The derlin Dfm1 couples retrotranslocation of a folded protein domain to its proteasomal degradation.

Endoplasmic reticulum (ER) proteins are degraded by proteasomes in the cytosol through ER-associated degradation (ERAD). This process involves the retrotranslocation of substrates across the ER membrane, their ubiquitination, and membrane extraction by the Cdc48/Npl4/Ufd1 ATPase complex prior to delivery to proteasomes for degradation. How the presence of a folded luminal domain affects substrate retrotranslocation and this event is coordinated with subsequent ERAD steps remains unknown. Here, using a model substrate with a folded luminal domain, we showed that Cdc48 ATPase activity is sufficient to drive substrate retrotranslocation independently of ERAD membrane components. However, the complete degradation of the folded luminal domain required substrate-tight coupling of retrotranslocation and proteasomal degradation, which was ensured by the derlin Dfm1. Mutations in Dfm1 intramembrane rhomboid-like or cytosolic Cdc48-binding regions resulted in partial degradation of the substrate with accumulation of its folded domain. Our study revealed Dfm1 as a critical regulator of Cdc48-driven retrotranslocation and highlights the importance of coordinating substrate retrotranslocation and degradation during ERAD.

Hydrogenosomal tail-anchored proteins are targeted to both mitochondria and ER upon their expression in yeast cells.

Some organisms, like Trichomonas vaginalis, contain mitochondria-related hydrogen-producing organelles, called hydrogenosomes. The protein targeting into these organelles is proposed to be similar to the well-studied mitochondria import. Indeed, S. cerevisiae mitochondria and T. vaginalis hydrogenosomes share some components of protein import complexes. However, it is still unknown whether targeting signals directing substrate proteins to hydrogenosomes can support in other eukaryotes specific mitochondrial localization. To address this issue, we investigated the intracellular localization of three hydrogenosomal tail-anchored proteins expressed in yeast cells. We observed that these proteins were targeted to both mitochondria and ER with a variable dependency on the mitochondrial MIM complex. Our results suggest that the targeting signal of TA proteins are only partially conserved between hydrogenosomes and yeast mitochondria.

The Biogenesis of Mitochondrial Outer Membrane Proteins Show Variable Dependence on Import Factors.

Biogenesis of mitochondrial outer membrane proteins involves their integration into the lipid bilayer. Among these proteins are those that form a single-span topology, but our understanding of their biogenesis is scarce. In this study, we found that the MIM complex is required for the membrane insertion of some single-span proteins. However, other such proteins integrate into the membrane in a MIM-independent manner. Moreover, the biogenesis of the studied proteins was dependent to a variable degree on the TOM receptors Tom20 and Tom70. We found that Atg32 C-terminal domain mediates dependency on Tom20, whereas the cytosolic domains of Atg32 and Gem1 facilitate MIM involvement. Collectively, our findings (1) enlarge the repertoire of MIM substrates to include also tail-anchored proteins, (2) provide new mechanistic insights to the functions of the MIM complex and TOM import receptors, and (3) demonstrate that the biogenesis of MOM single-span proteins shows variable dependence on import factors.

Pex19 is involved in importing dually targeted tail-anchored proteins to both mitochondria and peroxisomes.

Tail-anchored (TA) proteins are embedded into their corresponding membrane via a single transmembrane segment at their C-terminus whereas the majority of the protein is facing the cytosol. So far, cellular factors that mediate the integration of such proteins into the mitochondrial outer membrane were not found. Using budding yeast as a model system, we identified the cytosolic Hsp70 chaperone Ssa1 and the peroxisome import factor Pex19 as import mediators for a subset of mitochondrial TA proteins. Accordingly, deletion of PEX19 results in: (1) growth defect under respiration conditions, (2) alteration in mitochondrial morphology, (3) reduced steady-state levels of the mitochondrial TA proteins Fis1 and Gem1, and (4) hampered in organello import of the TA proteins Fis1 and Gem1. Furthermore, recombinant Pex19 can bind directly the TA proteins Fis1 and Gem1. Collectively, this work identified the first factors that are involved in the biogenesis of mitochondrial TA proteins and uncovered an unexpected function of Pex19.

Independent evolution of functionally exchangeable mitochondrial outer membrane import complexes.

Assembly and/or insertion of a subset of mitochondrial outer membrane (MOM) proteins, including subunits of the main MOM translocase, require the fungi-specific Mim1/Mim2 complex. So far it was unclear which proteins accomplish this task in other eukaryotes. Here, we show by reciprocal complementation that the MOM protein pATOM36 of trypanosomes is a functional analogue of yeast Mim1/Mim2 complex, even though these proteins show neither sequence nor topological similarity. Expression of pATOM36 rescues almost all growth, mitochondrial biogenesis, and morphology defects in yeast cells lacking Mim1 and/or Mim2. Conversely, co-expression of Mim1 and Mim2 restores the assembly and/or insertion defects of MOM proteins in trypanosomes ablated for pATOM36. Mim1/Mim2 and pATOM36 form native-like complexes when heterologously expressed, indicating that additional proteins are not part of these structures. Our findings indicate that Mim1/Mim2 and pATOM36 are the products of convergent evolution and arose only after the ancestors of fungi and trypanosomatids diverged.

The GET pathway can increase the risk of mitochondrial outer membrane proteins to be mistargeted to the ER.

Tail-anchored (TA) proteins are anchored to their corresponding membrane via a single transmembrane segment (TMS) at their C-terminus. In yeast, the targeting of TA proteins to the endoplasmic reticulum (ER) can be mediated by the guided entry of TA proteins (GET) pathway, whereas it is not yet clear how mitochondrial TA proteins are targeted to their destination. It has been widely observed that some mitochondrial outer membrane (MOM) proteins are mistargeted to the ER when overexpressed or when their targeting signal is masked. However, the mechanism of this erroneous sorting is currently unknown. In this study, we demonstrate the involvement of the GET machinery in the mistargeting of suboptimal MOM proteins to the ER. These findings suggest that the GET machinery can, in principle, recognize and guide mitochondrial and non-canonical TA proteins. Hence, under normal conditions, an active mitochondrial targeting pathway must exist that dominates the kinetic competition against other pathways.