The molecular machinery responsible for the generation of transport carriers moving from the Golgi complex to the plasma membrane relies on a tight interplay between proteins and lipids. Among the lipid-binding proteins of this machinery, we previously identified the four-phosphate adaptor protein FAPP2, the pleckstrin homology domain of which binds phosphatidylinositol 4-phosphate and the small GTPase ARF1. FAPP2 also possesses a glycolipid-transfer-protein homology domain. Here we show that human FAPP2 is a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids, key structural and signalling components of the plasma membrane. The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to regulation by phosphatidylinositol 4-phosphate and ARF1. Thus, by coupling the synthesis of glycosphingolipids with their export to the cell surface, FAPP2 emerges as crucial in determining the lipid identity and composition of the plasma membrane.

Glycosphingolipids are controlled by the spatial organization of their metabolism and by transport specificity. Using immunoelectron microscopy, we localize to the Golgi stack the glycosyltransferases that produce glucosylceramide (GlcCer), lactosylceramide (LacCer), and GM3. GlcCer is synthesized on the cytosolic side and must translocate across to the Golgi lumen for LacCer synthesis. However, only very little natural GlcCer translocates across the Golgi in vitro. As GlcCer reaches the cell surface when Golgi vesicular trafficking is inhibited, it must translocate across a post-Golgi membrane. Concanamycin, a vacuolar proton pump inhibitor, blocks translocation independently of multidrug transporters that are known to translocate short-chain GlcCer. Concanamycin did not reduce LacCer and GM3 synthesis. Thus, GlcCer destined for glycolipid synthesis follows a different pathway and transports back into the endoplasmic reticulum (ER) via the late Golgi protein FAPP2. FAPP2 knockdown strongly reduces GM3 synthesis. Overall, we show that newly synthesized GlcCer enters two pathways: one toward the noncytosolic surface of a post-Golgi membrane and one via the ER toward the Golgi lumen LacCer synthase.

Newly synthesized proteins and lipids are transported across the Golgi complex via different mechanisms whose respective roles are not completely clear. We previously identified a non-vesicular intra-Golgi transport pathway for glucosylceramide (GlcCer)--the common precursor of the different series of glycosphingolipids-that is operated by the cytosolic GlcCer-transfer protein FAPP2 (also known as PLEKHA8) (ref. 1). However, the molecular determinants of the FAPP2-mediated transfer of GlcCer from the cis-Golgi to the trans-Golgi network, as well as the physiological relevance of maintaining two parallel transport pathways of GlcCer--vesicular and non-vesicular--through the Golgi, remain poorly defined. Here, using mouse and cell models, we clarify the molecular mechanisms underlying the intra-Golgi vectorial transfer of GlcCer by FAPP2 and show that GlcCer is channelled by vesicular and non-vesicular transport to two topologically distinct glycosylation tracks in the Golgi cisternae and the trans-Golgi network, respectively. Our results indicate that the transport modality across the Golgi complex is a key determinant for the glycosylation pattern of a cargo and establish a new paradigm for the branching of the glycosphingolipid synthetic pathway.

To understand the structural basis for the apparent strong interaction between cholesterol and sphingomyelin (SPM), we have synthesized an analogue of SPM, 3-deoxy-2-O-stearoyl-SPM, in which an ester-linked acyl chain replaces the amide-linked acyl chain at C-2 and a hydrogen replaces the hydroxy group at C-3. We have compared the behavior of this analogue with that of 3-deoxy-N-stearoyl-SPM in monolayers and vesicles, both as pure phospholipids and in mixtures with cholesterol. The force-area isotherm of 3-deoxy-2-O-stearoyl-SPM was similar to that of 3-deoxy-N-stearoyl-SPM. The surface potential across the pure SPM monolayer at the air-water interface was larger for 3-deoxy-2-O-stearoyl-SPM than for 3-deoxy-N-stearoyl-SPM (about 430 mV and 330 mV, respectively, at 50 A2). The overall dipole moment of 3-deoxy-2-O-stearoyl-SPM was almost constant at 570 mD (between a mean molecular area range of 45-85 A2), whereas that of 3-deoxy-N-stearoyl-SPM was about 420 mD. Cholesterol appeared to be equally miscible in both SPM monolayers, as determined from the condensing effect cholesterol had on the lateral packing of the two SPMs. The oxidation of monolayer cholesterol by cholesterol oxidase was also determined using both SPMs. The stoichiometry at which free cholesterol clusters disappeared in monolayers, when going from high to low cholesterol content, was 2:1 (mol sterol/mol SPM) for both SPMs.(ABSTRACT TRUNCATED AT 250 WORDS)

The sphingomyelin-ceramide pathway is an evolutionarily conserved ubiquitous signal transduction system that regulates many cell functions including apoptosis. Sphingomyelin (SM) is hydrolyzed to ceramide by different sphingomyelinases. Ceramide serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase) activity was induced by UVA in normal JY lymphoblasts but was not detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. We also provide evidence that UVA can induce apoptosis by activating acid SMase in normal JY cells. In contrast, UVA-induced apoptosis was inhibited in MS1418 cells. Exogenous SMase and its product, ceramide (10 -40 M), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20 -80 M), induced apoptosis only in JY cells. These results suggest that UVA-induced apoptosis by SM is dependent on acid SMase activity. We also provide evidence that induction of apoptosis by UVA may occur through activation of JNKs via the acid SMase pathway.