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Home , Glycosphingolipid, Sphingolipid

Topology of modified by the addition of a phosphorylcholine moiety to form or by the stepwise addition of residues to form glycosphingo- . The formation of sphingomyelin occurs at the lumenal face of the . In glycosphingo- degradation 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 (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 after internalization through the are endocytosed and traffic through the endosomal endocytic pathway is achieved through the concerted actions of compartments to the . Here, hydrolysing cleave sugar residues sequentially to pro- hydrolysing enzymes and activator . The latter duce , which is deacylated to . 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 affect GSL degradation, and none has form water-soluble complexes, which lift out of the membrane and been described that affects 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 and lysosomal digestion, which is Topology of endocytosis and lysosomal digestion discussed in this article. 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 (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 , 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 , 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 takes place in membrane proteins)l3. Chemie und the (ER) and the Golgi appar- In an alternative model for the topology of endo- Biochemie der atus. The initial steps, converting L- 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

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, 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 , fusion and fission. When the vesicles reach the lyso- not only is the degradation block abolished, but some, 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- A is deficient. This Multivesicular storage bodies also occur in cells (e.g. causes accumulation of the 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- (-containing GSLs). In this bined activator protein deficiency2Q2. Analysis of autosomal recessive, lethal disease, the neuronal cultivated fibroblasts taken from this patient shows cells swell and eventually diez3sz4.

trends in CELL BIOLOGY (Vol. 6) March 1996 99 iizzicq GMl+galactosidase OH

Globoside ”

HO

a-Galactosidase A, SAP-B

~;~$!~+&;3~27

. I 0 GalCer-p-galactosidase GM1 +-galactosidase SAP-B and SAP-C I OH p p O-p-- cl 3H27 0-?+-‘C13H27 HNvC1 7H35 Ci 7H35 0 Digalactosylceramide

a-Galactosidase A f3-Galactosyl- SAP-B 1 OH Sphingomyelinase s OH 9H H”-&‘-i3”z7 ‘$c17H35 Sphingomyelin 0 Ceramide Metachromatic Farber Acid ceramidase -I SAP-D OH

HOwcH3 “J-b Sphingosine SulphatideO

FIGURE 2

Lysosomal sphingolipid degradation (modified, with permission, from Ref. 7). Sphingolipid activator proteins, exohydrolases and the eponyms of known storage diseases are shown. AB variant, AB variant of GM2 gangliosidosis (deficiency of GM2 activator protein); SAP, sphingolipid activator protein. Heterogeneity within the ceramide moiety due to varying degrees of saturation, hydroxylation and chain length is not shown.

For glycosphingolipids with chains analysis of , such as metachromatic of more than four sugar residues, the presence of an and Tay-Sachs disease, demonstrate enzymatically active exohydrolase is sufficient for that the in viva degradation of some glycolipids degradation in vivo. However, detailed biochemical involves not only exohydrolases but also a second

100 trends in CELL BIOLOGY (Vol. 6) March 1996 type of effector moleculez5. In these cases, lipid storage is observed in patients with- out any deficiency, but deficient P-hexosaminidase A in a protein cofactor. The degradation of membrane-bound GSLs possessing short oligosaccharide chains requires the cooperation of an exohydrolase and a SAP (for sphingolipid activator protein). By using two components in this way, the plasma membrane may be protected from inappropriate degradation. Any mis- sorted hydrolases that end up in the extracellular space, albeit at a low con- centration, are prevented from damag- ing the cell by the low extracellular concentration of lysosomal activator proteins. Furthermore, the neutral pH on the cell surface is not optimal for lyso- somal hydrolases as they mainly require a low pH for full activity. Several SAPSare FIGURE 3 now known, including the GM2 acti- Model for the CMZ-activator-stimulated degradation of ganglioside GM2 by human vator and the saposins. A detailed study B-hexosaminidase A (modified, with permission, from Ref. 14). Water-soluble of these proteins has illuminated some B-hexosaminidase A does not degrade membrane-bound ganglioside CM2, which has a short of their main features and has indicated carbohydrate chain, in the absence of GM2 activator or appropriate detergents. The GM2 the mechanisms by which they facilitate activator binds one molecule of ganglioside GM2 and lifts it a few angstroms out of the digestion of GSLs in the lysosome. membrane. In this position, this activator-lipid complex can be reached and recognized by water-soluble p-hexosaminidase A, which cleaves the substrate (mode 1). Alternatively, the The GM2 activator and its role in water-soluble activator-lipid complex may leave the membrane completely and the enzymatic lysosomal digestion reaction may take place in free solution (mode 2). The enzymatic degradation of ganglio- side GM2, the main storage material in Tay-Sachs disease, requires B-hexosaminidase A and a Other activator proteins lysosomal ganglioside-binding protein, the GM2 acti- The first activator protein was identified in 1964 as vatorz6. The GM2 activator binds ganglioside GM2, a protein that is necessary for the hydrolytic degra- as well as related gangliosides, and forms water- dation of glycosphingolipids carrying a sulphuric soluble complexes (mostly in a 1:l molar ratio). It group (sulphatides) by lysosomal arylsulphatase functions in vitro as a ganglioside-transfer protein, A (Ref. 29). This sulphatide activator, SAP-B (saposin transferring gangliosides from donor membranes B), is a small, lysosomal glycoprotein consisting of 80 to acceptor membranes. The GM2 activator behaves amino acids, with an N-linked carbohydrate chain as a ‘liftase’, recognizing GM2 within the mem- and three disulphide bridges30. Like the GM2 acti- brane and, by binding to it, lifting the lipid out of vator, it binds GSLs but has a broader specificity. In the bilayer and presenting it to the water-soluble some respects, it behaves similarly in vitro to the GM2 B-hexosaminidase A for degradation (Fig. 3, mode 1). activator; that is, it recognizes and binds several However, it is also possible that the activator-lipid different GSLs on the surface of micelles by forming complex leaves the membrane and the enzymatic a stoichiometric complex and then transfers them to reaction takes place in free solution (Fig. 3, mode 2). the membranes of acceptor liposomes14J5. SAP-B can It is unknown whether the GM2 activator acts in vivo also present GSLs as substrates to water-soluble according to mode 1 or mode 2, as depicted in Figure enzyme+. The inherited deficiency of the sulphatide 3. Also, formation of the ternary complex presum- activator leads to a lysosomal storage disease that ably involves a protein-protein interaction between resembles metachromatic leukodystrophy. However, the GM2 activator and p-hexosaminidase A (Ref. 27). unlike typical metachromatic leukodystrophy, not Point mutations within the structural gene encod- only sulphatide, but also additional glycolipids (e.g. ing the GM2 activator have been identified in two globotriaosyl ceramide) accumulate owing to a block- patients with the AB variant of GM2 gangliosidosis age of degradation at several points in the catabolic (reviewed in Refs 24 and 25). The mutated proteins pathway (Ref. 25; Fig. 2). are proteolytically labile, resulting in some of them The sulphatide activator has sequence homology being degraded in the ER and others in the Golgi with three further activator proteins, or saposins: the compartment. The resulting loss of the GM2 acti- Gaucher factor (SAP-C), SAP-Aand SAP-D.Molecular- vator causes a block to GM2 degradation in the biological analysis has revealed that these four acti- lysosomes, as demonstrated in metabolic studies of vator proteins are formed by proteolytic processing cultivated fibroblasts taken from the patients. The of a common precursor protein, the SAP precursor, block can be bypassed by feeding native or recombi- or (Refs 32-35; Fig. 4). The domain struc- nant GM2 activator to the culture medium of the ture of the precursor protein suggests that the acti- mutant fibroblastsz8. vators may have evolved by gene duplication. The

