A dileucine signal situated in the C-terminal tail of the lysosomal membrane protein p40 is responsible for its targeting to lysosomes.
Transport of newly synthesized lysosomal membrane proteins from the TGN (trans-Golgi network) to the lysosomes is due to the presence of specific signals in their cytoplasmic domains that are recognized by cytosolic adaptors. p40, a hypothetical transporter of 372 amino acids localized in the lysosomal membrane, contains four putative lysosomal sorting motifs in its sequence: three of the YXXphi-type (Y(6)QLF, Y(106)VAL, Y(333)NGL) and one of the [D/E]XXXL[L/I]-type (EQERL(360)L(361)). To test the role of these motifs in the biosynthetic transport of p40, we replaced the most critical residues of these consensus sequences, the tyrosine residue or the leucine-leucine pair, by alanine or alanine-valine respectively. We analysed the subcellular localization of the mutated p40 proteins in transfected HeLa cells by confocal microscopy and by biochemical approaches (subcellular fractionation on self-forming Percoll density gradients and cell surface biotinylation). The results of the present study show that p40 is mistargeted to the plasma membrane when its dileucine motif is disrupted. No role of the tyrosine motifs could be put forward. Taken together, our results provide evidence that the sorting of p40 from the TGN to the lysosomes is directed by the dileucine EQERL(360)L(361) motif situated in its C-terminal tail.
Boonen
Marielle
M
URPhiM, Laboratoire de Chimie Physiologique, FUNDP, B-5000 Namur, Belgium.
Rezende de Castro
Roberta
R
Cuvelier
Gaëlle
G
Hamer
Isabelle
I
Jadot
Michel
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007930:Leucine / Q000737:chemistry* / Q000235:genetics; D051907:Lysosomal Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D051907:Lysosomal Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D007930:Leucine