Analysis of intracellular distribution and trafficking of the CLN3 protein in fusion with the green fluorescent protein in vitro
Analysis of intracellular distribution and trafficking of the CLN3 protein in fusion with the green fluorescent protein in vitro
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DOI：
10.1006/mgme.1999.2837
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发表时间：
1999-04-01
影响因子：
3.8
通讯作者：
Wisniewski, KE
中科院分区：
文献类型：
--
作者：
Kida, E;Kaczmarski, W;Wisniewski, KE
CLN 3基因与幼年型神经元蜡样质脂褐质沉积症相关，编码一种新的蛋白质，预测为438个氨基酸残基。我们已经在中国仓鼠卵巢和人神经母细胞瘤细胞系中表达了全长CLN 3蛋白及其片段与绿色荧光蛋白的融合，以研究其亚细胞定位和细胞内运输模式。通过使用激光扫描共聚焦显微镜，我们证明了全长CLN 3融合蛋白是针对溶酶体隔室。衣霉素处理不改变CLN 3蛋白的溶酶体靶向，这表明全长CLN 3融合蛋白的广泛N-糖基化不参与其溶酶体分选。莫能菌素使CLN 3融合蛋白保留在核周空间中高尔基体的囊泡结构中，表明CLN 3融合蛋白通过高尔基体池转运到溶酶体区室。在溶酶体区室中没有公开包含其1-138、1-322和138-438个氨基酸残基的截短的CLN 3融合蛋白。然而，在氨基酸残基425和426处，因此在其推定的二亮氨酸溶酶体信号传导基序处显示双点突变的CLN 3融合蛋白仍然靶向溶酶体，表明单独的二亮氨酸基序不足以用于CLN 3融合蛋白的溶酶体分选，（C）1999 Academic Press。
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, (C) 1999 Academic Press.