title: 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.
authors.affiliation: IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
authors.forename: Bebiana
authors.initials: B
authors.lastname: Sá-Moura
authors.affiliation: IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
authors.forename: Ana Marisa
authors.initials: AM
authors.lastname: Simões
authors.affiliation: IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Fraga
authors.affiliation: Centre for Molecular and Structural Biomedicine, CBME/IBB, LA, Portugal ; Current address: Biophysics Section, Department of Life Sciences, Imperial College, London, UK.
authors.forename: Humberto
authors.initials: H
authors.lastname: Fernandes
authors.affiliation: IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal ; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal.
authors.forename: Isabel A
authors.initials: IA
authors.lastname: Abreu
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal ; Current address: BioFIG - Centre for Biodiversity, Functional and Integrative Genomics, Faculty of Sciences, University of Lisboa, Campo Grande 1749-016 Lisboa, Portugal.
authors.forename: Hugo M
authors.initials: HM
authors.lastname: Botelho
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal.
authors.forename: Cláudio M
authors.initials: CM
authors.lastname: Gomes
authors.affiliation: Centre for Molecular and Structural Biomedicine, CBME/IBB, LA, Portugal ; Current address: Immunodiagnostic Systems, Core Technology, 10 Didcot Way, Boldon, NE35 9PD, UK.
authors.forename: António J
authors.initials: AJ
authors.lastname: Marques
authors.affiliation: Institute for Genetics, University of Cologne, Zülpicher Str. 47, D-50674 Cologne, Germany.
authors.forename: R Jürgen
authors.initials: RJ
authors.lastname: Dohmen
authors.affiliation: Centre for Molecular and Structural Biomedicine, CBME/IBB, LA, Portugal ; Institute for Genetics, University of Cologne, Zülpicher Str. 47, D-50674 Cologne, Germany.
authors.forename: Paula C
authors.initials: PC
authors.lastname: Ramos
authors.affiliation: IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
authors.forename: Sandra
authors.initials: S
authors.lastname: Macedo-Ribeiro
keywords: Circular dichroism; dynamic light scattering; intrinsically disordered; protein structure