| Products/Services Used | Details | Operation |
|---|---|---|
| PCR Cloning and Subcloning | The human CERT PH domain-containing residues 20–122 and START domain-containing residues 360–598 were cloned into the prokaryotic overexpression vector pHis6-GB1 as previously described (8). DNA molecules encoding the CERT PH domain with an N-terminal c-Myc epitope tag and the START domain with a Cys residue added to the N terminus were purchased from GenScript in the pHis6-GB1 vector. The site-specific biotinylated START domain was produced using the EZ-Link Maleimide-PEG2-Biotin reagent (Thermo Scientific) according to the manufacturer’s instructions | Get A Quote |
De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT’s N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two... More
