Cell Biology and Metabolism Program, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2011 Jan 21;286(3):2022-30. doi: 10.1074/jbc.M110.197178. Epub 2010 Nov 19.
The clathrin-associated, heterotetrameric adaptor protein (AP) complexes, AP-1, AP-2, and AP-3, recognize signals in the cytosolic domains of transmembrane proteins, leading to their sorting to endosomes, lysosomes, lysosome-related organelles, and/or the basolateral membrane of polarized epithelial cells. One type of signal, referred to as "dileucine-based," fits the consensus motif (D/E)XXXL(L/I). Previous biochemical analyses showed that (D/E)XXXL(L/I) signals bind to a combination of two subunits of each AP complex, namely the AP-1 γ-σ1, AP-2 α-σ2, and AP-3 δ-σ3 hemicomplexes, and structural studies revealed that an imperfect variant of this motif lacking the (D/E) residue binds to a site straddling the interface of α and σ2. Herein, we report mutational and binding analyses showing that canonical (D/E)XXXL(L/I) signals bind to this same site on AP-2, and to similar sites on AP-1 and AP-3. The strength and amino acid requirements of different interactions depend on the specific signals and AP complexes involved. We also demonstrate the occurrence of diverse AP-1 heterotetramers by combinatorial assembly of various γ and σ1 subunit isoforms encoded by different genes. These AP-1 variants bind (D/E)XXXL(L/I) signals with marked preferences for certain sequences, implying that they are not functionally equivalent. Our results thus demonstrate that different AP complexes share a conserved binding site for (D/E)XXXL(L/I) signals. However, the characteristics of the binding site on each complex vary, providing for the specific recognition of a diverse repertoire of (D/E)XXXL(L/I) signals.
网格蛋白相关的、异四聚体衔接蛋白（AP）复合物，AP-1、AP-2 和 AP-3，识别跨膜蛋白胞质域中的信号，导致它们分选到内体、溶酶体、溶酶体相关细胞器和/或极化上皮细胞的基底外侧膜。一种信号类型，称为“双亮氨酸基”，符合共有基序（D/E）XXXL（L/I）。以前的生化分析表明，（D/E）XXXL（L/I）信号与每个 AP 复合物的两个亚基结合，即 AP-1γ-σ1、AP-2α-σ2 和 AP-3δ-σ3 半复合物，结构研究表明，该基序的不完美变体，缺少（D/E）残基，与横跨α和σ2 界面的位点结合。在此，我们报告突变和结合分析表明，典型的（D/E）XXXL（L/I）信号与 AP-2 上的相同位点结合，并与 AP-1 和 AP-3 上的类似位点结合。不同相互作用的强度和氨基酸要求取决于特定的信号和参与的 AP 复合物。我们还通过不同基因编码的各种γ和σ1 亚基同工型的组合，证明了不同 AP-1 异四聚体的存在。这些 AP-1 变体对不同序列具有明显的（D/E）XXXL（L/I）信号结合偏好，表明它们在功能上并不等同。因此，我们的结果表明，不同的 AP 复合物共享（D/E）XXXL（L/I）信号的保守结合位点。然而，每个复合物上的结合位点的特征不同，为不同的（D/E）XXXL（L/I）信号提供了特定的识别。
