Summary Lysosome-associated membrane protein-1 and -2 (LAMP-1 and LAMP-2, respectively) are type I transmembrane proteins. LAMP-2 comprises three splice isoforms (LAMP-2A, -B, and-C) with different cytoplasmic tails (CTs). These three CTs possess different tyrosine-based motifs (GYXXΦ, where Φ is a bulky hydrophobic amino acid) at their C-termini. Interactions between tyrosine-based motifs and μ-subunits of four tetrameric adaptor protein (AP) complexes are necessary for their vesicular transport to lysosomes. Little is known about how the interaction strengths of these tyrosine motifs with μ-subunits affect the localization of isoforms to lysosomes. The interactions were first investigated using a yeast two-hybrid system to address this question. LAMP-2A-CT interacted with all four μ-subunits (μ1, μ2, μ3A, and μ4 of AP-1, AP-2, AP-3, and AP-4, respectively). The interaction with μ3A was more robust than that with other μ-subunits. LAMP-2B-CT interacted exclusively and moderately with μ3A. LAMP-2C-CT did not detectably interact with any of the four μ-subunits. Immunofluorescence microscopy showed that all isoforms were localized in late endosomes and lysosomes. LAMP-2C was present in the plasma membrane and early endosomes; however, LAMP-2A and -2B were barely detectable in these organelles. In cell fractionation, LAMP-2A was the most abundant in the dense lysosomes, whereas LAMP-2C was significantly present in the low-density fraction containing the plasma membrane and early endosomes, in addition to the dense lysosomes. LAMP-2B considerably existed in the low-density late endosomal fraction. These data strongly suggest that the LAMP-2 isoforms are distributed differently in endocytic organelles depending on their interaction strengths with AP-3.

中文翻译：

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溶酶体相关膜糖蛋白 2 (LAMP-2) 亚细胞稳态亚细胞分布的比较研究，其与 GYXXΦ 型基于酪氨酸的基序与四种接头蛋白 (AP) 复合物的相互作用不同

摘要 溶酶体相关膜蛋白-1 和-2（分别为 LAMP-1 和 LAMP-2）是 I 型跨膜蛋白。LAMP-2 包含具有不同胞质尾部 (CT) 的三种剪接亚型（LAMP-2A、-B 和-C）。这三个 CT 在其 C 末端具有不同的基于酪氨酸的基序（GYXXΦ，其中 Φ 是一个大的疏水氨基酸）。基于酪氨酸的基序和四个四聚体衔接蛋白 (AP) 复合物的 μ 亚基之间的相互作用对于它们的囊泡转运至溶酶体是必需的。关于这些酪氨酸基序与μ亚基的相互作用强度如何影响异构体在溶酶体中的定位，人们知之甚少。为了解决这个问题，首先使用酵母双杂交系统研究了相互作用。LAMP-2A-CT 与所有四个 μ 亚基（分别为 AP-1、AP-2、AP-3 和 AP-4 的 μ1、μ2、μ3A 和 μ4）相互作用。与μ3A的相互作用比与其他μ亚基的相互作用更强。LAMP-2B-CT 与 μ3A 发生专门且适度的相互作用。LAMP-2C-CT 未检测到与四个 μ 亚基中的任何一个相互作用。免疫荧光显微镜显示所有亚型都位于晚期内体和溶酶体中。LAMP-2C 存在于质膜和早期内体中；然而，在这些细胞器中几乎检测不到 LAMP-2A 和 -2B。在细胞分级分离中，LAMP-2A 在致密溶酶体中最丰富，而除了致密溶酶体之外，LAMP-2C 在含有质膜和早期内体的低密度级分中也显着存在。LAMP-2B大量存在于低密度晚期内体部分中。这些数据强烈表明，LAMP-2 亚型在内吞细胞器中的分布不同，具体取决于它们与 AP-3 的相互作用强度。