RESUMO
Mitochondrial ribosomes (mitoribosomes) synthesize proteins encoded within the mitochondrial genome that are assembled into oxidative phosphorylation complexes. Thus, mitoribosome biogenesis is essential for ATP production and cellular metabolism1. Here we used cryo-electron microscopy to determine nine structures of native yeast and human mitoribosomal small subunit assembly intermediates, illuminating the mechanistic basis for how GTPases are used to control early steps of decoding centre formation, how initial rRNA folding and processing events are mediated, and how mitoribosomal proteins have active roles during assembly. Furthermore, this series of intermediates from two species with divergent mitoribosomal architecture uncovers both conserved principles and species-specific adaptations that govern the maturation of mitoribosomal small subunits in eukaryotes. By revealing the dynamic interplay between assembly factors, mitoribosomal proteins and rRNA that are required to generate functional subunits, our structural analysis provides a vignette for how molecular complexity and diversity can evolve in large ribonucleoprotein assemblies.
Assuntos
Microscopia Crioeletrônica , Ribossomos Mitocondriais , Ribonucleoproteínas , Subunidades Ribossômicas Menores , Saccharomyces cerevisiae , Humanos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Ribossomos Mitocondriais/química , Ribossomos Mitocondriais/metabolismo , Ribossomos Mitocondriais/ultraestrutura , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/ultraestrutura , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , RNA Ribossômico , GTP Fosfo-Hidrolases , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestrutura , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/ultraestrutura , Subunidades Ribossômicas Menores/química , Subunidades Ribossômicas Menores/metabolismo , Subunidades Ribossômicas Menores/ultraestruturaRESUMO
The PINK1 protein kinase activates the PARK2 ubiquitin ligase to promote mitochondrial ubiquitylation and recruitment of ubiquitin-binding mitophagy receptors typified by OPTN and TAX1BP1. Here, we combine proximity biotinylation of OPTN and TAX1BP1 with CRISPR-Cas9-based screens for mitophagic flux to develop a spatial proteogenetic map of PARK2-dependent mitophagy. Proximity labeling of OPTN allowed visualization of a "mitochondrial-autophagosome synapse" upon mitochondrial depolarization. Proximity proteomics of OPTN and TAX1BP1 revealed numerous proteins at the synapse, including both PARK2 substrates and autophagy components. Parallel mitophagic flux screens identified proteins with roles in autophagy, vesicle formation and fusion, as well as PARK2 targets, many of which were also identified via proximity proteomics. One protein identified in both approaches, HK2, promotes assembly of a high-molecular weight complex of PINK1 and phosphorylation of ubiquitin in response to mitochondrial damage. This work provides a resource for understanding the spatial and molecular landscape of PARK2-dependent mitophagy.
