Differential Effects of Sphingomyelin Hydrolysis and Cholesterol Transport on Oxysterol-binding Protein Phosphorylation and Golgi Localization

Abstract

The deposition of de novo synthesized and lipoproteinderived cholesterol at the plasma membrane and transport to the endoplasmic reticulum is dependent on sphingomyelin (SM) content. Here we show that hydrolysis of plasma membrane SM in Chinese hamster ovary cells by exogenous bacterial sphingomyelinase resulted in enhanced cholesterol esterification at the endoplasmic reticulum and rapid dephosphorylation of the oxysterol-binding protein (OSBP), a cytosolic/Golgi receptor for oxysterols such as 25-hydroxycholesterol. After sphingomyelinase treatment, restoration of OSBP phosphorylation closely paralleled resynthesis of SM and down-regulation of cholesterol ester synthesis. SM hydrolysis activated an okadaic acid-sensitive phosphatase that was not stimulated in Chinese hamster ovary cells by short chain ceramides. Agents that specifically blocked sphingomyelinase-mediated delivery of cholesterol to acyl-CoA:cholesterol acyltransferase (U18666A) or promoted cholesterol efflux to the medium (cyclodextrin) did not inhibit OSBP dephosphorylation. SM hydrolysis also promoted OSBP translocation from a vesicular compartment to the Golgi apparatus. Cyclodextrin and U18666A also caused OSBP translocation to the Golgi apparatus, suggesting that OSBP movement is coupled to changes in the cholesterol content of the plasma membrane or Golgi apparatus. These results identify OSBP as a potential target of SM turnover and cholesterol mobilization at the plasma membrane and/or Golgi apparatus.Studies in cultured cell models have identified three organelles that figure prominently in cholesterol trafficking: the ER, 1 the major site for cholesterol synthesis, regulation, and esterification; the plasma membrane, a prominent storage site for unesterified cholesterol; and lysosomes, where lipoproteinderived cholesterol is liberated (reviewed in Ref.

The Golgi Localization of Phosphatidylinositol Transfer Protein β Requires the Protein Kinase C-dependent Phosphorylation of Serine 262 and Is Essential for Maintaining Plasma Membrane Sphingomyelin Levels

“…Because the mutant protein expresses full lipid transfer activity in vitro, we infer that the association of PI-TP␤ with the Golgi is a prerequisite for PI-TP␤ to stimulate rapid SM replenishment. SM and cholesterol regulation in the Golgi has also been linked to the Golgi localization and phosphorylation of the oxysterol-binding protein (29). Similar to its yeast analog Sec14p, PI-TP␤ may play a role in the budding of SM-containing vesicles from the Golgi (10,30).…”

Role of sphingomyelin and ceramide in the regulation of the activity and fatty acid specificity of group V secretory phospholipase A2

“…The release of 16:0 occurred only in the presence of both SMase C and sPLA 2 V. These results suggest that the excess 16:0 came from the hydrolysis of lyso PC generated by the sPLA 2 activity. Further studies are required to characterize the putative lysophospholipase activity of the commercial preparations of SMase C, because this enzyme is extensively used as a probe to specifically deplete SM from cell membranes [22][23][24], and to induce LDL aggregation [25]. It is, however, unlikely that the overall activity or specificity of sPLA 2 V was substantially influenced by the lysophospholipase activity of SMase C because quantitatively, the amount of 16:0 released is much lower than that of the unsaturated fatty acids (Fig.…”

Sphingolipid Homeostasis in the Endoplasmic Reticulum and Beyond

“…For example, a reduction in plasma membrane sphingolipids leads to flux of sterols from the plasma membrane to the ER, where they are detected by sterol sensors such as SREBP and OSBP (Chang et al 2006;Brown and Goldstein 2009). These proteins initiate a variety of downstream responses, including OSBP-dependent activation of CERT (Ridgway et al 1998;Perry and Ridgway 2006).…”