Ogata S, Fukuda M
Glycobiology Program, La Jolla Cancer Research Foundation, California 92037.
J Biol Chem. 1994 Feb 18;269(7):5210-7.
A class of integral membrane glycoproteins specific to lysosomes has been identified, and they are classified into two separate groups depending on whether or not their cytoplasmic sequence contains a tyrosine residue. Lamp-1 and lamp-2 have a tyrosine-containing motif in their cytoplasmic segments, and this motif was found to direct the glycoproteins to lysosomes. Limp II glycoprotein, on the other hand, lacks a tyrosine in its cytoplasmic segment and it must be directed to lysosomes by a different signal (Fukuda, M. (1991) J. Biol. Chem. 266, 21327-21330). In order to elucidate the targeting signal of Limp II, a cDNA encoding its cytoplasmic segment was fused with a reporter molecule, a chimeric protein of human gonadotropin alpha chain-vesicular stomatitis G-protein transmembrane. After various mutations its expression was examined by immunofluorescence. First it was shown that a chimeric protein with a Limp II wild-type tail is transported to lysosomes. Deletion of the three amino acids of the cytoplasmic tail at the carboxyl terminus abolished this sorting to lysosomes. Substitution of individual amino acids revealed that the Leu-Ile motif in the Leu-Ile-Arg-Thr sequence at the carboxyl terminus is crucial to the sorting signal. When this motif was brought closer to the transmembrane domain by deletion of nine amino acids next to the transmembrane domain, this sorting function was abolished. In addition, substitution of alanine for the serine, which is at 5 residues from the transmembrane also abolished the sorting capacity, although there was no evidence that the phosphorylation of serine is involved in sorting. Altered proteins that were not transported to lysosomes were found to accumulate at the cell surface and, unlike proteins with a wild-type cytoplasmic tail, were unable to undergo endocytosis. These results indicate that the carboxyl-terminal amino acid sequence, including the Leu-Ile motif and the sequence that connects the motif to the transmembrane domain, is critical for the sorting of Limp II to lysosomes.
一类溶酶体特有的整合膜糖蛋白已被鉴定出来，根据其胞质序列是否含有酪氨酸残基，它们被分为两个不同的组。Lamp-1和Lamp-2在其胞质段含有一个含酪氨酸的基序，并且发现该基序可将糖蛋白导向溶酶体。另一方面，Limp II糖蛋白在其胞质段缺乏酪氨酸，它必须通过不同的信号被导向溶酶体（深田，M.（1991年）《生物化学杂志》266，21327 - 21330）。为了阐明Limp II的靶向信号，编码其胞质段的cDNA与一种报告分子融合，该报告分子是人类促性腺激素α链 - 水泡性口炎G蛋白跨膜的嵌合蛋白。在进行各种突变后，通过免疫荧光检查其表达。首先表明，具有Limp II野生型尾部的嵌合蛋白被转运到溶酶体。在羧基末端缺失胞质尾部的三个氨基酸消除了这种向溶酶体的分选。单个氨基酸的替换表明，羧基末端Leu - Ile - Arg - Thr序列中的Leu - Ile基序对分选信号至关重要。当通过缺失跨膜结构域旁边的九个氨基酸使该基序更靠近跨膜结构域时，这种分选功能被消除。此外，用丙氨酸替换距跨膜结构域5个残基处的丝氨酸也消除了分选能力，尽管没有证据表明丝氨酸的磷酸化参与分选。未被转运到溶酶体的改变后的蛋白质被发现积聚在细胞表面，并且与具有野生型胞质尾部的蛋白质不同，它们无法进行内吞作用。这些结果表明，包括Leu - Ile基序以及将该基序与跨膜结构域连接的序列在内的羧基末端氨基酸序列对于Limp II分选到溶酶体至关重要。
