Specific binding of the peroxisomal protein targeting sequence to glyoxysomal membranes.
It has been demonstrated that the carboxyl-terminal amino acid sequence, serine-lysine-leucine (SKL), is sufficient to direct a polypeptide to peroxisomes in vivo, and that this sequence is functional in plants, animals, and yeasts. Furthermore, many peroxisomal proteins have SKL carboxyl termini, including rat acyl-CoA oxidase. We have synthesized a 125I-peptide with the sequence of the last 12 amino acids of acyl-CoA oxidase, D-Tyr-HKHLKPLQSKL (SKLp), and used it to detect a receptor that recognizes SKL containing proteins targeted to glyoxysomes. SKLp binding to alkali-stripped glyoxysomal membranes was saturable and 80% of the binding could be displaced by 1 microM unlabeled SKLp or 8 micrograms/ml glyoxysomal matrix proteins. Very little specific binding was associated with endoplasmic reticulum or mitochondrial membranes. Specific binding was affected by the ionic composition of the medium; the binding was optimal at pH 6.5 and was inhibited by mono- and divalent cations. Scatchard analysis of SKLp binding to glyoxysomal membranes indicated that there were two binding sites with Kd values of 160 and 1450 nM and abundances of 17 and 43 nmol/mg glyoxysomal membrane protein, respectively. Protease treatment of the alkali-stripped glyoxysomal membranes lowered the number of high affinity sites and destroyed all the low affinity sites. These results demonstrate, for the first time, that there is an integral membrane protein in glyoxysomes that has the characteristics of a receptor for protein import.
Wolins
N E
NE
George Washington University, Department of Biological Sciences, Washington, D.C. 20052.
Donaldson
R P
RP
D000595:Amino Acid Sequence; D002413:Cations, Divalent; D002451:Cell Compartmentation; D007887:Fabaceae; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008830:Microbodies / Q000378:metabolism*; D008969:Molecular Sequence Data; D015388:Organelles / Q000378:metabolism*; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010946:Plants, Medicinal; D011956:Receptors, Cell Surface / Q000378:metabolism*
D002413:Cations, Divalent; D008565:Membrane Proteins; D010455:Peptides; D011956:Receptors, Cell Surface; C085380:peroxisomal targeting sequence receptor