Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, USA.
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, USA.
J Biol Chem. 2021 Nov;297(5):101328. doi: 10.1016/j.jbc.2021.101328. Epub 2021 Oct 22.
Human apoptosis-linked gene-2 interacting protein X (ALIX), a versatile adapter protein, regulates essential cellular processes by shuttling between late endosomal membranes and the cytosol, determined by its interactions with Src kinase. Here, we investigate the molecular basis of these transitions and the effects of tyrosine phosphorylation on the interplay between structure, assembly, and intramolecular and intermolecular interactions of ALIX. As evidenced by transmission electron microscopy, fluorescence and circular dichroism spectroscopy, the proline-rich domain of ALIX, which encodes binding epitopes of multiple cellular partners, formed rope-like β-sheet-rich reversible amyloid fibrils that dissolved upon Src-mediated phosphorylation and were restored on protein-tyrosine phosphatase 1B-mediated dephosphorylation of its conserved tyrosine residues. Analyses of the Bro1 domain of ALIX by solution NMR spectroscopy elucidated the conformational changes originating from its phosphorylation by Src and established that Bro1 binds to hyperphosphorylated proline-rich domain and to analogs of late endosomal membranes via its highly basic surface. These results uncover the autoinhibition mechanism that relocates ALIX to the cytosol and the diverse roles played by tyrosine phosphorylation in cellular and membrane functions of ALIX.
人类凋亡连接基因 2 相互作用蛋白 X（ALIX），一种多功能衔接蛋白，通过在晚期内体膜和细胞质之间穿梭来调节重要的细胞过程，这取决于它与Src 激酶的相互作用。在这里，我们研究了这些转变的分子基础以及酪氨酸磷酸化对 ALIX 结构、组装以及分子内和分子间相互作用的相互作用的影响。正如透射电子显微镜、荧光和圆二色性光谱所证明的那样，ALIX 的富含脯氨酸结构域，它编码了多个细胞伴侣的结合表位，形成了类似绳索的富含β-片层的可逆淀粉样纤维，这些纤维在 Src 介导的磷酸化后溶解，并在保守的酪氨酸残基被蛋白酪氨酸磷酸酶 1B 去磷酸化后恢复。通过溶液 NMR 光谱分析 ALIX 的 Bro1 结构域，阐明了其 Src 磷酸化引起的构象变化，并证实 Bro1 通过其高度碱性表面与富含脯氨酸的结构域和晚期内体膜的类似物结合。这些结果揭示了将 ALIX 重新定位到细胞质的自动抑制机制，以及酪氨酸磷酸化在 ALIX 的细胞和膜功能中的多种作用。
