Proteasome Activity Is Influenced by the HECT_2 Protein Ipa1 in Budding Yeast

Abstract

23The ubiquitin-proteasome system (UPS) controls cellular functions by maintenance of a 24 functional proteome and degradation of key regulatory proteins. Central to the UPS is the 25 proteasome that adjusts the abundance of numerous proteins, thereby safeguarding their 26 activity or initiating regulatory events. Here, we demonstrate that the essential 27Saccharomyces cerevisiae protein Yjr141w/Ipa1 (Important for cleavage and 28 PolyAdenylation) belongs to the HECT_2 (homologous to E6-AP carboxyl terminus_2) 29 family. We found that five cysteine residues within the HECT_2 family signature and the C-30 terminus are essential for Ipa1 activity. Furthermore, Ipa1 interacts with several ubiquitin-31 conjugating enzymes in vivo and localizes to the cytosol and nucleus. Importantly, Ipa1 32 has impact on proteasome activity, which is indicated by the activation of the Rpn4 regulon 33 as well as by decreased turnover of destabilized proteasome substrates in an IPA1 mutant. 34These changes in proteasome activity might be connected to reduced maturation or 35 modification of proteasomal core particle proteins. Our results highlight the influence of 36Ipa1 on the UPS. The conservation within the HECT_2 family and the connection of the 37 human HECT_2 family member to an age-related degeneration disease might suggest that 38

Regulation of the Ysh1 endonuclease of the mRNA cleavage/polyadenylation complex by ubiquitin-mediated degradation

“…We have also identified Ubc4 as a ubiquitin conjugase that participates in Ysh1 modification. The sequence of Ipa1 indicates that like Mpe1, it is a ubiquitin ligase, but of the HECT family [41,59].…”

“…While Ipa1 possesses homology to the E2 interaction domain of this group of proteins, it does not have an active site cysteine [59], suggesting it does not function like a conventional HECT-type E3, which covalently receives ubiquitin from a partner E2 and transfers it to the target protein.…”

“…Since Ipa1 is found in both nucleus and cytoplasm [59], another role for Ipa1 could be as a co-chaperone to help Ysh1 fold correctly during or after translation (Fig. 5).…”

“…3F). Thus, even though Ipa1 has been shown to physically interact with other E2 proteins [59], including Ubc1, Ubc5, Ubc7, Ubc8, Ubc11, Ubc13, and Pex4, the presence of wild-type genes encoding these enzymes or that of the close Ubc4 paralog, Ubc5, is not sufficient to cover for loss of Ubc4 in the ipa1-1 mutant.…”

Controlling Cells with Light and LOV

“…Compared to light‐inducible protein interaction‐based tools, light‐dependent allostery is a more direct mode for controlling protein activity and particularly powerful for proteins that cannot be (easily) split or controlled by recruitment. In principle, even endogenous proteins can be equipped with allostery‐based light control using genomic knock‐in technology …”