Mitochondrial carrier protein overloading and misfolding induce aggresomes and proteostatic adaptations in the cytosol

Previous studies in yeast showed that mitochondrial stressors not directly targeting the protein import machinery can cause mitochondrial precursor overaccumulation stress (mPOS) in the cytosol independent of bioenergetics. Here, we demonstrate mPOS and stress responses in human cells. We show that overloading of mitochondrial membrane carrier, but not matrix proteins, is sufficient to induce cytosolic aggresomes and apoptosis. The aggresomes appear to triage unimported mitochondrial proteins. Interestingly, expression of highly unstable mutant variants of the mitochondrial carrier protein, Ant1, also induces aggresomes despite a greater than 20-fold reduction in protein level compared to wild type. Thus, overloading of the protein import machinery, rather than protein accumulation, is critical for aggresome induction. The data suggest that the import of mitochondrial proteins is saturable and that the cytosol is limited in degrading unimported mitochondrial proteins. In addition, we found that EGR1, eEF1a, and ubiquitin C are up-regulated by Ant1 overloading. These proteins are known to promote autophagy, protein targeting to aggresomes, and the processing of protein aggregates, respectively. Finally, we found that overexpression of the misfolded variants of Ant1 induces additional cytosolic responses including proteasomal activation. In summary, our work captured a profound effect of unimported mitochondrial proteins on cytosolic proteostasis and revealed multiple anti-mPOS mechanisms in human cells.

Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria

SCL25A46 is a mitochondrial carrier protein that surprisingly localizes to the outer membrane and is distantly related to Ugo1. Here we show that a subset of SLC25A46 interacts with mitochondrial dynamics components and the MICOS complex. Decreased expression of SLC25A46 results in increased stability and oligomerization of MFN1 and MFN2 on mitochondria, promoting mitochondrial hyperfusion. A mutation at L341P causes rapid degradation of SLC25A46, which manifests as a rare disease, pontocerebellar hypoplasia. The E3 ubiquitin ligases MULAN and MARCH5 coordinate ubiquitylation of SLC25A46 L341P, leading to degradation by organized activities of P97 and the proteasome. Whereas outer mitochondrial membrane–associated degradation is typically associated with apoptosis or a specialized type of autophagy termed mitophagy, SLC25A46 degradation operates independently of activation of outer membrane stress pathways. Thus SLC25A46 is a new component in mitochondrial dynamics that serves as a regulator for MFN1/2 oligomerization. Moreover, SLC25A46 is selectively degraded from the outer membrane independently of mitophagy and apoptosis, providing a framework for mechanistic studies in the proteolysis of outer membrane proteins.

The non-canonical mitochondrial inner membrane presequence translocase of trypanosomatids contains two essential rhomboid-like proteins

Mitochondrial protein import is essential for all eukaryotes. Here we show that the early diverging eukaryote Trypanosoma brucei has a non-canonical inner membrane (IM) protein translocation machinery. Besides TbTim17, the single member of the Tim17/22/23 family in trypanosomes, the presequence translocase contains nine subunits that co-purify in reciprocal immunoprecipitations and with a presequence-containing substrate that is trapped in the translocation channel. Two of the newly discovered subunits are rhomboid-like proteins, which are essential for growth and mitochondrial protein import. Rhomboid-like proteins were proposed to form the protein translocation pore of the ER-associated degradation system, suggesting that they may contribute to pore formation in the presequence translocase of T. brucei. Pulldown of import-arrested mitochondrial carrier protein shows that the carrier translocase shares eight subunits with the presequence translocase. This indicates that T. brucei may have a single IM translocase that with compositional variations mediates import of presequence-containing and carrier proteins.

Proteins and lipids of glycosomal membranes from Leishmania tarentolae and Trypanosoma brucei

In kinetoplastid protists, several metabolic pathways, including glycolysis and purine salvage, are located in glycosomes, which are microbodies that are evolutionarily related to peroxisomes. With the exception of some potential transporters for fatty acids, and one member of the mitochondrial carrier protein family, proteins that transport metabolites across the glycosomal membrane have yet to be identified. We show here that the phosphatidylcholine species composition of Trypanosoma brucei glycosomal membranes resembles that of other cellular membranes, which means that glycosomal membranes are expected to be impermeable to small hydrophilic molecules unless transport is facilitated by specialized membrane proteins. Further, we identified 464 proteins in a glycosomal membrane preparation from Leishmania tarentolae. The proteins included approximately 40 glycosomal matrix proteins, and homologues of peroxisomal membrane proteins - PEX11, GIM5A and GIM5B; PXMP4, PEX2 and PEX16 - as well as the transporters GAT1 and GAT3. There were 27 other proteins that could not be unambiguously assigned to other compartments, and that had predicted trans-membrane domains. However, no clear candidates for transport of the major substrates and intermediates of energy metabolism were found. We suggest that, instead, these metabolites are transported via pores formed by the known glycosomal membrane proteins.