AbstractWe have previously identified 55 nonribosomal proteins in PAB1‐mRNP complexes in Saccharomyces cerevisiae using mass spectrometric analysis. Because one of the inherent limitations of mass spectrometry is that it does not inform as to the size or type of complexes in which the proteins are present, we consequently used analytical ultracentrifugation with fluorescent detection system (AU‐FDS) to determine which proteins are present in the 77S monosomal translation complex that contains minimally the closed‐loop structure components (eIF4E, eIF4G, and PAB1), mRNA, and the 40S and 60S ribosomes. We assayed by AU‐FDS analysis 33 additional PAB1‐mRNP factors but found that only five of these proteins were present in the 77S translation complex: eRF1, SLF1, SSD1, PUB1, and SBP1. eRF1 is involved in translation termination, SBP1 is a translational repressor, and SLF1, SSD1, and PUB1 are known mRNA binding proteins. Many of the known P body/stress granule proteins that associate with the PAB1‐mRNP were not present in the 77S translation complex, implying that P body/stress granules result from significant protein additions after translational cessation. These data inform that AU‐FDS can clarify protein complex identification that remains undetermined after typical immunoprecipitation and mass spectrometric analyses.