Substrate-Specific Effects of Natural Genetic Variation on Proteasome Activity

The bulk of targeted cellular protein degradation is performed by the proteasome, a multi-subunit complex consisting of the 19S regulatory particle, which binds, unfolds, and translocates substrate proteins, and the 20S core particle, which degrades them. Protein homeostasis requires precise, dynamic control of proteasome activity. To what extent genetic variation creates differences in proteasome activity is almost entirely unknown. Using the ubiquitin-independent degrons of the ornithine decarboxylase and Rpn4 proteins, we developed reporters that provide high-throughput, quantitative measurements of proteasome activity in vivo in genetically diverse cell populations. We used these reporters to characterize the genetic basis of variation in proteasome activity in the yeast Saccharomyces cerevisiae. We found that proteasome activity is a complex, polygenic trait, shaped by variation throughout the genome. Genetic influences on proteasome activity were predominantly substrate-specific, suggesting that they primarily affect the function or activity of the 19S regulatory particle. Our results demonstrate that individual genetic differences create heritable variation in proteasome activity and suggest that genetic effects on proteasomal protein degradation may be an important source of variation in cellular and organismal traits.

A Trio of Ubiquitin Ligases Sequentially Drive Ubiquitylation and Autophagic Degradation of Dysfunctional Yeast Proteasomes

As central effectors of ubiquitin (Ub)-mediated proteolysis, proteasomes are regulated at multiple levels, including degradation of unwanted or dysfunctional particles via autophagy (termed proteaphagy). In yeast, inactive proteasomes are exported from the nucleus, sequestered into cytoplasmic aggresomes via the Hsp42 chaperone, extensively ubiquitylated, and then tethered to the expanding phagophore by the autophagy receptor Cue5. Here, we demonstrate the need for ubiquitylation driven by the trio of Ub ligases (E3s) San1, Rsp5 and Hul5, which, together with their corresponding E2s, work sequentially to promote nuclear export and Cue5 recognition. Whereas San1 functions prior to nuclear export, Rsp5 and Hul5 likely decorate aggresome-localized proteasomes in concert. Ultimately, topologically complex Ub chain(s) containing both K48 and K63 Ub-Ub linkages are assembled, mainly on the regulatory particle, to generate autophagy-competent substrates. As San1, Rsp5, Hul5, Hsp42, and Cue5 also participate in general proteostasis, proteaphagy likely engages an essential mechanism for eliminating inactive/misfolded proteins.

Analysis of the Dynamic Proteasome Structure by Cross-Linking Mass Spectrometry

The 26S proteasome is a macromolecular complex that degrades proteins maintaining cell homeostasis; thus, determining its structure is a priority to understand its function. Although the 20S proteasome’s structure has been known for some years, the highly dynamic nature of the 19S regulatory particle has presented a challenge to structural biologists. Advances in cryo-electron microscopy (cryo-EM) made it possible to determine the structure of the 19S regulatory particle and showed at least seven different conformational states of the proteasome. However, there are still many questions to be answered. Cross-linking mass spectrometry (CLMS) is now routinely used in integrative structural biology studies, and it promises to take integrative structural biology to the next level, answering some of these questions.

Gametophyte-specific DEAD-box RNA helicase 29 is required for functional maturation of male and female gametophytes in Arabidopsis

The maturation of male and female gametophytes together with its impact on plant sexual reproduction has not received much attention, and the molecular mechanisms underlying the process are largely unknown. Here, we show that Arabidopsis DEAD-box RNA helicase 29 (RH29) is critical for the functional maturation of both male and female gametophytes. Homozygous rh29 mutants could not be obtained, and heterozygous mutant plants were semi-sterile. Progression of the cell cycle in rh29 female gametophytes was delayed. Delayed pollination experiments showed that rh29 female gametophytes underwent cell-fate specification but were unable to develop into functional gametophytes. Functional specification but not morphogenesis was also disrupted in rh29 male gametophytes, causing defective pollen tube growth in the pistil. RH29 was highly and specifically expressed in gametophytic cells. RH29 shares high amino acid sequence identity with yeast Dbp10p, which partially rescues the aborted-ovules phenotype of rh29/RH29 plants. RH29 is essential for the synthesis of REGULATORY PARTICLE TRIPLE A ATPase 5a (RPT5a), a subunit of the regulatory particle of the 26S proteasome. Our results suggest that gametophyte functional maturation is a necessary process for successful fertilization and that RH29 is essential for the functional maturation of both male and female gametophytes.

In-depth Analysis of the Lid Subunits Assembly Mechanism in Mammals

The 26S proteasome is a key player in the degradation of ubiquitinated proteins, comprising a 20S core particle (CP) and a 19S regulatory particle (RP). The RP is further divided into base and lid subcomplexes, which are assembled independently from each other. We have previously demonstrated the assembly pathway of the CP and the base by observing assembly intermediates resulting from knockdowns of each proteasome subunit and the assembly chaperones. In this study, we examine the assembly pathway of the mammalian lid, which remains to be elucidated. We show that the lid assembly pathway is conserved between humans and yeast. The final step is the incorporation of Rpn12 into the assembly intermediate consisting of two modular complexes, Rpn3-7-15 and Rpn5-6-8-9-11, in both humans and yeast. Furthermore, we dissect the assembly pathways of the two modular complexes by the knockdown of each lid subunit.