An unconventional dileucine-based motif and a novel cytosolic motif are required for the lysosomal and melanosomal targeting of OA1.
The protein product of the gene responsible for ocular albinism type 1, named OA1, is a pigment-cell-specific membrane glycoprotein, displaying features of G-protein-coupled receptors, yet exclusively localized to late endosomes, lysosomes and melanosomes. To dissect the signals responsible for the intracellular localization of OA1, we generated chimeric proteins consisting of the cytosolic domains of OA1 fused to the lumenal and transmembrane domains of LAMP1; in addition, we generated missense and deletion mutants of full-length OA1. Using this approach, we identified two separate sorting signals that are both necessary and sufficient for intracellular retention, as well as lysosomal and melanosomal localization, in melanocytic and non-melanocytic cells. These sorting signals are an unconventional dileucine motif within the third cytosolic loop and a novel motif, characterized by a tryptophan-glutamic acid doublet, within the C-terminal tail. Both motifs must be mutated to promote the plasma membrane localization of OA1, suggesting that they can independently drive its intracellular targeting. In addition, both motifs act similarly as lysosomal sorting signals in non-melanocytic cells, but appear to carry different specificities in melanocytic cells. Our findings indicate that OA1 contains multiple unconventional signals responsible for its lysosomal and melanosomal localization, and reveal a remarkable and unforeseen complexity in the regulation of polytopic protein sorting to specialized secretory organelles.
Piccirillo
Rosanna
R
San Raffalele Scientific Institute, DIBIT, Via Olgettina 58, 20132 Milan, Italy.
Palmisano
Ilaria
I
Innamorati
Giulio
G
Bagnato
Paola
P
Altimare
Domenico
D
Schiaffino
Maria Vittoria
MV
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000378:metabolism; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D007930:Leucine / Q000235:genetics* / Q000378:metabolism; D052118:Lysosomal-Associated Membrane Protein 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D020460:Melanosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
D005136:Eye Proteins; C095276:GPR143 protein, human; C463459:GPRASP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; D008562:Membrane Glycoproteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D007930:Leucine