Abstract
The intracellular site of sphingomyelin (SM) synthesis was examined in subcellular fractions from rat liver using a radioactive ceramide analog N-([1-14C]hexanoyl)-D-erythro-sphingosine. This lipid readily transferred from a complex with bovine serum albumin to liver fractions without disrupting the membranes, and was metabolized to radioactive SM. To prevent degradation of the newly synthesized SM to ceramide, all experiments were performed in the presence of EDTA to minimize neutral sphingomyelinase activity and at neutral pH to minimize acid sphingomyelinase activity. An intact Golgi apparatus fraction gave an 85-98-fold enrichment of SM synthesis and a 58-83-fold enrichment of galactosyltransferase activity. Controlled trypsin digestion demonstrated that SM synthesis was localized to the lumen of intact Golgi apparatus vesicles. Although small amounts of SM synthesis were detected in plasma membrane and rough microsome fractions, after accounting for contamination by Golgi apparatus membranes, their combined activity contributed less than 13% of the total SM synthesis in rat liver. Subfractions of the Golgi apparatus were obtained and characterized by immunoblotting and biochemical assays using cis/medial (mannosidase II) and trans (sialyltransferase and galactosyltransferase) Golgi apparatus markers. The specific activity of SM synthesis was highest in enriched cis and medial fractions but far lower in a trans fraction. We conclude that SM synthesis in rat liver occurs predominantly in the cis and medial cisternae of the Golgi apparatus and not at the plasma membrane or endoplasmic reticulum as has been previously suggested.
Highlights
Sphingomyelin Synthesis in Rat Liver Occurs Predominantly at the cis and medial Cisternae of the Golgi Apparatus*
Most of the [‘*C]hexanoyl Cer and [i4C]hexanoyl SM, but only a small amount of Sulforhodamine-conjugated bovine serum albumin (SRhBSA), was associated with the pellet obtained from Golgi apparatus membranes (Table I) or with the pellet obtained from the liver homogenate and the enriched plasma membrane (PM) fraction
Since the amount of [‘4C]hexanoyl sphingolipids transferred was similar for all three fractions, we assume that the synthesis of [‘4C]hexanoyl sphingolipids described below was not limited by the transfer of substrate from BSA to membranes.* To determine if incorporation of [‘4C]hexanoyl Cer disrupted the membranes of the Golgi apparatus, the trypsin sensitivity of galactosyltransferase, a lumenal Golgi enzyme
Summary
Affinity-purified rabbit IgG directed against rat liver a2.6~sialvltransferase [22] was a gift from Dr G. Radioactive Lipids ester of l-[‘%]hexanoic from Sigma or was prepared acid D-erythro-sphingosylphosphorylcholine ( [14C]hexanoyl SM) were prepared by N-acylation of sphingosine and sphingosylphosphorylcholine, respectively [23]. Defatted BSA-[‘4C]hexanoyl lipid complexes were prepared as described [25], except that 50 mM Tris (pH 7.4) was used; complexes contained an eauimolar ratio of defatted BSA and V4Clhexanovl lipid. After 20 min at room temperature, complexes were dialyzed against 50 mM Tris (pH 7.4) for 12-18 h at 4 “C. SM were prepared, complexes were not dialyzed.
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