The residues Leu(Ile)475-Ile(Leu, Val, Ala)476, contained in the extended carboxyl cytoplasmic tail, are critical for targeting of the resident lysosomal membrane protein LIMP II to lysosomes.
LIMP II, a type II lysosomal integral membrane protein, and the CD36/LIMP II construct are targeted to lysosomes by means of a signal expressed in the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II (Vega, M. A., Rodriguez, F., Seguí, B., Calés, C., Alcalde, J., and Sandoval, I. V. (1991) J. Biol. Chem. 266, 16269-16272; Vega, M. A., Seguí-Real, B., Garcia, J. A., Calés, C., Rodriguez, F., Vandekerckhove, J., and Sandoval, I. V. (1991) J. Biol. Chem. 266, 16818-16824). Substitution of Leu475 with Ile resulted in a decreased efficiency of targeting. Mutant forms produced by substituting Leu475 by hydrophobic residues with either large (Val) or small (Ala, Gly) side chains, or by a charged residue (Asp), showed inhibited targeting. In contrast, the contiguous Ile476 residue could be replaced by either Leu, without loss in the efficiency of targeting, or by Val or Ala, with some impediment. Substitution of Ile476 by either Gly or Asp inhibited completely the targeting. The addition of the sequence Ser-Trp-Asp to the carboxyl end of the construct did not interfere with targeting. Data from 1H NMR analysis of the icosapeptide corresponding to the carboxyl cytoplasmic tail of LIMP II indicated the predominance of structures with extended random coil conformations, suggesting that the targeting signal is contained in a domain with an extended configuration.
Sandoval
I V
IV
Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma de Madrid, Spain.
Arredondo
J J
JJ
Alcalde
J
J
Gonzalez Noriega
A
A
Vandekerckhove
J
J
Jimenez
M A
MA
Rico
M
M
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001483:Base Sequence; D018955:CD36 Antigens; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D007532:Isoleucine; D007930:Leucine; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D051116:Receptors, Scavenger; D017384:Sequence Deletion; D012795:Sialoglycoproteins; D014162:Transfection; D014633:Valine
D015703:Antigens, CD; D018955:CD36 Antigens; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D051116:Receptors, Scavenger; C074299:SCARB2 protein, human; D012795:Sialoglycoproteins; D007532:Isoleucine; D007930:Leucine; D014633:Valine; D000409:Alanine