Involvement of the Molecular Chaperone Ydj1 in the Ubiquitin-Dependent Degradation of Short-Lived and Abnormal Proteins in Saccharomyces cerevisiae

Abstract

In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38؇C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain ␤-galactosidase (␤-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-␤-gal and leucine-␤-gal) and the B-type cyclin Clb5-␤-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-␤-gal (Ub-P-␤-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-␤-gal, the ␤-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-␤-gal and not with stable ␤-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-␤-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitinligating enzymes.

Ubiquitin-dependent Degradation of Certain Protein Substrates in Vitro Requires the Molecular Chaperone Hsc70

“…A Saccharomyces cerevisiae cell that harbors a temperature-sensitive mutant of Ydj1 demonstrated a large defect in the overall breakdown of short-lived and abnormal proteins that is mediated by the ubiquitin system. Degradation of long-lived proteins that is mediated by the vacuole and proceeds in a ubiquitin-independent manner was unaffected (25). Interestingly, the effect was specific.…”

“…Concomitantly, the ubiquitination of the Ydj1-dependent substrates was significantly reduced. Researchers also found that the chaperone generates a complex with its target substrate, but due to the limitations of the in vivo studies, they could not demonstrate that the complex serves as an essential intermediate in the proteolytic process (25). Degradation of the yeast cyclin Cln3 is also stimulated by Ydj1.…”

Effects of Ubiquitin System Alterations on the Formation and Loss of a Yeast Prion

“…Mechanisms of the chaperone effects on prions are not yet completely understood. As some of the chaperones of Hsp70 and Hsp40 families were implicated in targeting misfolded proteins for Ubdependent degradation (30,31), it is possible that some of the effects of chaperones on prions could be mediated by UPS.…”

Mass Spectrometry Analysis of Proteome-Wide Proteolytic Post-Translational Degradation of Proteins

“…11 Lysosomes, on the other hand, are membrane bound organellelike structures containing multiple proteases that degrade protein aggregates and membrane proteins. 3,12 Proteins to-be-degraded are transported to the lysosome through e.g., the ATGs/ signaling/regulating systems 13,14 and the following proteolyses are catalyzed by endoproteinases 15,16 that are matured and activated within the lysosome. The resultant peptides continue to break down into amino acids by the lysosomal aminopeptidases and carboxypeptidases, 16,17 and the products (e.g., amino acids and others such as sugars, cholesterols) are then transported back out into the cytosol by the solute transporters in the Requests for materials should be addressed to Yufeng Shen (E-mail: yufeng.shen@pnl.gov) or Richard D. Smith (E-mail: dick.smith@pnl.gov).…”

“…13,14 Protein degradation has been primarily studied using the methods based on radioactivity, 3 which have proven to be informative on an overall level, but provided little information regarding the degradation steps occurring to individual proteins. Such information currently has been gained by performing in vitro experiments 6,7,[13][14][15][16][17] with individual model proteins/ peptides and proteases, but evidences provided from these in vitro experiments do not represent the true in vivo scenarios due to the inability of a few single proteins to truly mimic the regulatory processes and complexity of degradation occurring to multiple proteins at the same time within a cell.…”