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

Markus Pech,Thomas Spreter,Roland Beckmann,Birgitta Beatrix

doi:10.1074/jbc.m109.092536

Abstract

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of betaNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of betaNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, alphaNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.

Highlights

Nascent polypeptide-associated complex (NAC) Is Associated with Ribosomes via the N Terminus of ␤NAC—To characterize the binding partner(s) of NAC on the ribosome we first verified that the amino-terminal 11 amino acids of ␤NAC are critical for ribosome association in yeast as described before [43]
For subsequent assays we sedimented the ribosomes through a sucrose cushion at a physiological salt concentration and again comparable amounts of NAC sedimented with the yeast ribosomes in the WT situation as well as when NAC was expressed from a plasmid in a NAC knock-out strain
In the yeast S. cerevisiae, NAC is quantitatively associated with the ribosome [35, 46]

Summary

NAC Binds to Ribosome at a Novel Universal Adapter Site

NAC does not show similarity to other proteins and its cellular function is still poorly understood. Both NAC subunits seem to differ with respect to their function Both subunits contact the nascent chain on the ribosome, as was shown by crosslinking, ␤NAC alone was sufficient for binding to the ribosome and prevention of ribosome interaction with the translocon [15, 35]. The central NAC domain exhibits a unique novel protein fold It resembles a flattened ␤ barrel that exposes several hydrophobic residues on one of its concave surfaces. This domain is structurally conserved from archaea to humans emphasizing its importance for NACs function. We show that ␣NAC contacts Rpl, the direct neighbor of Rpl on the ribosomal surface and propose a model for NAC interaction with this novel universal docking site at the ribosomal tunnel exit

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION