Analysis of intracellular distribution and trafficking of the CLN3 proteinin fusion with the green fluorescent protein in vitro

CLN3 gene, associated with juvenile neuronal ceroid lipofuscinosis, encodes a novel protein of a predicted 438 amino acid residues. We have expressed a full-length CLN3 protein and fragments thereof in fusion with green fluor escent protein in Chinese hamster ovary and human neuroblastoma cell lines to study its subcellular localization and intracellular trafficking pattern . By using laser scanning confocal microscopy, we demonstrate that the full -length CLN3 fusion protein is targeted to lysosomal compartments. Tunicamy cin treatment did not alter the lysosomal targeting of the CLN3 protein, wh ich indicates that extensive N-glycosylation of the full-length CLN3 fusion protein is not engaged in its lysosomal sorting. Monensin produced retenti on of CLN3 fusion protein in vesicular structure of the Golgi apparatus in the perinuclear space, suggesting that CLN3 fusion protein is transported t o the lysosomal compartments through the trans-Golgi cisternae, Neither of the truncated CLN3 fusion proteins encompassing its 1-138, 1-322, and 138-4 38 amino acid residues was disclosed in lysosomal compartments. However, CL N3 fusion protein showing double-point mutations at amino acid residues 425 and 426, thus at its putative dileucine lysosomal signaling motif, was sti ll targeted to lysosomes, suggesting that a dileucine motif alone is not su fficient for lysosomal sorting of the CLN3 fusion protein, (C) 1999 Academi c Press.