Scp160p interacts in an mRNA-dependent manner with translating ribosomes via multiple RNA-binding heterogeneous nuclear ribonucleoprotein K-homology (KH) domains. In the present study, we show by protein–protein cross-linking that Scp160p is in close proximity to translation elongation factor 1A and the WD40 (Trp-Asp 40)-repeat containing protein Asc1p at ribosomes. Analysis of a truncation mutant revealed that the C-terminus of Scp160p is essential for ribosome binding and that Cys1067 at the C-terminus of Scp160p is required to obtain these cross-links. The interaction of Scp160p with ribosomes depends on Asc1p. In fast-growing yeast cells, nearly all Asc1p is tightly bound to ribosomes, but it can also be present in a ribosome-free form depending on growth conditions. The functional homologue of Asc1p, mammalian RACK1 (receptor of activated C kinase), was previously characterized as an adaptor protein bridging activated signalling molecules with their substrates. Our results suggest that Scp160p connects specific mRNAs, ribosomes and a translation factor with an adaptor for signalling molecules. These interactions might regulate the translation activity of ribosomes programmed with specific mRNAs.
- heterogeneous nuclear ribonucleoprotein K-homology domain (KH domain)
- RNA-binding protein
- signalling complex
Abbreviations used: BMH, bis-maleimidohexane; BMOE, bis-maleimidoethane; DTT, dithiothreitol; eEF1A, translation elongation factor 1A; eIF, eukaryotic translation-initiation factor; ER, endoplasmic reticulum; KH, heterogeneous nuclear ribonucleoprotein K-homology; LS, low salt; nano-ESI-Q-TOF MS, nanoelectrospray ionization-quadrupole-time-of-flight MS; PKC, protein kinase C; RACK1, receptor of activated C kinase; UTR, untranslated region; YPD, yeast extract, peptone, dextrose.
- The Biochemical Society, London ©2004