Hille A, Klumperman J, Geuze H J, Peters C, Brodsky F M, von Figura K
Biochemie II, Universität Göttingen, Deutschland.
Eur J Cell Biol. 1992 Oct;59(1):106-15.
The presence of lysosomal acid phosphatase (LAP) in coated pits at the plasma membrane was investigated by immunocytochemistry in thymidine kinase negative mouse L-cells (Ltk-) and baby hamster kidney (BHK) cells overexpressing human LAP (Ltk-LAP and BHK-LAP cells). Double immunogold labeling showed that at various stages of invaginating coated pits LAP colocalized with clathrin and plasma membrane adaptors (HA-2 adaptors). Quantitation of the immunogold label showed similar density of wild-type LAP in coated over non-coated areas of the plasma membrane, whereas an internalization-deficient, truncated mutant of LAP which lacks the cytoplasmic tail was less efficiently included into coated pits. Internalization of anti-LAP antibodies into endosomal vesicles was accompanied by rapid dissociation of the coat proteins as shown by an immunofluorescence assay. The role of clathrin-coated vesicles in internalization of LAP was further corroborated by microinjecting monoclonal antibodies against clathrin or HA-2 adaptors into BHK-LAP cells. Internalization of LAP as detected by an immunofluorescence assay was transiently blocked by microinjected antibodies against clathrin or HA-2 adaptors, whereas unrelated antibodies did not affect internalization. These data suggest that LAP is included into clathrin-coated pits of the plasma membrane for rapid internalization.
通过免疫细胞化学方法，在胸苷激酶阴性的小鼠L细胞（Ltk-）和过表达人溶酶体酸性磷酸酶（LAP）的幼仓鼠肾（BHK）细胞（Ltk-LAP和BHK-LAP细胞）中，研究了质膜包被小窝中溶酶体酸性磷酸酶（LAP）的存在情况。双重免疫金标记显示，在包被小窝内陷的各个阶段，LAP与网格蛋白和质膜衔接蛋白（HA-2衔接蛋白）共定位。免疫金标记的定量分析表明，野生型LAP在质膜包被区域和非包被区域的密度相似，而缺乏细胞质尾巴的内化缺陷型截短突变体LAP则较难被纳入包被小窝。免疫荧光分析显示，抗LAP抗体内化到内体小泡中时，包被蛋白会迅速解离。通过向BHK-LAP细胞显微注射抗网格蛋白或HA-2衔接蛋白的单克隆抗体，进一步证实了网格蛋白包被小泡在LAP内化过程中的作用。免疫荧光分析检测到的LAP内化被显微注射的抗网格蛋白或HA-2衔接蛋白抗体短暂阻断，而无关抗体则不影响内化。这些数据表明，LAP被纳入质膜的网格蛋白包被小窝以实现快速内化。
