Mitochondrial Protein Quality Control by the Proteasome Involves Ubiquitination and the Protease Omi

Abstract

We report here that blocking the activity of the 26 S proteasome results in drastic changes in the morphology of the mitochondria and accumulation of intermembrane space (IMS) proteins. Using endonuclease G (endoG) as a model IMS protein, we found that accumulation of wild-type but to a greater extent mutant endoG leads to changes in the morphology of the mitochondria similar to those observed following proteasomal inhibition. Further, we show that wildtype but to a greater extent mutant endoG is a substrate for ubiquitination, suggesting the presence of a protein quality control. Conversely, we also report that wild-type but not mutant endoG is a substrate for the mitochondrial protease Omi but only upon inhibition of the proteasome. These findings suggest that although elimination of mutant IMS proteins is strictly dependent on ubiquitination, elimination of excess or spontaneously misfolded wild-type IMS proteins is monitored by ubiquitination and as a second checkpoint by Omi cleavage when the proteasome function is deficient. One implication of our finding is that in the context of attenuated proteasomal function, accumulation of IMS proteins would contribute to the collapse of the mitochondrial network such as that observed in neurodegenerative diseases. Another implication is that such collapse could be accelerated either by mutations in IMS proteins or by mutations in Omi itself.The elimination of misfolded proteins represents an important mechanism for the maintenance of cellular viability. Such protein quality controls (PQC) 4 involve the binding of a chaperone to the misfolded protein and its presentation to the ubiquitin-dependent proteasome degradation pathway (1, 2). Linkage of ubiquitin to a protein is a highly organized process involving the sequential action of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-ligase (E3) (3). Most of the regulation of the ubiquitination pathway occurs at the level of the ubiquitin ligase. This enzymatic cascade results in the attachment of a polyubiquitin chain onto specific lysine residues of a substrate. Although several types of polyubiquitin chains have now been reported, chains that are built on lysine 48 of ubiquitin serve as a signal for degradation by the 26 S proteasome. The 20 S catalytic core of the proteasome consists of three distinct proteolytic activities within a cylindrical barrel that is flanked by two 19 S regulatory caps. The proteasomes are localized both in the nucleus and in the cytoplasm, where they exist free or in association with organelles including the endoplasmic reticulum (4, 5).PQC in the cytoplasm involves chaperones of the heat shock protein (hsp) family that act in concert with ubiquitin ligases (2). A similar PQC acting in the nucleus has recently been reported (6). Ubiquitin-dependent PQC was also found to monitor the folding of proteins present in the lumen of the endoplasmic reticulum, a result that was at first surprising since proteins that are translocated across the ER membrane ...

SirT3 Regulates the Mitochondrial Unfolded Protein Response

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Degradation of a Cytosolic Protein Requires Endoplasmic Reticulum-associated Degradation Machinery

“…10 l of each dilution was spotted onto the indi- 4 Kreft and Hochstrasser, unpublished data. cated media using a multichannel Pipetman, and plates were incubated for 2 days at 30°C unless otherwise indicated.…”

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“…Proteasome-mediated proteolysis is known to be a key checkpoint by which excess or misfolded mitochondrial proteins can be eliminated (27). Therefore, we attempted to assess the ubiquitination level of Mitofilin proteins in the mDISC1 knockdown or truncated mDISC1 overexpression conditions.…”