A Conserved Motif Is Prerequisite for the Interaction of NAC with Ribosomal Protein L23 and Nascent Chains

• Published in: The Journal of biological chemistry Vol. 281; no. 5; pp. 2847 - 2857
• Main Authors: Wegrzyn, Renee D., Hofmann, Diana, Merz, Frieder, Nikolay, Rainer, Rauch, Thomas, Graf, Christian, Deuerling, Elke
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.02.2006; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Motifs; Binding Sites; Conserved Sequence; DNA-Binding Proteins; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Molecular Chaperones; Mutation; Peptidylprolyl Isomerase; Protein Biosynthesis; Ribosomal Proteins - genetics; Ribosomal Proteins - metabolism; Ribosomes - metabolism; Trans-Activators - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M511420200

In eukaryotes, newly synthesized proteins interact co-translationally with a multitude of different ribosome-bound factors and chaperones including the conserved heterodimeric nascent polypeptide-associated complex (NAC) and a Hsp40/70-based chaperone system. These factors are thought to play an important role in protein folding and targeting, yet their specific ribosomal localizations, which are prerequisite for their functions, remain elusive. This study describes the ribosomal localization of NAC and the molecular details by which NAC is able to contact the ribosome and gain access to nascent polypeptides. We identified a conserved RRK(X)nKK ribosome binding motif within the β-subunit of NAC that is essential for the entire NAC complex to attach to ribosomes and allow for its interaction with nascent polypeptide chains. The motif localizes within a potential loop region between two predicted α-helices in the N terminus of βNAC. This N-terminal βNAC ribosome-binding domain was completely portable and sufficient to target an otherwise cytosolic protein to the ribosome. NAC modified with a UV-activatable cross-linker within its ribosome binding motif specifically cross-linked to L23 ribosomal protein family members at the exit site of the ribosome, providing the first evidence of NAC-L23 interaction in the context of the ribosome. Mutations of L23 reduced NAC ribosome binding in vivo and in vitro, whereas other eukaryotic ribosome-associated factors such as the Hsp70/40 chaperones Ssb or Zuotin were unaffected. We conclude that NAC employs a conserved ribosome binding domain to position itself on the L23 ribosomal protein adjacent to the nascent polypeptide exit site.