Ancestral sequence reconstruction of Mic60 reveals a residue signature supporting respiration in yeast.

In eukaryotes, the essential process of cellular respiration takes place in the cristae of mitochondria. The protein Mic60 is known to stabilize crista junctions; however, how the C-terminal Mitofilin domain of Mic60 mediates cristae-supported respiration remains elusive. Here, we used ancestral sequence reconstruction to generate Mitofilin ancestors up to and including the last opisthokont common ancestor (LOCA). We found that yeast-lineage derived Mitofilin ancestors as far back as the LOCA rescue respiration. By comparing Mitofilin ancestors with different respiratory phenotypes, we identify four residues that explain the difference between respiration functional yeast- and non-functional animal-derived common Mitofilin ancestors. Our results imply that Mitofilin-supported respiration in yeast stems from a conserved mechanism, and provide a foundation for investigating the divergence of candidate crista junction interactions present during the emergence of eukaryotes.

Yeast derlin Dfm1 employs a chaperone-like function to resolve misfolded membrane protein stress.

Protein aggregates are a common feature of diseased and aged cells. Membrane proteins comprise a quarter of the proteome, and yet, it is not well understood how aggregation of membrane proteins is regulated and what effects these aggregates can have on cellular health. We have determined in yeast that the derlin Dfm1 has a chaperone-like activity that influences misfolded membrane protein aggregation. We establish that this function of Dfm1 does not require recruitment of the ATPase Cdc48 and it is distinct from Dfm1's previously identified function in dislocating misfolded membrane proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Additionally, we assess the cellular impacts of misfolded membrane proteins in the absence of Dfm1 and determine that misfolded membrane proteins are toxic to cells in the absence of Dfm1 and cause disruptions to proteasomal and ubiquitin homeostasis.

HRD Complex Self-Remodeling Enables a Novel Route of Membrane Protein Retrotranslocation.

ER-associated degradation (ERAD) targets misfolded ER proteins for degradation. Retrotranslocation, a key feature of ERAD, entails removal of ubiquitinated substrates into the cytosol for proteasomal destruction. Recently, it has been shown that the Hrd1 E3 ligase forms a retrotranslocation channel for luminal (ERAD-L) substrates. Conversely, our studies found that integral membrane (ERAD-M) substrates exit the ER through a distinct pathway mediated by the Dfm1 rhomboid protein. Those studies also revealed a second, Hrd1-dependent pathway of ERAD-M retrotranslocation can arise in dfm1Δ null. Here we show that, in the dfm1Δ null, the HRD complex undergoes remodeling to a form that mediates ERAD-M retrotranslocation. Specifically, Hrd1's normally present stochiometric partner Hrd3 is efficiently removed during suppressive remodeling, allowing Hrd1 to function in this novel capacity. Neither Hrd1 autoubiquitination nor its cytosolic domain is required for suppressive ERAD-M retrotranslocation. Thus, the HRD complex displays remarkable functional flexibility in response to ER stress.