Topology of modified by the addition of a phosphorylcholine moiety to form sphingomyelin or by the stepwise glycosphingolipid addition of sugar residues to form glycosphingo- lipids. The formation of sphingomyelin occurs at the lumenal face of the Golgi apparatus. In glycosphingo- degradation lipid synthesis, the first glucosyl residue is added at the cytosolic face of the Golgi apparatu9. The step- wise introduction of other sugar residues seems to be restricted to the lumenal face of Golgi membranes. The products of these reactions are then transported Glycosphingolipids (GSLs) form cell-type-specific patterns on the to the plasma membrane by exocytosis. Degradation of GSLs occurs in lysosomal compart- surface of eukaryotic cells. Degradation ofplasma-membrane- ments within the ce117. Fragments of the plasma- membrane-containing GSLs destined for degradation derived GSLs in the lysosomes after internalization through the are endocytosed and traffic through the endosomal endocytic pathway is achieved through the concerted actions of compartments to the lysosome. Here, hydrolysing enzymes cleave sugar residues sequentially to pro- hydrolysing enzymes and sphingolipid activator proteins. The latter duce ceramide, which is deacylated to sphingosine. are proteins necessary for the degradation of GSLs possessing short This can leave the lysosome and re-enter the bio- synthetic pathway or can be degraded further. To oligosaccharide chains. Some activator proteins bind to GSLs and date, all characterized, inherited diseases of GSL metabolism affect GSL degradation, and none has form water-soluble complexes, which lift out of the membrane and been described that affects biosynthesis of GSL. give the water-soluble hydrolysing enzymes access to the regions of Presumably, severe disorders affecting GSL biosyn- thesis would result in incorrect GSL patterns on the the GSL that would otherwise be obscured by the membrane. The cell surface, and these in turn would affect -morpho- genesis and embryogenesis and result in embryonic inherited deficiency of both lysosomal hydrolases and sphingolipid lethality. A study of the inherited disorders in GSL activator proteins gives rise to sphingolipid storage diseases. degradation, the lysosomal storage diseases, has provided insights into the processes involved in An analysis of these diseases suggests a new model for the topology GSL degradation, of endocytosis and lysosomal digestion, which is Topology of endocytosis and lysosomal digestion discussed in this article. Catabolism of complex GSLs derived from the plasma membrane occurs in the lysosomes after endocytosis. In the conventional model, com- ponents of the plasma membrane reach the lysosomal compartment by endocytic membrane flow via the Glycosphingolipids (GSLs) are components of the early and late endosomes (Ref. 8; Fig. 1). During this plasma membrane of eukaryotic cell+. Each GSL con- vesicular flow, molecules in the membrane are sub- tains a hydrophobic ceramide moiety, which acts as jected to a sorting process that directs some of the a membrane anchor, and a hydrophilic, extracellular molecules to the lysosomal compartment, others to oligosaccharide chain. Variations in the type, num- the Golgi apparatus and yet others back to the plasma ber and linkage of sugar residues in the oligosac- membrane9-lz. After a series of vesicle-budding and charide chain give rise to the wide range of naturally -fusion events through the endosomal compartment, occurring GSLs. These form cell-type-specific patterns the membrane fragments reach the lysosomal com- at the cell surface that change upon cell growth, dif- partment. As a consequence of endocytic membrane ferentiation, viral transformation and oncogenesis2. flow, constituents of the plasma membrane are incor- GSLs interact at the cell surface with toxins, viruses porated into the lysosomal membrane. Lysosomal and bacteria, as well as with membrane-bound recep- degradation of former components of the plasma tors and enzymes”. They are also involved in cell- membrane must then proceed selectively within the type-specific adhesion processes, and, in addition, lysosomal membrane, without destroying it. It is lipophilic products of GSL metabolism, such as difficult to see how this could occur, especially as sphingosine and ceramide, play a role in signal- the inner leaflet of the lysosomal membrane is cov- transduction events4. Finally, GSLs form a protective ered with a thick glycocalix composed of glyco- The authors are at layer on biological membranes, protecting them proteins, the so-called limps (lysosomal integral the lnstitut fiir from inappropriate degradation. membrane proteins) and lamps (lysosomal associated Organische The biosynthesis5 of sphingolipids takes place in