Obermüller Stefanie, Kiecke Christina, von Figura Kurt, Höning Stefan
Institute for Biochemistry II, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany.
J Cell Sci. 2002 Jan 1;115(Pt 1):185-94. doi: 10.1242/jcs.115.1.185.
Lamp 1 and lysosomal acid phosphatase (LAP) are lysosomal membrane proteins that harbour a tyrosine-based sorting motif within their short cytoplasmic tails. Lamp 1 is delivered from the trans-Golgi network (TGN) via endosomes directly to lysosomes bypassing the plasma membrane, whereas LAP is indirectly transported to lysosomes and recycles between endosomes and the plasma membrane before being delivered to lysosomes. By analysing truncated forms of LAP and chimeras in which the cytoplasmic tail or part of the cytoplasmic tails of LAP and Lamp 1 were exchanged, we were able to show that the YRHV tyrosine motif of LAP is necessary and sufficient to mediate recycling between endosomes and the plasma membrane. When peptides corresponding to the cytoplasmic tails of LAP and Lamp 1 and chimeric or mutant forms of these tails were assayed for in vitro binding of AP1 and AP2, we found that AP2 bound to LAP- and Lamp-1-derived peptides, whereas AP1 bound only to peptides containing the YQTI tyrosine motif of Lamp 1. Residues +2 and +3 of the tyrosine motif were critical for the differential binding of adaptors. LAP in which these residues (-HV) were substituted for those of Lamp 1 (-TI) was transported directly to lysosomes, whereas a chimera carrying the Lamp 1 tail in which residues +2 and +3 were substituted for those of LAP (-HV) gained the ability to recycle. In conclusion, the residues +2 and +3 of the tyrosine motifs determine the sorting of Lamp 1 and LAP in endosomes, mediating either the direct or the indirect pathway to lysosomes.
Lamp 1和溶酶体酸性磷酸酶（LAP）是溶酶体膜蛋白，在其短细胞质尾巴内含有基于酪氨酸的分选基序。Lamp 1从反式高尔基体网络（TGN）经内体直接输送到溶酶体，绕过质膜，而LAP则间接转运到溶酶体，并在内体和质膜之间循环，然后再被输送到溶酶体。通过分析LAP的截短形式以及LAP和Lamp 1的细胞质尾巴或部分细胞质尾巴进行交换的嵌合体，我们能够证明LAP的YRHV酪氨酸基序对于介导内体和质膜之间的循环是必要且充分的。当检测与LAP和Lamp 1的细胞质尾巴相对应的肽以及这些尾巴的嵌合或突变形式在体外与AP1和AP2的结合时，我们发现AP2与源自LAP和Lamp-1的肽结合，而AP1仅与含有Lamp 1的YQTI酪氨酸基序的肽结合。酪氨酸基序的+2和+3位残基对于衔接蛋白的差异结合至关重要。将这些残基（-HV）替换为Lamp 1的残基（-TI）的LAP被直接转运到溶酶体，而携带Lamp 1尾巴且其+2和+3位残基被替换为LAP的残基（-HV）的嵌合体获得了循环能力。总之，酪氨酸基序的+2和+3位残基决定了Lamp 1和LAP在内体中的分选，介导了通向溶酶体的直接或间接途径。
