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The assembly of large multimeric complexes in the crowded cytoplasm is challenging. Here we reveal a mechanism that ensures accurate production of the yeast proteasome, involving ribosome pausing and cotranslational assembly of Rpt1 and Rpt2. Interaction of nascent Rpt1 and Rpt2 then lifts ribosome pausing. We show that the N-terminal disordered domain of Rpt1 is required to ensure efficient ribosome pausing and association of nascent Rpt1 protein complexes into heavy particles, wherein the nascent protein complexes escape ribosome quality control.Immunofluorescence and in situ hybridization studies indicate that Rpt1-and Rpt2encoding mRNAs colocalize in these particles that contain and depend upon Not1, the scaffold of the Ccr4-Not complex. We refer to these particles as Not1-Containing Assemblysomes (NCA), as they are smaller and distinct from other RNA granules such as stress granules, GW-or P-bodies. Synthesis of Rpt1 with ribosome pausing and NCA induction is conserved from yeast to human cells.

In this study we determine that the Not4 E3 ligase is important for proteasome integrity. Consequently, deletion of Not4 leads to an accumulation of polyubiquitinated proteins and reduced levels of free ubiquitin. In the absence of Not4, the proteasome regulatory particle (RP) and core particle (CP) form salt-resistant complexes, and all other forms of RPs are unstable. Not4 can associate with RP species present in purified proteasome holoenzyme but not with purified RP. Additionally, Not4 interacts with Ecm29, a protein that stabilizes the proteasome. Interestingly, Ecm29 is identified in RP species that are inactive and not detectable in cells lacking Not4. In the absence of Not4, Ecm29 interacts less well with the proteasome and becomes ubiquitinated and degraded. Our results characterize Ecm29 as a proteasome chaperone whose appropriate interaction with the proteasome requires Not4.In eukaryotes, short-lived proteins are degraded primarily by the ubiquitin-proteasome system (UPS) (25). The multicatalytic protease, the 26S proteasome, is responsible for this degradation. Most proteasome substrates are modified by polyubiquitin chains that are recognized by the proteasome. The UPS controls a diverse array of biologically important processes, including cell cycle progression, DNA repair, signal transduction, and protein quality control.The 26S proteasome consists of 2 major subcomplexes: a proteolytically active 20S core particle (CP) bound at one or both ends by a 19S regulatory particle (RP; also called PA700 in mammals). The CP has a hollow cylindrical shape and consists of a stack of 4 heptameric rings. The 2 outer rings contain ␣-type subunits, and the 2 inner rings contain ␤-type subunits. The proteolytic sites of the proteasome are located in its central cavity on specific ␤ subunits (19). Free CP exists in an autoinhibited state in which the N termini of ␣ subunits form a gate to block substrate entry. Activation of CP occurs upon opening of this gate by a proteasome activator, 19S. In mammals, two additional activators have been identified: the PA28 (or PA26)/11S regulator and PA200. In Saccharomyces cerevisiae, Blm10, which is similar to mammalian PA200, can function as an alternative activator (49).RP consists of 2 subcomplexes: the base, which binds directly to CP, and a peripheral lid. The base includes 6 ATPase subunits (Rpt1 to -6) that facilitate gate opening, substrate unfolding, and translocation into CP using ATP (47). The lid consists of 9 non-ATPase subunits (Rpn3, -5 to -9, -11, and -12 and Sem1) and is required for the recognition and deubiquitination of substrates (50).Several recent investigations have focused on how this highly abundant complex of about 2,500 kDa is assembled. CP assembly requires the assistance of CP chaperones (23). Similarly RP assembly is realized by several RP chaperones: Nas2, Hsm3, Nas6, and Rpn14 (36,46). Once RP is assembled, the base binds to the lid and all chaperones are released prior to, or during, RP-CP association (12,48). Recent data suggest that lid,...

SummaryIn this study, we determine that Saccharomyces cerevisiae Not4 E3 ligase ubiquitinates Rps7A in vivo and in vitro, but not its paralogue, Rps7B. Ubiquitinated Rps7A is detectable only in 80S and polysomes, but not in free 40S fractions. A different role of the Rps7 paralogues in vivo is supported by the observation that the deletion of Rps7A but not Rps7B is sensitive to translational inhibitors and leads to an accumulation of aggregated proteins. An important accumulation of aggregated proteins that include ribosomal proteins and ribosome-associated chaperones is also observed in cells lacking Not4. A contribution of Not4 to ribosomal function extending beyond Rps7A ubiquitination is supported by the observation that the deletion of Not4 displays a synthetic slow growth phenotype when combined with the deletion of either one of the two Rps7 paralogues. Not4 is detectable in polysome fractions, as are other subunits of the Ccr4-Not complex such as Not5. The optimal presence of Not5 in polysomes is dependent upon Not4 and the deletion of Not5 leads to a dramatic reduction of polysomes. These results lead us to suggest that Not4 contributes to normal polysome levels and is important for cellular protein solubility maybe in part by ubiquitination of Rps7A.

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