The accuracy of tail-anchored (TA) protein targeting to the endoplasmic reticulum (ER) depends on the Guided Entry of Tail-Anchored (Get) protein targeting machinery. The fate of TA proteins that become inappropriately inserted into other organelles, such as mitochondria, is unknown. Here, we identify Msp1, a conserved, membrane-anchored AAA-ATPase (ATPase associated with a variety of cellular activities) that localizes to mitochondria and peroxisomes, as a critical factor in a quality control pathway that senses and degrades TA proteins mistargeted to the outer mitochondrial membrane (OMM). Pex15 is normally targeted by the Get pathway to the ER, from where it travels to peroxisomes. Loss of Msp1 or loss of the Get pathway results in the redistribution of Pex15 to mitochondria. Cells lacking both a functional Get pathway and Msp1 accumulate increased amounts of Pex15 on the OMM and display severely dysfunctional mitochondrial morphology. In addition, Msp1 binds and promotes the turnover of a Pex15 mutant that is misdirected to the OMM. Our data suggest that Msp1 functions in local organelle surveillance by extracting mistargeted proteins, ensuring the fidelity of organelle specific-localization of TA proteins.

The endoplasmic reticulum (ER) protein folding capacity is balanced with the protein folding burden to prevent accumulation of un- or misfolded proteins. The ER membrane-resident kinase/RNase Ire1 maintains ER protein homeostasis through two fundamentally distinct processes. First, Ire1 can initiate a transcriptional response through a non-conventional mRNA splicing reaction to increase the ER folding capacity. Second, Ire1 can decrease the ER folding burden through selective mRNA decay. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two Ire1 functions have been evolutionarily separated. Here, we show that the respective Ire1 orthologs have become specialized for their functional outputs by divergence of their RNase specificities. In addition, RNA structural features separate the splicing substrates from the decay substrates. Using these insights, we engineered an S. pombe Ire1 cleavage substrate into a splicing substrate, which confers S. pombe with both Ire1 functional outputs.

Mitochondrial DNA (mtDNA) is essential for mitochondrial and cellular function. In Saccharomyces cerevisiae, mtDNA is organized in nucleoprotein structures termed nucleoids, which are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle. How the cell distributes and inherits mtDNA is incompletely understood although an involvement of mitochondrial fission and fusion has been suggested. We developed a LacO-LacI system to noninvasively image mtDNA dynamics in living cells. Using this system, we found that nucleoids are nonrandomly spaced within the mitochondrial network and observed the spatiotemporal events involved in mtDNA inheritance. Surprisingly, cells deficient in mitochondrial fusion and fission distributed and inherited mtDNA normally, pointing to alternative pathways involved in these processes. We identified such a mechanism, where we observed fission-independent, but F-actin-dependent, tip generation that was linked to the positioning of mtDNA to the newly generated tip. Although mitochondrial fusion and fission were dispensable for mtDNA distribution and inheritance, we show through a combination of genetics and next-generation sequencing that their absence leads to an accumulation of mitochondrial genomes harboring deleterious structural variations that cluster at the origins of mtDNA replication, thus revealing crucial roles for mitochondrial fusion and fission in maintaining the integrity of the mitochondrial genome.