Abstract：
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.
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包含三个剪接亚型（LAMP-2A，-B和-C）具有不同的细胞质尾巴（CT）。这三个CT具有不同的基于酪氨酸的基序(GYXXΦ，其中Φ是大体积的疏水氨基酸）在其C末端。酪氨酸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同种型在溶酶体中的定位知之甚少。首先使用酵母双杂交系统研究相互作用以解决这个问题。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大。LAMP-2B-CT与μ3A完全和适度地相互作用。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，所有同工型均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在致密溶酶体中含量最高,而LAMP-2C显著存在于含有质膜和早期内体的低密度组分中，除了致密的溶酶体。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同工型在内吞细胞器中的分布不同，这取决于它们与AP-3的相互作用强度。
