Highlights d Dfm1 selectively binds ERAD-targeted membrane substrates d Polyubiquitin chains bind directly to Cdc48 recruited by Dfm1 d Derlin lipid thinning facilitates removal of integral membrane substrates in the ER d Substrate engagement and lipid thinning are conserved derlin features
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.
Nearly one-third of proteins are initially targeted to the endoplasmic reticulum (ER) membrane where they are correctly folded, assembled, and then delivered to their final cellular destinations. In order to prevent the accumulation of misfolded membrane proteins, ER associated degradation (ERAD) moves these clients from the ER membrane to the cytosol; a process known as retrotranslocation. Our recent work in S. cerevisiae has revealed a derlin rhomboid pseudoprotease Dfm1 is involved in the retrotranslocation of ubiquitinated ERAD membrane substrates. In this study we sought to understand the mechanism associated with Dfm1's actions and found that Dfm1's conserved rhomboid residues are critical for membrane protein retrotranslocation. Specifically, we identified several retrotranslocation-deficient Loop 1 mutants that display impaired binding to membrane substrates. Furthermore, Dfm1 has retained the lipid thinning functions of its rhomboid protease predecessors to facilitate in the removal of ER membrane substrates. We find this substrate engagement and lipid thinning feature is conserved in its human homolog, Derlin-1. Utilizing interaction studies and molecular dynamics simulations, this work reveals that rhomboid pseudoprotease derlins employ novel mechanisms of substrate engagement and lipid thinning for catalyzing extraction of multi-spanning membrane substrates.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.