Mitochondrial import deficiency causes cellular toxicity due to the accumulation of non-imported mitochondrial precursor proteins, termed mitoprotein-induced stress. Despite the burden mis-localized mitochondrial precursors place on cells, our understanding of the systems that dispose of these proteins is incomplete. Here, we cataloged the location and steady-state abundance of mitochondrial precursor proteins during mitochondrial impairment in S. cerevisiae. We found that a number of non-imported mitochondrial proteins localize to the nucleus, where they are subjected to proteasome-dependent degradation through a process we term nuclear-associated mitoprotein degradation (mitoNUC). Recognition and destruction of mitochondrial precursors by the mitoNUC pathway requires the presence of an N-terminal mitochondrial targeting sequence (MTS) and is mediated by combined action of the E3 ubiquitin ligases San1, Ubr1, and Doa10. Impaired breakdown of precursors leads to alternative sequestration in nuclear-associated foci. These results identify the nucleus as an important destination for the disposal of non-imported mitochondrial precursors.

Deficiencies in mitochondrial import cause the toxic accumulation of non-imported mitochondrial precursor proteins. Numerous fates for non-imported mitochondrial precursors have been identified in budding yeast, including proteasomal destruction, deposition into protein aggregates, and mistargeting to other organelles. Amongst organelles, the ER has emerged as a key destination for a subset of non-imported mitochondrial proteins. However, how ER targeting of various types of mitochondrial proteins is achieved remains incompletely understood. Here, we show that the ER delivery of endogenous mitochondrial transmembrane proteins, especially those belonging to the SLC25A mitochondrial carrier family, is dependent on the guided entry of tail-anchored proteins (GET) complex. Without a functional GET pathway, non-imported mitochondrial proteins destined for the ER are alternatively sequestered into Hsp42-dependent protein foci. Loss of the GET pathway is detrimental to yeast cells experiencing mitochondrial import failure and prevents re-import of mitochondrial proteins from the ER via the ER-SURF pathway. Overall, this study outlines an important role for the GET complex in ER targeting of non-imported mitochondrial carrier proteins.

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Mitochondria are dynamic organelles with essential roles in signaling and metabolism. We recently identified a cellular structure called the mitochondrial-derived compartment (MDC) that is generated from mitochondria in response to amino acid overabundance stress. How cells form MDCs is unclear. Here, we show that MDCs are dynamic structures that form and stably persist at sites of contact between the ER and mitochondria. MDC biogenesis requires the ER–mitochondria encounter structure (ERMES) and the conserved GTPase Gem1, factors previously implicated in lipid exchange and membrane tethering at ER–mitochondria contacts. Interestingly, common genetic suppressors of abnormalities displayed by ERMES mutants exhibit distinct abilities to rescue MDC formation in ERMES-depleted strains and are incapable of rescuing MDC formation in cells lacking Gem1. Thus, the function of ERMES and Gem1 in MDC biogenesis may extend beyond their conventional role in maintaining mitochondrial phospholipid homeostasis. Overall, this study identifies an important function for ER–mitochondria contacts in the biogenesis of MDCs.

SUMMARYDeficiencies in mitochondrial import cause the toxic accumulation of non-imported mitochondrial precursor proteins. Numerous fates for non-imported mitochondrial precursors have been identified, including proteasomal destruction, deposition into protein aggregates, and mis-targeting to other organelles. Amongst organelles, the endoplasmic reticulum (ER) has emerged as a key destination for non-imported mitochondrial proteins, but how ER-targeting of these proteins is achieved remains unclear. Here, we show that the guided entry of tail-anchored proteins (GET) complex is required for ER-targeting of endogenous mitochondrial multi-transmembrane proteins. Without a functional GET pathway, non-imported mitochondrial proteins destined for the ER are alternatively sequestered into Hsp42-dependent protein foci. The ER targeting of non-imported mitochondrial proteins by the GET complex prevents cellular toxicity and facilitates re-import of mitochondrial proteins from the ER via the recently identified ER-SURF pathway. Overall, this study outlines an important and unconventional role for the GET complex in mitigating stress associated with non-imported mitochondrial proteins.