Lysosomal targeting of P-selectin is mediated by a novel sequence within its cytoplasmic tail.
Signals controlling the intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains. P-selectin is a type I membrane protein receptor for leukocytes, acting during the inflammation response. Heterologous expression experiments have demonstrated that its 35-residue cytoplasmic tail contains signals for targeting to synaptic-like microvesicles, dense-cored granules, and lysosomes. We have examined the lysosomal targeting information present within the cytoplasmic tail by site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras followed by transient transfection in H.Ep.2 cells. Assaying lysosomal targeting by subcellular fractionation as well as intracellular proteolysis, we have discovered a novel lysosomal targeting signal, KCPL, located within the C1 domain of the cytoplasmic tail. Alanine substitution of this tetrapeptide reduced lysosomal targeting to the level of a tailless horseradish peroxidase-P-selectin chimera, which was previously found to be deficient in both internalization and delivery to lysosomes. A proline residue within this lysosomal targeting signal makes a major contribution to the efficiency of lysosomal targeting. A diaminobenzidine density shift procedure established that chimeras with an inactivated KCPL sequence are present within transferrin-positive compartments. Such a mutant also displays an increased level of expression at the plasma membrane. Our results indicate that the sequence KCPL within the cytoplasmic tail of P-selectin is a structural element that mediates sorting from endosomes to lysosomes.
Blagoveshchenskaya
A D
AD
Medical Research Council Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Norcott
J P
JP
Cutler
D F
DF
D000595:Amino Acid Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004252:DNA Mutational Analysis; D004705:Endocytosis / Q000235:genetics*; D011992:Endosomes / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D019007:P-Selectin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry
D019007:P-Selectin; D011993:Recombinant Fusion Proteins; D006735:Horseradish Peroxidase