Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching

Abstract

Edited by George CarmanSingle-molecule photobleaching has emerged as a powerful non-invasive approach to extract the stoichiometry of multimeric membrane proteins in their native cellular environment. However, this method has mainly been used to determine the subunit composition of ion channels and receptors at the plasma membrane. Here, we applied single-molecule photobleaching to analyze the oligomeric state of an endoplasmic reticulum (ER) resident candidate ceramide sensor protein, SMSr/SAMD8. Coimmunoprecipitation and chemical cross-linking studies previously revealed that the N-terminal sterile alpha motif (or SAM) domain of SMSr drives self-assembly of the protein into oligomers and that SMSr oligomerization is promoted by curcumin, a drug known to perturb ER ceramide and calcium homeostasis. Application of cell spreading surface-active coating materials in combination with total internal reflection fluorescence (TIRF) microscopy allowed us to image GFP-tagged SMSr proteins as single fluorescent spots in the ER of HeLa cells in which expression of endogenous SMSr was abolished. In line with our biochemical analysis, we find that the number of bleaching steps in SMSr-GFP-positive spots displays a substantial drop after removal of the SAM domain. In contrast, treatment of cells with curcumin increased the number of bleaching steps. Our results document the first successful application of single-molecule photobleaching to resolve drug-induced and domain-dependent changes in the oligomeric state of an ER-resident membrane protein, hence establishing a complementary method to unravel the mechanism by which SMSr controls ceramide levels in the ER.

New Era of Diacylglycerol Kinase, Phosphatidic Acid and Phosphatidic Acid-Binding Protein

“…Sphingomyelin synthase-related protein (SMSr) is a six-transmembrane protein in the endoplasmic reticulum (ER), which generates DG and ceramide phosphoethanolamine (CPE) by utilizing phosphatidylethanolamine (PE) and ceramide ( Figure 4 ) [ 269 ]. A SAM domain in SMSr form a homo-oligomer [ 270 , 271 ]. DGKδ also possesses a SAM domain and forms homo-oligomers via the domains [ 112 , 113 ].…”

“…DGKδ also possesses a SAM domain and forms homo-oligomers via the domains [ 112 , 113 ]. Intriguingly, the SAM domains in SMSr and DGKδ are primary structurally similar to each other [ 270 , 271 ]. It is noteworthy that we recently found that SMSr and DGKδ interacted with each other through their SAM domains ( Figure 4 ) [ 272 ].…”

Self-assembly of robust gold nanoparticle monolayer architectures for quantitative protein interaction analysis by LSPR spectroscopy

“…By controlling the ratio of PEGs graft to the lysine moieties of PLL, 30% of the ε-amine moieties in poly-L-lysine are conjugated with PEG-OPSS and the remaining are left unmodified for electrostatic interaction. Such PEG-tolysine ratio yields high biocompatibility while maintaining sufficient amine density to warrant stable adsorption onto glass substrates [36][37][38].…”

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

“…Dynamic changes in the stoichiometry of membrane proteins are hard to resolve by bulk biochemical approaches like chemical crosslinking or co-immunoprecipitation analysis. Therefore, our ongoing efforts focus on the application of single-molecule fluorescence microscopy as complementary method to monitor SMSr oligomerization in intact cells52.…”