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

Abstract

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) n KK ribosome binding motif within the ␤-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 ␣-helices in the N terminus of ␤NAC. This N-terminal ␤NAC 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.

Crystal structures of NAC domains of human nascent polypeptide-associated complex (NAC) and its αNAC subunit

“…3B). The latter conserved region is potentially responsible for conserved functions such as ribosome binding (Wegrzyn et al, 2006). It is quite probable that an ancestor gene coding a NAC-like domain was duplicated into two copies.…”

“…Either of the two NAC subunits can bind directly with nascent polypeptides emerging from the ribosome, albeit the binding of αNAC is rather weak. βNAC can directly bind ribosome protein near the exit site of nascent polypeptide using its N-terminal ribosome binding motif (Wegrzyn et al, 2006). In contrast, αNAC does not bind ribosome protein directly.…”

Dual Binding Mode of the Nascent Polypeptide-associated Complex Reveals a Novel Universal Adapter Site on the Ribosome

“…Notably, the N-terminal 23 amino acids of ␤NAC, which are sufficient to mediate ribosome binding, do not contain the recently identified "NAC signature" ( 24 RRK(X) n KK 30 ), which was suggested to be responsible for the NAC interaction with the ribosome via Rpl25 (L23 in eubacteria) (38). In contrast to the binding motif described here, which is part of the first ␣-helix of yeast ␤NAC, the previously observed "NAC signature" is located in an intrinsically disordered region between two ␣-helices at the N terminus of ␤NAC.…”

“…Extension of the ␤NAC part in the fusion protein to amino acid 39 did not significantly increase the amount of associated protein (lanes 7 and 8). Interestingly, these first 23 amino acids do not contain the motif (Arg 24 -Lys 30 ) that was recently identified by Wegrzyn et al (38) as being responsible for NACs association with the ribosome via Rpl25 (see "Discussion"). of the equivalent volume of total (T ϭ S100) and supernatant (S) were analyzed via Western blot.…”

“…Recently, Rpl25 was suggested as a NAC binding site on the ribosome based on a heterologous cross-linking assay using yeast NAC and Escherichia coli ribosomes (38).…”