UCLA

Matrix Processing Peptidase and Putative Roles in Mitochondrial Biogenesis

The mitochondrial protein import and proteolytic system are important for homeostasis of the mitochondrion. Typically, precursor proteins are targeted to the mitochondria by an N-terminal targeting sequence and are imported via TOM and TIM translocons of the outer and inner membrane. A series of proteases is required for the import and processing of precursors as well assembly and degradation of mitochondrial proteins. The matrix processing peptidase (MPP) coordinates the removal of presequences from precursors and is important in maintaining mitochondrial homeostasis. Furthermore, MPP is associated with neurodegenerative diseases. A mutation in PMPCA causes a form of non-progressive cerebellar ataxia, because the Friedreich’s ataxia protein is not processed correctly. The Parkinson’s associated protein PINK1 is processed by MPP and attenuation of β-MPP has been shown to induce mitophagy. To characterize the function of MPP in mammalian systems and develop probes to attenuate MPP function, we developed a small molecule screening strategy to develop MPP-specific chemical probes from a library of over 100,000 drug-like small molecules. The screen yielded two structurally distinct and specific MPP inhibitors deemed MitoBloCK-50 and MitoBloCK-51. Our results suggest in addition to playing a role in the processing of precursors, MPP also plays a role in the import of precursors. Here I present data that characterize the utility of MitoBloCK-50 and MitoBloCK-51 for MPP attenuation in various model systems. In addition, an in vivo tagging system known as BioID identified an interaction between MPP and core subunit Qcr2 of Complex III, suggesting an evolutionary conserved interaction with the respiratory chain.

Nuc1 and Porin Influence L-A Viral loads Co-dependently in Saccharomyces Cerevisiae

The yeast double-stranded RNA (dsRNA) killer viruses of the Totiviridae family are well-characterized and one of the most prolific in laboratory yeast strains. Infected yeast are able to tolerate high levels of the killer virus without displaying any growth defects. The complex symbiotic relationship between host yeast and dsRNA viruses are still being elucidated. Notably, deletion of mitochondrial genes NUC1, which encodes the major mitochondrial nuclease, and POR1, the voltage-dependent anion channel of the outer mitochondrial membrane, lead to overproduction of the capsid Gag protein encoded by the L-A dsRNA helper genome of Killer virus. In this work we explore mechanisms of Nuc1 and porin regulation of the L-A dsRNA genome of yeast Killer viruses. We observed strains with NUC1 deletion have higher L-A levels than POR1 deleted strains. Furthermore, deletion of both NUC1 and POR1 have comparable L-A levels to single NUC1 mutants. Plasmid expression of Nuc1 mutants that are nuclease deficient or cytosolic localized fail to rescue L-A viral loads in NUC1 deleted strains. In addition, plasmid expression of NUC1 appears to increase the L-A viral load in the NUC1 and POR1 double knockout. Nuclease protection experiments demonstrate L-A transcripts are protected in ΔNUC1 and ΔPOR1 mitochondria but not in wildtype. We propose a model where ss(+) L-A transcripts are imported into the mitochondrial IMS and are subsequently degraded by Nuc1. Meanwhile, POR1 deletion disrupts the L-A regulating function of Nuc1. Overall these results demonstrate a role for mitochondria in regulating dsRNA viruses in yeast.