Interaction of the Doa4 Deubiquitinating Enzyme with the Yeast 26S Proteasome

The Saccharomyces cerevisiae Doa4 deubiquitinating enzyme is required for the rapid degradation of protein substrates of the ubiquitin–proteasome pathway. Previous work suggested that Doa4 functions late in the pathway, possibly by deubiquitinating (poly)-ubiquitin-substrate intermediates associated with the 26S proteasome. We now provide evidence for physical and functional interaction between Doa4 and the proteasome. Genetic interaction is indicated by the mutual enhancement of defects associated with a deletion of DOA4 or a proteasome mutation when the two mutations are combined. Physical association of Doa4 and the proteasome was investigated with a new yeast 26S proteasome purification procedure, by which we find that a sizeable fraction of Doa4 copurifies with the protease. Another yeast deubiquitinating enzyme, Ubp5, which is related in sequence to Doa4 but cannot substitute for it even when overproduced, does not associate with the proteasome. DOA4-UBP5 chimeras were made by a novel PCR/yeast recombination method and used to identify an N-terminal 310-residue domain of Doa4 that, when appended to the catalytic domain of Ubp5, conferred Doa4 function, consistent with Ubp enzymes having a modular architecture. Unlike Ubp5, a functional Doa4-Ubp5 chimera associates with the proteasome, suggesting that proteasome binding is important for Doa4 function. Together, these data support a model in which Doa4 promotes proteolysis through removal of ubiquitin from proteolytic intermediates on the proteasome before or after initiation of substrate breakdown.

Functions of the DNA damage response pathway target Ho endonuclease of yeast for degradation via the ubiquitin-26S proteasome system

Ho endonuclease of Saccharomyces cerevisiae is a homing endonuclease that makes a site-specific double-strand break in the MAT gene in late G1. Here we show that Ho is rapidly degraded via the ubiquitin-26S proteasome system through two ubiquitin-conjugating enzymes UBC2Rad6 and UBC3Cdc34. UBC2Rad6 is complexed with the ring finger DNA-binding protein Rad18, and we find that Ho is stabilized in rad18 mutants. We show that the Ho degradation pathway involving UBC3Cdc34 goes through the Skp1/Cdc53/F-box (SCF) ubiquitin ligase complex and identify a F-box protein, Yml088w, that is required for Ho degradation. Components of a defined pathway of the DNA damage response, MEC1, RAD9, and CHK1, are also necessary for Ho degradation, whereas functions of the RAD24 epistasis group and the downstream effector RAD53 have no role in degradation of Ho. Our results indicate a link between the endonuclease function of Ho and its destruction.

RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: A negative feedback circuit

The RPN4 (SON1, UFD5) protein of the yeast Saccharomyces cerevisiae is required for normal levels of intracellular proteolysis. RPN4 is a transcriptional activator of genes encoding proteasomal subunits. Here we show that RPN4 is required for normal levels of these subunits. Further, we demonstrate that RPN4 is extremely short-lived (t1/2 ≈2 min), that it directly interacts with RPN2, a subunit of the 26S proteasome, and that rpn4Δ cells are perturbed in their cell cycle. The degradation signal of RPN4 was mapped to its N-terminal region, outside the transcription–activation domains of RPN4. The ability of RPN4 to augment the synthesis of proteasomal subunits while being metabolically unstable yields a negative feedback circuit in which the same protein up-regulates the proteasome production and is destroyed by the assembled active proteasome.

Mammalian Sug1 and c-Fos in the nuclear 26S proteasome.

In a search for regulatory proteins that interact with the leucine zipper motif of c-Fos in the yeast two-hybrid screen, we have identified a protein (FZA-B) that has extensive sequence similarity to SUG1 of Saccharomyces cerevisiae. Here we show that FZA-B can functionally substitute for SUG1 in yeast and that FZA-B interacts with Fos proteins in vitro through their leucine zippers. In rat liver and in HeLa cells, FZA-B is present in the 26S proteasome complex, as is c-Fos. Immobilized antibody raised against an FZA-B-specific peptide depleted peptidase activity, proteasomal proteins, FZA-B, and c-Fos from a 26S proteasome preparation. FZA-B is found predominantly in the nuclear fraction of COS cells expressing an FZA-B transgene and in the nuclear 26S proteasome of HeLa cells. We conclude that FZA-B is the mammalian homolog of SUG1 (mSug1) and that it is present in the nuclear 26S proteasome of cells. Our results suggest that mSug1 may be involved in the degradation of c-Fos and other transcription factors.

Arabidopsis MBP1 gene encodes a conserved ubiquitin recognition component of the 26S proteasome.

Multiubiquitin chain attachment is a key step leading to the selective degradation of abnormal polypeptides and many important regulatory proteins by the eukaryotic 26S proteasome. However, the mechanism by which the 26S complex recognizes this posttranslational modification is unknown. Using synthetic multiubiquitin chains to probe an expression library for interacting proteins, we have isolated an Arabidopsis cDNA, designated MBP1, that encodes a 41-kDa acidic protein exhibiting high affinity for chains, especially those containing four or more ubiquitins. Based on similar physical and immunological properties, multiubiquitin binding affinities, and peptide sequence, MBP1 is homologous to subunit 5a of the human 26S proteasome. Structurally related proteins also exist in yeast, Caenorhabditis, and other plant species. Given their binding properties, association with the 26S proteasome, and widespread distribution, MBP1, S5a, and related proteins likely function as essential ubiquitin recognition components of the 26S proteasome.