RESUMO
Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy.
Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia , Fosforilação Oxidativa , Proteína Sequestossoma-1/metabolismo , Animais , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/genética , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Sequestossoma-1/genética , Transdução de SinaisRESUMO
Autophagy is the degradation of cytoplasmic material through the lysosomal pathway. One of the most studied autophagy-related proteins is LC3. Despite growing evidence that LC3 is enriched in the nucleus, its nuclear role is poorly understood. Here, we show that Drosophila Atg8a protein, homologous to mammalian LC3, interacts with the transcription factor Sequoia in a LIR motif-dependent manner. We show that Sequoia depletion induces autophagy in nutrient-rich conditions through the enhanced expression of autophagy genes. We show that Atg8a interacts with YL-1, a component of a nuclear acetyltransferase complex, and that it is acetylated in nutrient-rich conditions. We also show that Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation to activate autophagy. Our results suggest a mechanism of regulation of the expression of autophagy genes by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1, and Sir2.
Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Chaperonas de Histonas/metabolismo , Histona Desacetilases/metabolismo , Sequoia/metabolismo , Sirtuínas/metabolismo , AnimaisRESUMO
p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 domain filament formation in vitro remain to be determined and the role of p62 filaments inside the cell is currently unclear. We here determine four high-resolution cryo-EM structures of different human and Arabidopsis PB1 domain assemblies and observed a filamentous ultrastructure of p62/SQSTM1 bodies using correlative cellular EM. We show that oligomerization or polymerization, driven by a double arginine finger in the PB1 domain, is a general requirement for lysosomal targeting of p62. Furthermore, the filamentous assembly state of p62 is required for autophagosomal processing of the p62-specific cargo KEAP1. Our results show that using such mechanisms, p62 filaments can be critical for cargo uptake in autophagy and are an integral part of phase-separated p62 bodies.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Transporte/química , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/química , Autofagia/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Células HeLa , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lisossomos/metabolismo , Polimerização , Conformação Proteica , Domínios Proteicos , Proteína Sequestossoma-1/genéticaRESUMO
Transmission Electron Microscopy (TEM) provides high resolution and accuracy at the subcellular level for observing and investigating cellular structures. This is essential for understanding a large variety of cellular processes. In this chapter, we describe a detailed protocol for preparing Drosophila follicles in order to be used as a specimen for transmission electron microscopy.