Related Experiment Video
Updated: May 19, 2026

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes
Published on: March 14, 2021
Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin
Asako Goto1, Xinwei Liu, Carolyn-Ann Robinson
1Department of Pediatrics, Atlantic Research Centre, Dalhousie University, Halifax, NS, Canada.
Abstract:
The endoplasmic reticulum (ER)-Golgi sterol transfer activity of oxysterol-binding protein (OSBP) regulates sphingomyelin (SM) synthesis, as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated, suggesting this modification regulates the directionality and/or specificity of transfer activity. In this paper, we report that phosphorylation on two serine-rich motifs, S381-S391 (site 1) and S192, S195, S200 (site 2), specifically controls OSBP activity at the ER. A phosphomimetic of the SM/cholesterol-sensitive phosphorylation site 1 (OSBP-S5E) had increased in vitro cholesterol and 25-hydroxycholesterol-binding capacity, and cholesterol extraction from liposomes, but reduced transfer activity. Phosphatidylinositol 4-phosphate (PI(4)P) and cholesterol competed for a common binding site on OSBP; however, direct binding of PI(4)P was not affected by site 1 phosphorylation. Individual site 1 and site 2 phosphomutants supported oxysterol activation of SM synthesis in OSBP-deficient CHO cells. However, a double site1/2 mutant (OSBP-S381A/S3D) was deficient in this activity and was constitutively colocalized with vesicle-associated membrane protein-associated protein A (VAP-A) in a collapsed ER network. This study identifies phosphorylation regulation of sterol and VAP-A binding by OSBP in the ER, and PI(4)P as an alternate ligand that could be exchanged for sterol in the Golgi apparatus.
Related Concept Videos
Regulation of Nuclear Protein Sorting
Phosphoinositides and PIPs
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane...
Allosteric Regulation
Calmodulin-dependent Signaling
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
Synthesis of Phosphatidylcholine in the ER Membrane
The major components of all eukaryotic cell...
Ligand Binding and Linkage
