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.

Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER), where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ERassociated degradation (ERAD) removes these client proteins from the ER membrane to the cytosol in a process known as retrotranslocation. Our previous work demonstrated that rhomboid pseudoprotease Dfm1 is involved in the retrotranslocation of ubiquitinated membrane integral ERAD substrates. Herein, we found that Dfm1 associates with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components that are critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. Furthermore, Dfm1 employs an ERADindependent role for facilitating the ER export and endosome-and Golgi-associated degradation (EGAD) of Orm2, which is a major antagonist of SPT activity. Given that the accumulation of human Orm2 homologs, ORMDLs, is associated with various pathologies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.

Accumulation of misfolded proteins is a known source of cellular stress and can be detrimental to cellular health. While protein aggregation is a known hallmark of many diseases the mechanisms by which protein aggregates cause toxicity and the molecular machines that prevent this toxicity are not completely understood. Here, we show that the accumulated misfolded membrane proteins form endoplasmic reticulum (ER) localized aggregates, impacting ubiquitin and proteasome homeostasis. Additionally, we have identified a chaperone ability of the yeast rhomboid pseudoprotease Dfm1 to influence the solubilization of misfolded membrane proteins and prevent toxicity from misfolded membrane proteins. We establish that this function of Dfm1 does not require recruitment of the ATPase Cdc48 and it is distinct from Dfm1s previously identified function in dislocating misfolded membrane proteins to the cytosol for degradation.

Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER) where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins ER-associated-degradation (ERAD) removes these clients from the ER membrane to the cytosol in a process known as retrotranslocation. Our recent work demonstrates that rhomboid pseudoprotease Dfm1 is involved in the retrotranslocation of ubiquitinated integral membrane ERAD substrates. To survey for potential interaction partners of Dfm1 we performed protein-proximity labeling by BioID (proximity-dependent biotin identification) followed by mass spectrometry and identified several interacting proteins known to play a role in the sphingolipid biosynthesis pathway. Specifically we found that Dfm1 physically interacts with the SPOTS complex which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components and is critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. We demonstrate for the first time that Dfm1 has a role in ER export a function that is independent of Dfm1s canonical ERAD retrotranslocation function. Specifically we show that loss of Dfm1 results in the accumulation of phosphorylated Orm2 at the ER suggesting a novel role for Dfm1 in controlling Orm2 export from the ER and its subsequent degradation by EGAD. Moreover recruitment of Cdc48 by Dfm1 which is critical for its role in ERAD retrotranslocation is dispensable for Dfm1s role in ER export. Given that the accumulation of human Orm2 homologs ORMDLs are associated with many maladies our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.