A multimeric assembly factor controls the formation of alternative 20S proteasomes

The proteasome is the central regulatory protease of eukaryotic cells. Heteroheptameric α-subunit and β-subunit rings stack to form the 20S proteasome, which associates with a 19S regulatory particle (RP). Here we show that two yeast proteins, Pba3 and Pba4, form a previously unidentified 20S proteasome–assembly chaperone. Pba3–Pba4 interacts genetically and physically with specific proteasomal α subunits, and loss of Pba3–Pba4 causes both a reduction and a remodeling of cellular proteasomes. Notably, mutant cells accumulate proteasomes in which a second copy of the α4 subunit replaces α3. 20S proteasome–assembly defects also are associated with altered RP assembly; this unexpected result suggests that the 20S proteasome can function as an RP-assembly factor in vivo. Our data demonstrate that Pba3–Pba4 orchestrates formation of a specific type of proteasome, the first example of a trans-acting factor that controls assembly of alternative proteasomal complexes.

[1]  J. Belote,et al.  Duplicated proteasome subunit genes in Drosophila melanogaster encoding testes-specific isoforms. , 1996, Genetics.

[2]  A. Ciechanover,et al.  The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. , 2002, Physiological reviews.

[3]  J. Bader,et al.  A DNA Integrity Network in the Yeast Saccharomyces cerevisiae , 2013, Cell.

[4]  A. Emili,et al.  β‐Subunit appendages promote 20S proteasome assembly by overcoming an Ump1‐dependent checkpoint , 2007, The EMBO journal.

[5]  R. Dohmen,et al.  The ultimate nanoscale mincer: assembly, structure and active sites of the 20S proteasome core. , 2004, Cellular and molecular life sciences : CMLS.

[6]  M. Hochstrasser,et al.  Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[7]  S. Gygi,et al.  The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle , 2005, Nature Structural &Molecular Biology.

[8]  M. Hochstrasser,et al.  Autocatalytic Subunit Processing Couples Active Site Formation in the 20S Proteasome to Completion of Assembly , 1996, Cell.

[9]  D. Wolf,et al.  The Active Sites of the Eukaryotic 20 S Proteasome and Their Involvement in Subunit Precursor Processing* , 1997, The Journal of Biological Chemistry.

[10]  J. Monaco,et al.  Beta 2 subunit propeptides influence cooperative proteasome assembly. , 2003, The Journal of biological chemistry.

[11]  M. Hochstrasser,et al.  Biogenesis, structure and function of the yeast 20S proteasome. , 1995, The EMBO journal.

[12]  J. Gavilanes,et al.  A permanent Zn2+ reverse staining method for the detection and quantification of proteins in polyacrylamide gels. , 1993, Analytical biochemistry.

[13]  P. Kloetzel,et al.  20S proteasome biogenesis. , 2001, Biochimie.

[14]  J. Monaco,et al.  Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor β subunits , 1997 .

[15]  S. Elsasser,et al.  Characterization of the proteasome using native gel electrophoresis. , 2005, Methods in enzymology.

[16]  R. Huber,et al.  A gated channel into the proteasome core particle , 2000, Nature Structural Biology.

[17]  Raphaël Guérois,et al.  20S proteasome assembly is orchestrated by two distinct pairs of chaperones in yeast and in mammals. , 2007, Molecular cell.

[18]  Erica S. Johnson,et al.  Ump1p Is Required for Proper Maturation of the 20S Proteasome and Becomes Its Substrate upon Completion of the Assembly , 1998, Cell.

[19]  Cassandra S Arendt,et al.  Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast , 2004, The EMBO journal.

[20]  M. Kasahara,et al.  Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes. , 2006, Molecular cell.

[21]  M. Hochstrasser.  Ubiquitin-dependent protein degradation. , 1996, Annual review of genetics.

[22]  Alexander Varshavsky,et al.  Regulated protein degradation. , 2005, Trends in biochemical sciences.

[23]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[24]  P. Thomas,et al.  Endoproteolytic Activity of the Proteasome , 2002, Science.

[25]  R. Dohmen,et al.  Biting the hand that feeds: Rpn4-dependent feedback regulation of proteasome function. , 2007, Biochimica et biophysica acta.

[26]  J. Monaco,et al.  β2 Subunit Propeptides Influence Cooperative Proteasome Assembly* , 2003, The Journal of Biological Chemistry.

[27]  Soo Chan Lee,et al.  A novel interaction between N‐myristoylation and the 26S proteasome during cell morphogenesis , 2007, Molecular microbiology.

[28]  M. Hochstrasser,et al.  Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways , 2006, The EMBO journal.

[29]  A. Rivett,et al.  Regulation of proteasome complexes by γ-interferon and phosphorylation , 2001 .

[30]  Sean R. Collins,et al.  Global landscape of protein complexes in the yeast Saccharomyces cerevisiae , 2006, Nature.

[31]  W. Saunders,et al.  Large-scale functional genomic analysis of sporulation and meiosis in Saccharomyces cerevisiae. , 2003, Genetics.

[32]  Tohru Natsume,et al.  A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes , 2005, Nature.

[33]  C. Slaughter,et al.  The Proteasome, a Novel Protease Regulated by Multiple Mechanisms* , 1999, The Journal of Biological Chemistry.

[34]  A. Nakano,et al.  The assembly pathway of the 19S regulatory particle of the yeast 26S proteasome. , 2006, Molecular biology of the cell.

[35]  R. Schiestl,et al.  Cadmium is an inducer of oxidative stress in yeast. , 1996, Mutation research.

[36]  M. Nishikimi,et al.  Increased degradation of oxidized proteins in yeast defective in 26 S proteasome assembly. , 2002, Archives of biochemistry and biophysics.

[37]  J. Yates,et al.  Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. , 2000, Molecular biology of the cell.

[38]  Gerald R. Fink,et al.  Guide to yeast genetics and molecular biology , 1993 .