A fundamental problem in proteomics is the identification of protein complexes and their components. We have used analytical ultracentrifugation with a fluorescence detection system (AU-FDS) to precisely and rapidly identify translation complexes in the yeast Saccharomyces cerevisiae. Following a one-step affinity purification of either poly(A)-binding protein (PAB1) or the large ribosomal subunit protein RPL25A in conjunction with GFP-tagged yeast proteins/RNAs, we have detected a 77S translation complex that contains the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G, and PAB1. This 77S structure, not readily observed previously, is consistent with the monosomal translation complex. The 77S complex abundance decreased with translational defects and following the stress of glucose deprivation that causes translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions that block translation initiation, we observed that the stress of glucose deprivation affected translation initiation primarily by operating through a pathway involving the mRNA cap binding protein eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1M KCl) and robust heat shock acted at other steps. The presumed sites of translational blockage caused by these stresses coincided with the types of stress granules, if any, which are subsequently formed.

Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1’s defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made.

The eukaryotic eRF1 translation termination factor plays an important role in recognizing stop codons and initiating the end to translation. However, which exact complexes contain eRF1 and at what abundance is not clear. We have used analytical ultracentrifugation with fluorescent detection system to identify the protein complexome of eRF1 in the yeast Saccharomyces cerevisiae. In addition to eRF1 presence in translating polysomes, we found that eRF1 associated with five other macromolecular complexes: 77S, 57S, 39S, 28S, and 20S in size. Generally equal abundances of each of these complexes were found. The 77S complex primarily contained the free 80S ribosome consistent with in vitro studies and did not appear to contain significant levels of the monosomal translating complex that co-migrates with the free 80S ribosome. The 57S and 39S complexes represented, respectively, free 60S and 40S ribosomal subunits bound to eRF1, associations not previously reported. The novel 28S and 20S complexes (containing minimal masses of 830 KDa and 500 KDa, respectively) lacked significant RNA components and appeared to be oligomeric, as eRF1 has a mass of 49 KDa. The majority of polysomal complexes containing eRF1 were both substantially deadenylated and lacking in closed-loop factors eIF4E and eIF4G. The thirteen percent of such translating polysomes that contained poly(A) tails had equivalent levels of eIF4E and eIF4G, suggesting these complexes were in a closed-loop structure. The identification of eRF1 in these unique and previously unrecognized complexes suggests a variety of new roles for eRF1 in the regulation of cellular processes.

The poly(A)-binding protein PAB1 from the yeast Saccharomyces cerevisiae plays an important role in controlling mRNA deadenylation rates. Deletion of either its RRM1 or proline-rich domain (P domain) severely restricts deadenylation and slows mRNA degradation. Because these large deletions could be having unknown effects on the structure of PAB1, different strategies were used to determine the importance of the RRM1 and P domains to deadenylation. Since the P domain is quite variable in size and sequence among eukaryotes, P domains from two human PABPCs and from Xenopus were substituted for that of PAB1. The resultant PAB1 hybrid proteins, however, displayed limited or no difference in mRNA deadenylation as compared to PAB1. In contrast to the P domain, the RRM1 domain is highly conserved across species, and a systematic mutagenesis of the RRM1 domain was undertaken to identify its functional regions. Several mutations along the RNA binding surface of RRM1 inhibited deadenylation whereas one set of mutations on its exterior non-RNA binding surface shifted deadenylation from a slow distributive process to a rapid processive deadenylation. These results suggest that the RRM1 domain is the more critical region of PAB1 for controlling deadenylation and consists of at least two distinguishable functional regions.

Prions, misfolded proteins that aggregate, cause an array of progressively deteriorating conditions to which, currently, there are no effective treatments. The presently accepted model indicates that the soluble non-prion forms of prion-forming proteins, such as the well-studied SUP35, do not exist in large aggregated molecular complexes. Here, we show using analytical ultracentrifugation with fluorescent detection that the non-prion form of SUP35 exists in a range of discretely sized soluble complexes (19S, 28S, 39S, 57S, and 70S-200S). Similar to the [PSI+] aggregated complexes, each of these [psi-] complexes associates at stoichiometric levels with a large variety of molecular chaperones: HSP70 proteins comprise the major component.Another yeast prion-forming protein, RNQ1 (known to promote the production of the prion SUP35 state), is also present in SUP35 complexes. These results establish that the non-prion SUP35, like its prion form, is predisposed to form large molecular complexes containing chaperones and other prion-forming proteins. These results agree with our previous studies on the huntingtin protein. That the normal forms for aggregation-prone proteins may preexist in large molecular complexes has important ramifications for the progression of diseases involving protein aggregation.