Biochemical and Biophysical Characterization of Recombinant Yeast Proteasome Maturation Factor Ump1

Abstract

Protein degradation is essential for maintaining cellular homeostasis. The proteasome is the central enzyme responsible for non-lysosomal protein degradation in eukaryotic cells. Although proteasome assembly is not yet completely understood, a number of cofactors required for proper assembly and maturation have been identified. Ump is a short-lived maturation factor required for the efficient biogenesis of the 20S proteasome. Upon the association of the two precursor complexes, Ump is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. In order to further understand the mechanisms behind proteasomal maturation, we expressed and purified yeast Ump in E. coli for biophysical and structural analysis.We show that recombinant Ump is purified as a mixture of different oligomeric species and that oligomerization is mediated by intermolecular disulfide bond formation involving the only cysteine residue present in the protein. Furthermore, a combination of bioinformatic, biochemical and structural analysis revealed that Ump shows characteristics of an intrinsically disordered protein, which might become structured only upon interaction with the proteasome subunits.

Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1–Pba2 chaperone

“…34) and its human orthologue 36 . The stretched out conformation of Ump1 and the lack of defined EM density along the projected path fit its description as an intrinsically unstructured protein with little secondary structure elements 37,38 . It also explains how Ump1 can be degraded by the nascent proteasome without dissolving its tertiary structure first.…”

The Intrinsically Disordered Sem1 Protein Functions as a Molecular Tether during Proteasome Lid Biogenesis

“…We expect that it will also contribute to the tethering function of Sem1 in proteasome assembly. Intriguingly, the dedicated CP chaperone Ump1 has also recently been shown to be an intrinsically disordered protein (Sá-Moura et al, 2013; Uekusa et al, 2013). Whether Ump1 serves a function similar to Sem1 during CP assembly remains to be tested.…”

Proteasome assembly

“…With the b-propeptide processing, the active site threonines are exposed and Ump1 is degraded by the nascent CP. Intriguingly, Ump1 has recently shown to be an intrinsically disordered protein [65]. Driven by a newly defined mechanism based on disordered protein domains, Ump1 may benefit from being an intrinsically disordered protein during degradation as the first substrate of the matured CP [66].…”

“…Sem1, an intrinsically disordered protein, further tethers lid subunits suggesting that RP lid assembly is facilitated by a newly defined mechanism involving intrinsically disordered protein domains [124]. Intriguingly, Ump1, the CP-dedicated chaperone, is an intrinsically disordered protein as well suggesting that a disorder-driven mechanism also applies for CP assembly [65,66].…”