Dimerization of mitophagy receptor BNIP3L/NIX is essential for recruitment of autophagic machinery

Mitophagy is a conserved intracellular catabolic process responsible for the selective removal of dysfunctional or superfluous mitochondria to maintain mitochondrial quality and need in cells. Here, we examine the mechanisms of receptor-mediated mitophagy activation, with the focus on BNIP3L/NIX mitophagy receptor, proven to be indispensable for selective removal of mitochondria during the terminal differentiation of reticulocytes. The molecular mechanisms of selecting damaged mitochondria from healthy ones are still very obscure. We investigated BNIP3L dimerization as a potentially novel molecular mechanism underlying BNIP3L-dependent mitophagy. Forming stable homodimers, BNIP3L recruits autophagosomes more robustly than its monomeric form. Amino acid substitutions of key transmembrane residues of BNIP3L, BNIP3L^G204A or BNIP3L^G208V, led to the abolishment of dimer formation, resulting in the lower LC3A-BNIP3L recognition and subsequently lower mitophagy induction. Moreover, we identified the serine 212 as the main amino acid residue at the C-terminal of BNIP3L, which extends to the intermembrane space, responsible for dimerization. In accordance, the phosphomimetic mutation BNIP3L^S212E leads to a complete loss of BNIP3L dimerization. Thus, the interplay between BNIP3L phosphorylation and dimerization indicates that the combined mechanism of LIR phosphorylation and receptor dimerization is needed for proper BNIP3L-dependent mitophagy initiation and progression. <b>Abbreviations:</b> AMBRA1: autophagy and beclin 1 regulator 1; Baf A1: bafilomycin A₁; BH3: BCL2 homology 3; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CoCl₂: cobalt (II) chloride; FKBP8: FKBP prolyl isomerase 8; FUNDC1: FUN14 domain containing 1; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OMM: outer mitochondrial membrane; PHB2: prohibitin 2; PI: propidium iodide; PINK1: PTEN induced kinase 1; TM: transmembrane domain; TOMM20: translocase of outer mitochondrial membrane 20

线粒体自噬（Mitophagy）是一种保守的胞内分解代谢过程，负责选择性清除功能异常或多余的线粒体，以维持细胞内线粒体的质量与稳态需求。本研究探讨了受体介导的线粒体自噬激活机制，重点聚焦于BNIP3L/NIX线粒体自噬受体——该受体已被证实为网织红细胞终末分化过程中选择性清除线粒体所不可或缺的因子。区分受损线粒体与健康线粒体的分子机制仍不甚明晰。我们研究了BNIP3L二聚化作为依赖BNIP3L的线粒体自噬的潜在新型分子机制。BNIP3L通过形成稳定的同源二聚体，相较于其单体形式，能够更高效地招募自噬体。对BNIP3L关键跨膜残基进行氨基酸替换，即引入BNIP3L^G204A或BNIP3L^G208V突变，会破坏其二聚体的形成，导致LC3A与BNIP3L的识别效率降低，进而减弱线粒体自噬的诱导效果。此外，我们鉴定出BNIP3L羧基端延伸至线粒体膜间隙的丝氨酸212为调控其二聚化的关键氨基酸残基。与之相符的是，磷酸化模拟突变体BNIP3L^S212E会完全丧失BNIP3L的二聚化能力。综上，BNIP3L磷酸化与二聚化之间的相互作用表明，要实现依赖BNIP3L的线粒体自噬的正常启动与进展，需要结合LC3相互作用区域（LC3-interacting region）磷酸化与受体二聚化的联合调控机制。
**缩写说明：** AMBRA1：自噬与Beclin 1调控因子1（autophagy and beclin 1 regulator 1）；Baf A1：巴弗洛霉素A₁（bafilomycin A₁）；BH3：BCL2同源结构域3（BCL2 homology 3）；BNIP3：BCL2相互作用蛋白3（BCL2 interacting protein 3）；BNIP3L/NIX：BCL2相互作用蛋白3样蛋白（BCL2 interacting protein 3 like）；CCCP：羰基氰化物3-氯苯腙（carbonyl cyanide 3-chlorophenylhydrazone）；CoCl₂：氯化钴(II)（cobalt (II) chloride）；FKBP8：FKBP脯氨酰异构酶8（FKBP prolyl isomerase 8）；FUNDC1：FUN14结构域包含蛋白1（FUN14 domain containing 1）；GABARAP：GABA型A受体相关蛋白（GABA type A receptor-associated protein）；GST：谷胱甘肽S-转移酶（glutathione S-transferase）；IMM：线粒体内膜（inner mitochondrial membrane）；LIR：LC3相互作用区域（LC3-interacting region）；MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）；OMM：线粒体外膜（outer mitochondrial membrane）；PHB2：抑制素2（prohibitin 2）；PI：碘化丙啶（propidium iodide）；PINK1：PTEN诱导激酶1（PTEN induced kinase 1）；TM：跨膜结构域（transmembrane domain）；TOMM20：线粒体外膜转位酶20（translocase of outer mitochondrial membrane 20）