Abstract
Ribosome-associated protein biogenesis factors (RPBs) act during a short but critical period of protein biogenesis. The action of RPBs starts as soon as a nascent polypeptide becomes accessible from the outside of the ribosome and ends upon termination of translation. In yeast, RPBs include the chaperones Ssb1/2 and ribosome-associated complex, signal recognition particle, nascent polypeptide-associated complex (NAC), the aminopeptidases Map1 and Map2, and the Nalpha-terminal acetyltransferase NatA. Here, we provide the first comprehensive analysis of RPB binding at the yeast ribosomal tunnel exit as a function of translational status and polypeptide sequence. We measured the ratios of RPBs to ribosomes in yeast cells and determined RPB occupation of translating and non-translating ribosomes. The combined results imply a requirement for dynamic and coordinated interactions at the tunnel exit. Exclusively, NAC was associated with the majority of ribosomes regardless of their translational status. All other RPBs occupied only ribosomal subpopulations, binding with increased apparent affinity to randomly translating ribosomes as compared with non-translating ones. Analysis of RPB interaction with homogenous ribosome populations engaged in the translation of specific nascent polypeptides revealed that the affinities of Ssb1/2, NAC, and, as expected, signal recognition particle, were influenced by the amino acid sequence of the nascent polypeptide. Complementary cross-linking data suggest that not only affinity of RPBs to the ribosome but also positioning can be influenced in a nascent polypeptide-dependent manner.
Highlights
Eubacteria possess trigger factor, a chaperone involved in cotranslational protein folding, which is restricted to eubacteria and signal recognition particle (SRP), a targeting factor involved in the translocation of membrane proteins with a hydrophobic signal-anchor sequence [1, 2]
We have employed a homologous system in which all Ribosome-associated protein biogenesis factors (RPBs), ribosomes, and ribosome nascent chain complexes (RNCs) including nascent polypeptides were derived from yeast
Total extracts derived from logarithmically growing yeast cells containing 3–5 ϫ 107 cells/ml were applied to quantitative immunoblotting with purified RPBs as standards
Summary
Construction of Plasmids—Genes encoding Rpl17a, Rpl, Rps9a, Asc, Srp, Ard, Nat, Map, Map, and Ssb were amplified from yeast genomic DNA. For ECL detection membranes were incubated for 1 min in 100 mM Tris-HCl, pH 8.6, either in the presence of 1ϫ reagent (0.2 mM p-cumaric acid in Me2SO, 1.2 mM luminol sodium salt in Me2SO, 0.01% H2O2) in case of quantifications of purified RNCs, or with 0.5ϫ reagent for all other immunoblots. Cross-linking reactions and immunoprecipitations under denaturing conditions were performed as previously described [20]. Purification of FLAG-tagged RNCs under Native Conditions— For a typical experiment 75-l translation reactions were performed at 20 °C for 80 min and were terminated by the addition of cycloheximide to a final concentration of 200 g/ml. Washed ␣FLAG beads were incubated in SDSPAGE sample buffer for 10 min at 95 °C, and aliquots and standard proteins were run on the same 10% Tris-Tricine gels. Gradients were fractionated from top to bottom with a density gradient fractionator (Teledyne Isco, Inc.) monitoring A254
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