Plant Physiol. Journal of Pharmacology and Experimental Therapeutics
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PLANT PHYSIOLOGY , Vol 113, Issue 3 943-949, Copyright © 1997 by American Society of Plant Biologists

CELL BIOLOGY AND SIGNAL TRANSDUCTION

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)

N. E. Wolins and R. P. Donaldson
Department of Biological Sciences, The George Washington University, Washington, DC 20052

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 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. Wolins, 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 YHKHLKPLQSKG 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 YHKHLKPLQSKG, SKLp bound to the low-affinity site with a dissociation constant (Kd) of 8.5 [mu]M. In an attempt to disrupt high-affinity binding, two of 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.




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Copyright © 1997 by the American Society of Plant Biologists