Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

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【标题】Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

【作者】 Breyan H. Ross  Yimo Lin  Esteban A. Corales  Patricia V. Burgos  Gonzalo A. Mardones 

【关键词】AP-4 Binding APP AP-1 YXX 

【摘要】Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXX?-sequences (? is a bulky hydrophobic residue); which are sorting signals in transmembrane proteins. A conserved; canonical region in μ subunits mediates recognition of YXX?-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXX?-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXX?-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site; in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXX?-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable; suggesting an explanation for its lower binding affinity to the APP signal. Finally; in contrast to overexpression of the D190A mutant; and acting in a dominant-negative manner; overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together; our analyses support that the functional recognition of the non-canonical YXX?-signal of APP is limited to the non-canonical site of μ4.