Maintaining the essential functions of mitochondria requires mechanisms to recognize and remove misfolded proteins. However, quality control (QC) pathways for misfolded mitochondrial proteins remain poorly defined. Here, we establish temperature-sensitive (ts-) peripheral mitochondrial outer membrane (MOM) proteins as novel model QC substrates in Saccharomyces cerevisiae. The ts- proteins sen2-1HAts and sam35-2HAts are degraded from the MOM by the ubiquitin-proteasome system. Ubiquitination of sen2-1HAts is mediated by the ubiquitin ligase (E3) Ubr1, while sam35-2HAts is ubiquitinated primarily by San1. Mitochondria-associated degradation (MAD) of both substrates requires the SSA family of Hsp70s and the Hsp40 Sis1, providing the first evidence for chaperone involvement in MAD. In addition to a role for the Cdc48-Npl4-Ufd1 AAA-ATPase complex, Doa1 and a mitochondrial pool of the transmembrane Cdc48 adaptor, Ubx2, are implicated in their degradation. This study reveals a unique QC pathway comprised of a combination of cytosolic and mitochondrial factors that distinguish it from other cellular QC pathways.
Keywords: MAD; S. cerevisiae; UPS; cell biology; misfolded; quality control; yeast.
Proteins are molecules that need to fold into the right shape to do their job. If proteins lose that shape, not only do they stop working but they risk clumping together and becoming toxic, potentially leading to disease. Fortunately, the cell has quality control systems that normally detect and remove misfolded proteins before they can cause damage to the cell. First, sets of proteins known as chaperones recognize the misfolded proteins, and then another class of proteins attaches a molecular tag, known as ubiquitin, to the misshapen proteins. When several ubiquitin tags are attached to a protein, forming chains of ubiquitin, it is transported to a large molecular machine within the cell called the proteasome. The proteasome unravels the protein and breaks it down into its constituent building blocks, which can then be used to create new proteins. Proteins are found throughout the different compartments of the cell and quality control processes have been well-studied in some parts of the cell but not others. Metzger et al. have now revealed how the process works on the surface of mitochondria, the compartment that provides the cell with most of its energy. To do this, they used baker’s yeast, a model laboratory organism that shares many fundamental properties with animal cells, but which is easier to manipulate genetically. The quality control process was studied using two mitochondrial proteins that had been mutated to make them sensitive to changes in temperature. This meant that, when the temperature increased from 25°C to 37°C, these proteins would begin to unravel and trigger the clean-up operation. This approach has been used previously to understand the quality control processes in other parts of the cell. By removing different quality control machinery in turn from the yeast cells, Metzger et al. could detect which were necessary for the process on mitochondria. This showed that there were many similarities with how this process happen in other parts of the cell but that the precise combination of chaperones and enzymes involved was distinct. Furthermore, when the proteasome was not working, the misfolded proteins remained on the mitochondria, showing that they are not transported to other parts of the cell to be broken down. In the future, understanding this process could help to find potential drug targets for mitochondrial diseases. The next steps will be to see how well these findings apply to human and other mammalian cells.