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The proteasome holoenzyme is a molecular machine that degrades most proteins in eukaryotes. In the holoenzyme, its heterohexameric ATPase injects protein substrates into the proteolytic core particle, where degradation occurs. The heterohexameric ATPase, referred to as 'Rpt ring', assembles through six ATPase subunits (Rpt1-Rpt6) individually binding to specific chaperones (Rpn14, Nas6, Nas2, and Hsm3). Here, our findings suggest that the onset of Rpt ring assembly can be regulated by two alternative mechanisms. Excess Rpt subunits relative to their chaperones are sequestered into multiple puncta specifically during early-stage Rpt ring assembly. Sequestration occurs during stressed conditions, for example heat, which transcriptionally induce Rpt subunits. When the free Rpt pool is limited experimentally, Rpt subunits are competent for proteasome assembly even without their cognate chaperones. These data suggest that sequestration may regulate amounts of individual Rpt subunits relative to their chaperones, allowing for proper onset of Rpt ring assembly. Indeed, Rpt subunits in the puncta can later resume their assembly into the proteasome. Intriguingly, when proteasome assembly resumes in stressed cells or is ongoing in unstressed cells, excess Rpt subunits are recognized by an alternative mechanism-degradation by the proteasome holoenzyme itself. Rpt subunits undergo proteasome assembly until the holoenzyme complex is generated at a sufficient level. The fully-formed holoenzyme can then degrade any remaining excess Rpt subunits, thereby regulating its own Rpt ring assembly. These two alternative mechanisms, degradation and sequestration of Rpt subunits, may help control the onset of chaperone-mediated Rpt ring assembly, thereby promoting proper proteasome holoenzyme formation. by guest on July 10, 2020 http://www.jbc.org/ Downloaded from Figure 1. Proteasome holoenzyme formation requires chaperone-mediated Rpt ring assembly during heat stress. A and B, upon heat stress, early-stage Rpt ring assembly requires the chaperones. Yeast strains were grown at 30°C (lanes 1 -4), and then exposed to heat stress at 37°C for 4 h (lanes 5-8). Proteasome activities were assessed by subjecting whole-cell lysates (60 g) to native PAGE and in-gel peptidase assays using the fluorogenic peptide substrate, LLVY-AMC (panel i). After imaging the native gels in panel i, 0.02% SDS was added to activate free CP in panel ii; SDS denatures the substrate entry gate of the CP (25). Native gels were then subjected to immunoblotting (IB) to detect levels of the Rpt5-Rpt4 modules (t5-t4 module) and proteasome holoenzymes (panel iii). Pgk1 is a loading control. B, Pgk1 blot in lanes 1-4 and 5-8 derives from two different gels, which were processed the same in parallel during immunoblotting and signal detection. C and D, ubiquitinated protein degradation requires chaperone-mediated proteasome assembly during heat stress. Whole-cell lysates (20 g) from samples as in A and B were subjected to 10% BisTris SDS-PAGE and immunoblotting for ubiquitin....

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Whether macrophage migration inhibitory factor (MIF) in the bovine oviduct is important for early embryogenesis has not been well substantiated. The aim of the present study was to test the hypothesis that bovine oviduct expresses higher levels of MIF during the post-ovulation phase. Both ampullary and isthmic samples were collected from Japanese black heifers during oestrus (Day 0; n=5), postovulation (Day 3; n=6) and luteal phase (Days 9-12; n=5). MIF mRNA and protein were extracted from the ampullary and isthmic samples and their levels measured by real-time polymerase chain reaction and western blot analysis respectively. Fluorescent immunohistochemistry was performed on frozen ampullary and isthmic sections using antibodies against MIF. MIF mRNA and protein expression was higher in the postovulatory phase than during oestrus and the luteal phase (P<0.05). Fluorescent immunohistochemistry confirmed that in all phases of the oestrous cycle evaluated, the primary site of MIF expression in the ampulla and isthmus was the tunica mucosa. In conclusion, the bovine ampulla and isthmus have higher MIF expression during the postovulatory phase. Further studies are needed to clarify the role of MIF in bovine oviducts.

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