ER residency of the ceramide phosphoethanolamine synthase SMSr relies on homotypic oligomerization mediated by its SAM domain

SMSr/SAMD8 is an ER-resident ceramide phosphoethanolamine synthase with a critical role in controlling ER ceramides and suppressing ceramide-induced apoptosis in cultured cells. SMSr-mediated ceramide homeostasis relies on the enzyme’s catalytic activity as well as on its N-terminal sterile α-motif or SAM domain. Here we report that SMSr-SAM is structurally and functionally related to the SAM domain of diacylglycerol kinase DGKδ, a central regulator of lipid signaling at the plasma membrane. Native gel electrophoresis indicates that both SAM domains form homotypic oligomers. Chemical crosslinking studies show that SMSr self-associates into ER-resident trimers and hexamers that resemble the helical oligomers formed by DGKδ-SAM. Residues critical for DGKδ-SAM oligomerization are conserved in SMSr-SAM and their substitution causes a dissociation of SMSr oligomers as well as a partial redistribution of the enzyme to the Golgi. Conversely, treatment of cells with curcumin, a drug disrupting ceramide and Ca2+ homeostasis in the ER, stabilizes SMSr oligomers and promotes retention of the enzyme in the ER. Our data provide first demonstration of a multi-pass membrane protein that undergoes homotypic oligomerization via its SAM domain and indicate that SAM-mediated self-assembly of SMSr is required for efficient retention of the enzyme in the ER

Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases

SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS)1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog, ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with Glu permitting SMS-catalyzed CPE production and Asp confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes

Molecular Dissection of a Candidate Ceramide Sensor

Sphingomyelin is an essential component of cellular membranes that contribute to the barrier function of the plasma membrane, signaling and molecular sorting. Ceramides are precursors of sphingomyelins and they are produced de novo in the ER. Ceramides are also associated with growth arrest, senescence and apoptosis. Thus, cells must control their ceramides to keep the balance between proliferation and growth arrest/apoptosis. The bulk of newly synthesized ceramides is converted to sphingomyelin in the Golgi by sphingomyelin synthases (SMS). Mammalian cells contain two SMS isoforms, SMS1 in the Golgi and SMS2 at the plasma membrane. A closely related third enzyme, sphingomyelin synthase-related protein (SMSr) is not a conventional SM synthase but synthesizes ceramide-phosphoethanolamine. SMSr orthologs are found in all members of the animal kingdom, even though some do not produce SM. We recently suggested that SMSr is a candidate ceramide sensor whose primary role is to monitor ER ceramide levels to protect cells against the potential risk of apoptosis during sphingolipid biosynthesis. The present thesis aims to investigate the molecular mechanisms by which SMSr regulates ceramide homeostasis. We found that SMSr is a novel and specific substrate of caspase-6, a non-conventional effector caspase implicated in Alzheimer’s Huntington’s disease. In cells treated with staurosporine or FasL, SMSr undergoes caspase-6-dependent cleavage at a conserved aspartate located between its N-terminal SAM domain and the first membrane span. Next, we uncovered a striking structural and functional similarity between SMSr-SAM and the SAM domain of diacylglycerol kinase DGKδ, a central regulator of lipid signaling at the plasma membrane. We demonstrated that SMSr-SAM drives self-assembly of the enzyme into ER-resident trimers and hexamers. Mutations that destabilize SMSr oligomers caused a partial redistribution of the enzyme to the Golgi. Conversely, treatment of cells with curcumin, a drug disrupting ceramide and calcium homeostasis in the ER, stabilizes SMSr oligomers and promotes retention of the enzyme in the ER. Moreover, we report on the successful application of single-molecule photobleaching as approach to monitor homo-typic oligomerization of SMSr in its native cellular environment. By imaging GFP-tagged SMSr proteins as single-fluorescent spots in the ER of intact cells, we were able to trace photobleaching of protein complexes to monitor their oligomeric states. In agreement with our biochemical data, this analysis shows that the SAM domain of SMSr drives self-assembly of the enzyme in the ER and that curcumin promotes SMSr oligomerization. Our study documents, for the first time, that single-molecule photobleaching can be used to resolve changes in the oligomeric state of an ER-resident membrane protein, hence establishing a valuable complementary method to unravel the mechanism by which SMSr mediates ceramide homeostasis in the ER. Collectively, our findings in this thesis have resulted in a better understanding and a toolbox for further investigation of the candidate ceramide sensor SMSr

Ceramide phosphoethanolamine synthase SMSr is a target of caspase-6 during apoptotic cell death

Ceramides are essential precursors of sphingolipids with a dual role as mediators of apoptotic cell death. Previous work revealed that the ER-resident ceramide phosphoethanolamine (CPE) synthase SMSr/SAMD8 is a suppressor of ceramide-mediated apoptosis in cultured cells. Anti-apoptotic activity of SMSr requires a catalytically active enzyme but also relies on the enzyme’s N-terminal sterile a-motif or SAM domain. Here, we demonstrate that SMSr itself is a target of the apoptotic machinery. Treatment of cells with staurosporine or the death receptor ligand FasL triggers caspase-mediated cleavage of SMSr at a conserved aspartate located downstream of the enzyme’s SAM domain and upstream of its first membrane span. Taking advantage of reconstitution experiments with SMSr produced in a cell-free expression system, specific caspase-inhibitors and gene silencing approaches, we show that SMSr is a novel and specific substrate of caspase-6, a non-conventional effector caspase implicated in Huntington’s and Alzheimer’s diseases. Our findings underscore a role of SMSr as negative regulator of ceramide-induced cell death and, in view of a prominent expression of the enzyme in brain, raise questions regarding its potential involvement in neurodegenerative disorders

SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport

The endolysosomal system fulfils a myriad of cellular functions predicated on regulated membrane identity progressions, collectively termed maturation. Mature or “late” endosomes are designated by small membrane-bound GTPases Rab7 and Arl8b, which can either operate independently or collaborate to form a joint compartment. Whether, and how, Rab7 and Arl8b resolve this hybrid identity compartment to regain functional autonomy is unknown. Here, we report that Arl8b employs its effector SKIP to instigate inactivation and removal of Rab7 from select membranes. We find that SKIP interacts with Rab7 and functions as its negative effector, delivering the cognate GAP, TBC1D15. Recruitment of TBC1D15 to SKIP occurs via the HOPS complex, whose assembly is facilitated by contacts between Rab7 and the KMI motif of SKIP. Consequently, SKIP mediates reinstatement of single identity Arl8b sub-compartment through an ordered Rab7-to-Arl8b handover, and, together with Rab7's positive effector RILP, enforces spatial, temporal and morphological compartmentalization of endolysosomal organelles