THE MAJORITY OF YEAST UPF1 COLOCALIZES WITH POLYRIBOSOMES IN THE CYTOPLASM

In Saccharomyces cerevisiae the UPF1 protein is required for nonsense- mediated mRNA decay, the accelerated turnover of mRNAs containing a no nsense mutation. Several lines of evidence suggest that translation pl ays an important role in the mechanism of nonsense mRNA decay, includi ng a previous report that nonsense mRNAs assemble in polyribosomes. In this study we show that UPF1 and ribosomal protein L1 co-localize in the cytoplasm and that UPF1 co-sediments with polyribosomes. To detect UPF1, three copies of the influenza hemagglutinin epitope were placed at the C-terminus. The tagged protein, UPF1-3EP, retains 86% (+/- 5%) of function. Using immunological detection, we found that UPF1-3EP is primarily cytoplasmic and was not detected either in the nucleus or i n the mitochondrion. UPF1-3EP and L1 co-distributed with polyribosomes fractionated in a 7-47% sucrose gradient. The sucrose sedimentation p rofiles for UPF1-3EP and L1 exhibited similar changes using three diff erent sets of conditions that altered the polyribosome profile. When p olyribosomes were disaggregated, UPF1-3EP and L1 accumulated in fracti ons coincident with 80S ribosomal particles. These results suggest tha t UPF1-3EP associates with polyribosomes. L3 and S3 mRNAs, which code for ribosomal proteins of the 60S and 40S ribosomal subunits, respecti vely, were on average about 100-fold more abundant than UPF1 mRNA. Ass uming that translation rates for L3, S3, and UPF1 mRNA are similar, th is result suggests that there are far fewer UPF1 molecules than riboso mes per cell. Constraints imposed by the low UPF1 abundance on the fun ctional relationships between UPF1, polyribosomes, and nonsense mRNA t urnover are discussed.