medicinal; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification*; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D002966:Clathrin; D003432:Cross-Linking Reagents; D004792:Enzyme Precursors; D008562:Membrane Glycoproteins; D010940:Plant Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D003546:Cysteine Endopeptidases; C076060:proaleurain