Determining the Mechanism of Sphingosine‐1‐Phosphate Transport Between Membranes

Abstract

Sphingosine‐1‐phosphate (S1P) is synthesized from sphingosine by the enzyme sphingosine kinase (SK). The main pathways of S1P degradation occur via the action of two important enzymes; S1P phosphatase (SPP) and S1P lyase (SPL) which are both bound to the endoplasmic reticulum (ER). So the ER plays a vital role in the metabolism of S1P.The goal of these studies is to determine the mechanism of S1P transport between membranes. We are hypothesizing that this is a protein mediated transfer. Initially we have defined the parameters of S1P extraction from membranes, which is the first step in intermembrane transfer. We have used a model lipid‐binding protein, bovine serum albumin (BSA). To measure S1P extraction from membranes, membranes were first loaded with 33P‐S1P by using membranes ectopically expressing a membrane‐anchored SK construct (Lck‐SK). Extraction of S1P was measured by incubating pre‐labeled membranes in the presence or absence of fatty acid‐free BSA for various lengths of time. With this system we find that extraction of S1P is protein‐dependent, but very rapid. We then tested the ability of cytosol harvested from Hela cells to replace BSA in extracting S1P from membranes. Interestingly, results indicated the presence of an S1P transport factor(s) in the cytosol. Size‐exclusion chromatography was then used to further fractionate the cytosol and various fractions were assayed for activity. S1P extraction activity was detected to be higher in higher molecular weight fractions away from bulk protein. Concentrates of these fractions demonstrated enhanced S1P extraction activity from membranes over whole cytosol.