Single-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. Co-immunoprecipitation 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.
