Lysosomal Targeting of the CLN7 Membrane Glycoprotein and Transport Via the Plasma Membrane Require a Dileucine Motif

Abstract

CLN7 is a polytopic lysosomal membrane protein deficient in variant late infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder. In this study fluorescence protease protection assays and mutational analyses revealed the N-and C-terminal tails of CLN7 in the cytosol and two N-glycosylation sites at N371 and N376. Both partially and non-glycosylated CLN7 were correctly transported to lysosomes. To identify lysosomal targeting motifs, we generated CD4-chimera fused to the N-and C-terminal domains of CLN7. Lysosomal localization of the chimeric proteins requires a consensus acidic dileucine-based motif in the N-terminus and two tandem tyrosine-based signals in the C-terminus. Mutation of these sorting motifs resulted in cell surface redistribution of CD4 chimeras. However, the dileucine-based motif is of critical importance for lysosomal localization of the full-length CLN7 in different cell lines. Cell surface biotinylation revealed that at equilibrium 22% of total CLN7 is localized at the plasma membrane. Mutation of the dileucine motif or the co-expression of dominantnegative mutant dynamin K44A led to a further increase of CLN7 at the plasma membrane. Our data demonstrate that CLN7 contains several cytoplasmic lysosomal targeting signals of which the N-terminal dileucine-based motif is required for the predominant lysosomal targeting along the indirect pathway and clathrin-mediated endocytosis of CLN7.

Plasma Membrane Association of p63 Rho Guanine Nucleotide Exchange Factor (p63RhoGEF) Is Mediated by Palmitoylation and Is Required for Basal Activity in Cells

“…3A). For many proteins, di-Leu-based motifs are known to play a role in trafficking and PM targeting (32,33). However, deletion of this motif in p63RhoGEF did not perturb PM localization (Fig.…”

Mutations in MFSD8, Encoding a Lysosomal Membrane Protein, Are Associated with Nonsyndromic Autosomal Recessive Macular Dystrophy

“…A main dileucine-based motif at the N-terminus is presumed to direct MFSD8 to the lysosomes. 39,46,47 Studies in gene-targeted mouse embryonic fibroblasts show that MFSD8 is asymmetrically double cleaved for proper function. 48 Analysis of 2 missense mutations demonstrates that the cleavage occurs in the luminal L9 loop, and the proteolytic cleavage is altered by these mutations.…”

The Endocytosis and Signaling of the γδ T Cell Coreceptor WC1 Are Regulated by a Dileucine Motif

“…In addition, WC1 dileucine mutations are associated with a higher overall CD4/WC1 protein level per CD4/WC1 gene copy number. The dileucine motif was shown to be a sorting signal for routing gp130 and CLN7 through the multivesicle bodies to the lysosomes (37,38); thus, the dileucine motif mutants may increase overall CD4/ WC1 protein by not routing it to lysosomes for degradation.…”