Cotranslational roles of NAC in protein biogenesis : a functional analysis in C. elegans

Abstract

Proteostasis, the spatiotemporal control of protein abundance, location, and folding status within a cell, depends on the continuous renewal of the proteome. Newly synthesised proteins undergo cotranslational processing by various protein biogenesis factors that exert different substrate-specific activities, including protein modification, folding, targeting, and degradation. These factors use the ribosome as a central binding platform to engage nascent polypeptides as soon as they emerge from the ribosomal exit tunnel. Most of these factors are expressed sub-stoichiometrically compared to ribosomes and compete for access to the ribosome exit site. How these factors gain timely and specific access to their nascent substrates and how they affect each other's binding properties is not well understood. The nascent polypeptide-associated complex (NAC) is an essential and highly abundant ribosome-associated factor in eukaryotes. The heterodimeric NAC complex (NACα and NACβ) is characterised by a distinctive structural architecture, defined by a central globular domain and long, flexible N- and C-termini, which are utilised for ribosome binding and factor recruitment. Functionally, it has been connected to cotranslational targeting to the ER and mitochondria, maturation of cyto-nuclear nascent polypeptides, and chaperoning of nascent polypeptides. Using a structure-guided in vivo approach in C. elegans, this study demonstrates that NAC employs conserved features within its flexible C-termini to recruit methionine aminopeptidase 1 (MetAP1) and N-terminal acetyl transferase A (NatA) to ribosomes, thereby facilitating initiator methionine excision and subsequent acetylation of the resulting N terminal primary amine. Moreover, it is shown that NAC globular domain attachment to the ribosomal tunnel exit is crucial for cotranslational initiator methionine excision by MetAP1. Hydrophobic signal sequences of ER-proteins, which escape modification by MetAP1 and NatA, destabilise this specific NAC conformation and disfavour MetAP1 enzymatic action. This explains how a subset of 30 % of the proteome bypasses subsequent modification by MetAP1 and NatA. Additionally, this thesis provides the first mechanistic insights into NatA regulation by the inhibitor Huntingtin Interacting Protein K (HYPK). Given HYPK’s high affinity to NatA and its approximately equimolar cellular concentration, the majority of NatA is likely inhibited. In a collaborative effort employing structural elucidation of ribosome bound NatA-HYPK in complex with NAC, in vitro N-terminal acetylation reconstitution assays, and a genetic C. elegans approach, it is revealed that HYPK inhibition is alleviated at the ribosome through an allosteric mechanism dependent on the NAC-NatA interaction. The mechanisms governing NAC binding to translating ribosomes are currently not well understood, particularly in the context of cotranslational folding and targeting to mitochondria. To address these questions, this thesis employs a NAC-selective ribosome profiling approach. A protocol was established to purify NAC-bound ribosome-nascent chain complexes (RNCs) from C. elegans. In a collaborative effort, ribosome-protected mRNA footprints were recovered and deep-sequenced, enabling a global and near-residue resolution analysis of the in vivo NAC-nascent chain interactome. The data reveal that NAC resides at RNCs during the translation of at least a third of the proteome. NAC interaction sites are observed throughout translation and can be categorised into three distinct regions: 1) soon after translation initiation, when the nascent chain is still buried deep within the ribosomal tunnel; 2) as the nascent chain emerges from the ribosomal tunnel exit; 3) and later in translation when nascent polypeptides start to fold. Interactions during tunnel exposure of nascent chains correspond well with NAC’s established function in ER targeting and cotranslational maturation of cyto-nuclear N-termini, as demonstrated in this thesis. Late interactions to nascent cyto-nuclear proteins support a cotranslational chaperone function of NAC. These interactions coincide with the emergence of hydrophobic α-helical structures with high amphipathic character, here defined as a NAC chaperone recognition motif. Additionally, NAC was found to contact RNCs translating transmembrane domains (TMDs) of mitochondrial proteins, but was notably absent during the exposure of mitochondrial targeting sequences (MTSs). A microscopy-based approach in C. elegans supports that NAC chaperones these TMDs to facilitate cotranslational import of mitochondrial membrane proteins.