membrane proteins)l3. Chemie und the endoplasmic reticulum (ER) and the Golgi appar- In an alternative model for the topology of endo- Biochemie der atus. The initial steps, converting L-serine into cytosis (Ref. 14; Fig. l), components of the plasma Universitlt, dihydroceramide, occur at the cytosolic face of membrane pass through the endosomal compart- Cerhard-Domagk- the ER. At present, the exact subcellular localiz- ment as intraendosomal vesicles that become intra- Str. 1, 53121 ation of the desaturation step leading from dihydro- lysosomal vesicles on reaching the lysosomes. The Bonn, Germany. ceramide to ceramide is not known. Ceramide is then vesicles are formed initially in the early endosome 98 0 1996 Elsevier Science Ltd trends in CELL BIOLOGY (Vol. 6) March 1996 \ \ J Late Golgi apparatus Golgi apparatus 4 ~ endosome ~ (3zY b Transient fusion Glycocalix Glycocalix membrane membrane FIGURE 1 Two models for the topology of endocytosis and lysosomal digestion of glycosphingolipids (CSLs) derived from the plasma membrane. (a) Conventional model: degradation of CSLs derived from the plasma membrane occurs selectively within the lysosomal membrane. (b) Alternative model: during endocytosis, glycolipids of the plasma membrane become incorporated into the membranes of intraendosomal vesicles (multivesicular bodies). The vesicles are transferred into the lysosomal compartment when the late endosome fuses with primary lysosomes. PM, plasma membrane; I, glycosphingolipid. by selective invagination (budding-in) of endosomal that the affected storage vesicles are in the late endo- membranes enriched for components of the plasma somal or lysosomal compartments. These compart- membrane. The surrounding endosome then passes ments are still functionally active, except that they along the endocytic pathway by way of the normal, are unable to degrade sphingolipids that have successive events of membrane fission and fusion. short oligosaccharide head groups. After comple- The intraendosomal vesicles, however, are carried mentation of the medium of these cells with the along as passengers and, normally, do not undergo missing precursor of sphingolipid activator protein, fusion and fission. When the vesicles reach the lyso- not only is the degradation block abolished, but some, glycoconjugates originating from the outer also the number and size of the storage organelles leaflet of the plasma membrane face the lysosol on are reduced (J. K. Burkhardt, S. Hiittler, A. Klein, the outer leaflet of intralysosomal vesicles and, thus, W. Mobius, A. Habermann, G. Griffiths and K. Sandhoff, are in the correct orientation for degradation. unpublished). According to this model, there is no glycocalix block- To date, budding-in of vesicles at the early endo- ing the action of hydrolases. This hypothesis is some stage has not been observed by microscopy. supported by a series of observations: This membrane fission event is thought to occur l Multivesicular bodies are observed at the level of since it would explain both the occurrence of intra- the early and late endosomal reticulum12~15-17. lysosomal vesicles and the selective degradation of * The epidermal growth factor receptor derived GSLs derived from the plasma membrane, in contrast from the plasma membrane and internalized into to those of the lysosomal membrane. lysosomes of hepatocytes is not integrated into the Within the lysosome, degradation of GSLs occurs lysosomal membrane18. by the stepwise action of specific acid exohydrolases * Spherical multivesicular bodies are the predomi- (Ref. 7; Fig. 2). More than ten different exohydro- nant endocytic compartments in HEp-2 cells. lases are involved in this process. If any of these Moreover, these multivesicular bodies mature within enzymes is deficient, the corresponding lipid sub- 60 to 90 min into lysosomes that still contain inter- strate accumulates and is stored in the lysosomal nal vesicles19. compartment. In the inherited sphingolipid storage * Multivesicular storage bodies accumulate in cells diseases, accumulation of lipids occurs mainly in from patients with sphingolipid storage diseases. those cell types and organs in which the lipids are They were observed almost 30 years ago in cerebral predominantly synthesized. In Tay-Sachs disease, cells from patients with GM1 gangliosidosiszO. for example, B-hexosaminidase A is deficient. This Multivesicular storage bodies also occur in cells (e.g. causes accumulation of the ganglioside GM2 in Kupffer cells and fibroblasts) of a patient with neuronal cells, the main sites for synthesis of another sphingolipid storage disease - giving a com- gangliosides (sialic acid-containing GSLs). In this bined activator protein deficiency2Q2. Analysis of autosomal recessive, lethal disease, the