Analysis of intracellular distribution and trafficking of the CLN3 protein in 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 fluorescent 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. Tunicamycin treatment did not alter the lysosomal targeting of the CLN3 protein, which indicates that extensive N-glycosylation of the full-length CLN3 fusion protein is not engaged in its lysosomal sorting. Monensin produced retention of CLN3 fusion protein in vesicular structure of the Golgi apparatus in the perinuclear space, suggesting that CLN3 fusion protein is transported to the lysosomal compartments through the trans-Golgi cisternae. Neither of the truncated CLN3 fusion proteins encompassing its 1-138, 1-322, and 138-438 amino acid residues was disclosed in lysosomal compartments. However, CLN3 fusion protein showing double-point mutations at amino acid residues 425 and 426, thus at its putative dileucine lysosomal signaling motif, was still targeted to lysosomes, suggesting that a dileucine motif alone is not sufficient for lysosomal sorting of the CLN3 fusion protein.
Kida
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Department of Pathological Neurobiology, NYS Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA.
Kaczmarski
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W
Golabek
A A
AA
Kaczmarski
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A
Michalewski
M
M
Wisniewski
K E
KE
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D016213:Cyclins; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000378:metabolism*; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018832:Molecular Chaperones / Q000378:metabolism*; D008985:Monensin / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014415:Tunicamycin / Q000378:metabolism
D015703:Antigens, CD; C079895:CLN3 protein, S cerevisiae; C105199:CLN3 protein, human; D016213:Cyclins; D008164:Luminescent Proteins; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014415:Tunicamycin; D049452:Green Fluorescent Proteins; D008985:Monensin