title: Binding of the peroxisomal targeting sequence SKL is specified by a low-affinity site in castor bean glyoxysomal membranes. A domain next to the SKL binds to a high-affinity site.
abstract: The carboxyl-terminal amino acid sequence serine-lysine-leucine (SKL) is the consensus peroxisomal targeting sequence 1 (PTS1) and is sufficient to direct a polypeptide to peroxisomes in vivo in plants, animals, and yeasts. However, there are also two sites on alkali-stripped glyoxysomal membranes from castor bean (Ricinus communis) endosperm that bind the peptide YHKHLKPLQSKL (SKLp), the sequence of the last 12 amino acids of acyl-coenzyme A oxidase (N.E. Wollins, R.P. Donaldson [1994] J Biol Chem 289: 1149-1153). It was hypothesized that one of these sites interacts with information other than the PTS1. To explore the sequence requirements for each SKLp binding site, we tested the peptides YHKHLKPQSKG and YHKHLKPLQS and found that they bound to the high-affinity site, but not to the low-affinity site. When the high-affinity site was blocked with YHKHLKPQSKG, SKLp bound to the low-affinity site with a dissociation constant (Kd) of 8.5 microM. In an attempt to disrupt high-affinity binding, two the upstream, positively charged residues were replaced with negatively charged residues to make the peptide YHKETEPLQSKL. YHKETEPLQSKL did not bind to either site on the glyoxysomal membranes. These results indicate that the PTS1 binds to the low-affinity site and that the adjacent, positively charged domain binds to the high-affinity site.
authors.affiliation: Department of Biological Sciences, George Washington University, Washington, DC 20052, USA.
authors.forename: N E
authors.initials: NE
authors.lastname: Wolins
authors.forename: R P
authors.initials: RP
authors.lastname: Donaldson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002367:Ricinus communis / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008830:Microbodies / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010947:Plants, Toxic; D011485:Protein Binding
chemical_list: D009842:Oligopeptides; C088449:seryl-lysyl-leucine