Abstract
Translation of mRNA into protein is a fundamental step in eukaryotic gene expression requiring the large (60S) and small (40S) ribosome subunits and associated proteins. By modern proteomic approaches, we previously identified a novel 40S-associated protein named Asc1p in budding yeast and RACK1 in mammals. The goals of this study were to establish Asc1p or RACK1 as a core conserved eukaryotic ribosomal protein and to determine the role of Asc1p or RACK1 in translational control. We provide biochemical, evolutionary, genetic, and functional evidence showing that Asc1p or RACK1 is indeed a conserved core component of the eukaryotic ribosome. We also show that purified Asc1p-deficient ribosomes have increased translational activity compared to that of wild-type yeast ribosomes. Further, we demonstrate that asc1Δ null strains have increased levels of specific proteins in vivo and that this molecular phenotype is complemented by either Asc1p or RACK1. Our data suggest that one of Asc1p's or RACK1's functions is to repress gene expression.
We thank Tracey Fleischer and David Powell for numerous discussions during this project. We thank Tony Weil, Kathy Friedman, and Elizabeth Link for comments during the preparation of the manuscript. We thank Jennifer Jennings for assistance with the MS experiments and Rodney Gabriel for medium preparation. We thank Corbin Williams for assistance with 2D DIGE experiments. We thank Linda Riles and Mark Johnston for yeast lambda clone 5992 (ATCC 70652), John Warner for antibodies to yeast ribosomal protein Rpl3p, David Amberg for antibody to yeast Aip1p, Alan Hinnebusch for GCN4 reporter plasmid p180, Gerhard Braus for plasmid pME1867, Tony Weil for yeast vectors pRS416 and p426GPD, and Alan Sachs for plasmid T3 lucpA and in vitro translation protocols.
We thank the Vanderbilt Academic Venture Capital Fund. This project was supported by NIH grant GM64779 to A.J.L. V.R.G. is supported by NIH training grant T32 CA009385. C.W. is supported by NIH grants GM64779 and HL68744. A.J.L. is supported by NIH grants GM64779, HL68744, NS43952, ES11993, and CA098131.