Characterization of the ubiquitin-associated domain of αNAC

Abstract

Protein biogenesis in cells is carried out by cytosolic ribosomes, which translate proteins from all cellular compartments, including the cytosol, mitochondria, and endoplasmic reticulum (ER). While cytosolic proteins must be folded directly, mitochondrial and ER proteins must first be transported to the corresponding cell organelle to become functionally active. The newly synthesized proteins are co-translationally sorted into the correct biogenesis pathway by ribosome-associated factors that bind to the tunnel exit of the ribosome, where the nascent chains are released into the cytosol. The first factor that interacts with translated proteins in eukaryotes is the nascent polypeptide-associated complex (NAC), a heterodimeric complex consisting of an α and β subunit (αNAC and βNAC). NAC is an essential factor that prevents the ER targeting factor signal recognition particle (SRP) from binding nonspecifically to ribosomes that translate mitochondrial or cytosolic substrates to prevent mislocalization of proteins in cells. The mechanism of how NAC inhibits the ER targeting machinery and how this inhibition is overcome on ribosomes translating ER proteins is unknown. The main objective of the present work was to gain a deeper understanding of the interaction mechanism of NAC with ribosomes and other co-translational protein biogenesis factors. Focus was placed on a conserved structured domain, the ubiquitin-associated domain (UBA),