Characterization of a Fourth Adaptor-related Protein Complex

Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat. To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (ε, β4, μ4, and ς4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 is localized to ∼10–20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF. Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The μ4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.

衔接蛋白复合物（adaptor protein complexes，APs）在不同膜运输通路中作为囊泡被膜组分发挥功能；然而目前仍有多种通路尚未发现对应的被膜候选组分。为挖掘与AP复合物相关的新型被膜组分，我们检索了表达序列标签（expressed sequence tag，EST）数据库，鉴定、克隆并测序了四个AP亚基家族各自的新成员。我们通过免疫共沉淀与酵母双杂交分析联用实验证实，这四种蛋白（ε、β4、μ4和ς4）构成了一种新型类衔接蛋白异四聚体复合物，我们将其命名为AP-4。免疫荧光实验结果显示，AP-4定位于细胞的核周区域，呈现约10~20个离散斑点的分布模式。用布雷菲德菌素A处理细胞可破坏该分布模式，这表明与其他被膜蛋白类似，AP-4与膜结构的结合受小GTP酶ARF调控。免疫金标记电子显微镜实验表明，AP-4定位于反式高尔基体网络（trans-Golgi network，TGN）区域的无网格蛋白包被囊泡。该复合物的μ4亚基可特异性结合基于酪氨酸的分选信号，这提示AP-4与其他三类AP复合物一样，参与识别并分选携带酪氨酸基序的货物蛋白。AP-4的丰度相对较低，但呈遍在表达模式，这表明其参与一条特化的运输通路，而该通路在所有细胞类型中均为必需。