C termini of proteasomal ATPases play nonequivalent roles in cellular assembly of mammalian 26 S proteasome.
ABSTRACT The 26 S proteasome comprises two multisubunit subcomplexes as follows: 20 S proteasome and PA700/19 S regulatory particle. The cellular mechanisms by which these subcomplexes assemble into 26 S proteasome and the molecular determinants that govern the assembly process are poorly defined. Here, we demonstrate the nonequivalent roles of the C termini of six AAA subunits (Rpt1-Rpt6) of PA700 in 26 S proteasome assembly in mammalian cells. The C-terminal HbYX motif (where Hb is a hydrophobic residue, Y is tyrosine, and X is any amino acid) of each of two subunits, Rpt3 and Rpt5, but not that of a third subunit Rpt2, was essential for assembly of 26 S proteasome. The C termini of none of the three non-HbYX motif Rpt subunits were essential for cellular 26 S proteasome assembly, although deletion of the last three residues of Rpt6 destabilized the 20 S-PA700 interaction. Rpt subunits defective for assembly into 26 S proteasome due to C-terminal truncations were incorporated into intact PA700. Moreover, intact PA700 accumulated as an isolated subcomplex when cellular 20 S proteasome content was reduced by RNAi. These results indicate that 20 S proteasome is not an obligatory template for assembly of PA700. Collectively, these results identify specific structural elements of two Rpt subunits required for 26 S proteasome assembly, demonstrate that PA700 can be assembled independently of the 20 S proteasome, and suggest that intact PA700 is a direct intermediate in the cellular pathway of 26 S proteasome assembly.
SourceAvailable from: Rasmus Hartmann-Petersen[Show abstract] [Hide abstract]
ABSTRACT: Cells are regularly exposed to stress conditions that may lead to protein misfolding. To cope with this challenge, molecular chaperones selectively target structurally perturbed proteins for degradation via the ubiquitin-proteasome pathway. In mammals the co-chaperone BAG-1 plays an important role in this system. BAG-1 has two orthologues, Bag101 and Bag102, in the fission yeast Schizosaccharomyces pombe. We show that both Bag101 and Bag102 interact with 26S proteasomes and Hsp70. By epistasis mapping we identify a mutant in the conserved kinetochore component Spc7 (Spc105/Blinkin) as a target for a quality control system that also involves, Hsp70, Bag102, the 26S proteasome, Ubc4 and the ubiquitin-ligases Ubr11 and San1. Accordingly, chromosome missegregation of spc7 mutant strains is alleviated by mutation of components in this pathway. In addition, we isolated a dominant negative version of the deubiquitylating enzyme, Ubp3, as a suppressor of the spc7-23 phenotype, suggesting that the proteasome-associated Ubp3 is required for this degradation system. Finally, our data suggest that the identified pathway is also involved in quality control of other kinetochore components and therefore likely to be a common degradation mechanism to ensure nuclear protein homeostasis and genome integrity.PLoS Genetics 01/2014; 10(1):e1004140. DOI:10.1371/journal.pgen.1004140 · 8.17 Impact Factor
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ABSTRACT: The 26S proteasome is the major eukaryotic ATP-dependent protease, yet the detailed mechanisms used by the proteasomal heterohexameric AAA+ unfoldase to drive substrate degradation remain poorly understood. To perform systematic mutational analyses of individual ATPase subunits, we heterologously expressed the unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro. Our studies demonstrate that the six ATPases have distinct roles in degradation, corresponding to their positions in the spiral staircases adopted by the AAA+ domains in the absence or presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. Whereas the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits three-fold symmetry with contributions from alternate subunits. These diverse functional asymmetries highlight how the 26S proteasome deviates from simpler, homomeric AAA+ proteases.Nature Structural & Molecular Biology 09/2013; DOI:10.1038/nsmb.2659 · 11.63 Impact Factor
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ABSTRACT: We investigated molecular features and cellular roles of PI31 (PSMF1) on regulation of proteasome function. PI31 has a C-terminal HbYX motif characteristic of several proteasome activators. Peptides corresponding to the PI31 C-terminus also bind to and activate the 20S proteasome in an HbYX-dependent manner, but intact PI31protein inhibits in vitro 20S activity. Binding to and inhibition of the proteasome by PI31 are conferred by the HbYX-containing proline-rich C-terminal domain but do not require HbYX residues. Thus, multiple regions of PI31 bind independently to the proteasome and collectively determine effects on activity. PI31 blocks the ATP-dependent in vitro assembly of 26S proteasome from 20S proteasome and PA700 subcomplexes, but has no effect on in vitro activity of the intact 26S proteasome. To determine the physiologic significance of these in vitro effects, we assessed multiple aspects of cellular proteasome content and function after altering PI31 levels. We detected no change in overall cellular proteasome content or function when PI31 levels were either increased by moderate ectopic overexpression or decreased by RNAi. We also failed to identify a role of PI31 ADP-ribosylation as a mechanism for regulation of overall 26S proteasome content and function, as recently proposed. Thus, despite its in vitro effects on various proteasome activities and its structural relationship to established proteasome regulators, cellular roles and mechanisms of PI31 in regulation of proteasome function are remain unclear and require future definition.Journal of Biological Chemistry 04/2014; 289(25). DOI:10.1074/jbc.M114.561183 · 4.60 Impact Factor