An ERAD‐independent role for rhomboid pseudoprotease Dfm1 in mediating sphingolipid homeostasis

Abstract

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.

Utilizing a nanobody recruitment approach for assessing serine palmitoyltransferase activity in ER sub-compartments of yeast

“…In a recent study we have solved the structure of the Orm1 containing SPOTS complex and have proposed that a monomeric form is the predominant form in yeast cells (Schäfer et al , 2023). This could also explain why the two Orm proteins are differentially regulated by the EGAD pathway (Schmidt et al , 2019; Bhaduri et al , 2023). It is also noteworthy that the timing of the increase in LCB biosynthesis after 5 minutes of heat shock and the regulatory mechanism by the EGAD pathway are difficult to align.…”

“…The two Orm proteins also underlie different regulatory mechanisms. Only the Orm2 protein is a substrate for the endosome/Golgi associated degradation (EGAD) pathway that depends on its phosphorylation by Ypk1/2, export from the ER, ubiquitination and finally degradation via the proteasome (Schmidt et al , 2019; Bhaduri et al , 2023). In addition, it was suggested that upregulation of SPT activity is directly linked to the uptake of exogenous serine via the Gnp1 amino acid permease (Esch et al , 2020).…”

Lipid Biosynthesis Perturbation Impairs Endoplasmic Reticulum-Associated Degradation

“…Yeast and mammalian homologs of Hrd1 and Doa10 promote feedback-regulated degradation of sterol-biosynthetic enzymes as well as turnover of proteins implicated in triacylglycerol and low-density-lipoprotein synthesis (HAMPTON et al 1996;GARZA et al 2009;JO et al 2011;FORESTI et al 2013;STEVENSON et al 2016;HUANG AND CHEN 2023). Moreover, the ER-resident rhomboid pseudoprotease Dfm1 was recently shown to regulate the ER export and Golgi-associated degradation of Orm2, an inhibitor of sphingolipid biosynthesis (BHADURI et al 2022). Lipid bilayer stress activates the yeast and mammalian ER unfolded protein response (UPR) via a distinct mechanism than activation by unfolded proteins (JONIKAS et al 2009;PROMLEK et al 2011;THIBAULT et al 2012;VOLMER et al 2013;HALBLEIB et al 2017;HO et al 2020; XU AND TAUBERT 2021).…”

“…2016; H uang and C hen 2023). Moreover, the ER-resident rhomboid pseudoprotease Dfm1 was recently shown to regulate the ER export and Golgi-associated degradation of Orm2, an inhibitor of sphingolipid biosynthesis (B haduri et al . 2022).…”

The Dsc ubiquitin ligase complex identifies transmembrane degrons to degrade orphaned proteins at the Golgi

“…We termed this membrane protein degradation pathway e ndosome and G olgi a ssociated d egradation (EGAD) 7,20,28 . To reach the Golgi, Orm2 must no longer bind to the serine palmitoyl transferase complex (SPT, Lcb1/2) at the ER, and exit the ER in COPII carriers with the help of the derlin Dfm1 29 . EGAD of Orm2 contributes to the de-repression of SPT activity and thus helps to maintain sphingolipid homeostasis 7 .…”