trends in CELL BIOLOGY (Vol. 6) March 1996 101 0 0.5 1.0 1.5 2.0 2.5 kb 1 in vivo function of this activator protein39. As ex- pected, if the mutant cells are fed with the SAP pre- cursor in nanomolar concentrations, storage of all these glycolipids is abolished and the aberrant accumulation of intralysosomal membrane struc- ATG TAG AAA tures is prevented. These studies on cultured cells from patients with FIGURE 4 different storage diseases support our hypothesis con- Structure of the cDNA for SAP precursor (modified, with permission, from cerning the function of SAPS.In their absence, mem- Ref. 14). The cDNA encodes a sequence of 524 amino acids (or 527 amino acids, brane-bound GSLswith short oligosaccharide chains see Ref. 43), including a signal peptide of 16 amino acids. The four domains on are not readily accessible for digestion by water- the precursor, SAP-A, -6, -C and -D (Ref. 34), correspond to the mature proteins soluble exohydrolases. By analogy to a razor blade, found in human tissues. SAP-A is also known as saposin A; SAP-B is also known as the hydrolases can only attack oligosaccharide chains saposin 9, SAP-l or sulphatide activator; SAP-C is also known as SAP-2, saposin C of GSLsthat protrude far enough from the membrane or activator protein; SAP-D is also known as saposin D or into the aqueous phase. This is no problem for long- component C. The positions of the cysteine residues are marked by vertical bars chain oligosaccharides, but, for the degradation of and the positions of the N- sites by arrowheads. GSLs with short sugar chains, the assistance of acti- vator proteins is required. Some of them act as bind- ing proteins or liftases, forming a water-soluble com- four activator proteins, SAP-A, -B, -C and -D show plex with the lipid and hence raising the lipid out of homology to each other and have some similar the membrane. However, the function of activator properties, but differ in their function and their proteins is not necessarily restricted to binding lipids mechanism of action14J5. Their physiological func- in this way. For example, SAP-C can activate directly tions are only partially clarified to date; most of our glucosylceramide @glucosidase (Ref. 40) and the knowledge of them has emerged from studies of GM2 activator interacts specifically with p-hexo- patients with atypical lipid storage diseases. A de- saminidase A (Ref. 27). ficiency of SAP-C causesa juvenile variant of Gaucher The simultaneous deficiency of the four SAPS,as disease, in which glucosyl ceramide accumulates in seen in the patient described earlier, causes an spite of normal levels of glucosylceramidase36,37. In intraendosomal and intralysosomal accumulation of one patient with a complete deficiency of the whole vesicles and other membrane fragments (multi- SAP-precursor protein caused by a homoallelic mu- vesicular bodies) and supports our view of the topol- tation within the start codon (changing AUG to ogy of endocytosis and lysosomal digestion. Further- UUG; Ref. 22), there is simultaneous storage of many more, the occurrence of storage bodies in the sphingolipids, including ceramide, glucosylceramide, combined activator protein deficiency suggests a lactosylceramide, ganglioside GM3, galactosylcera- possible function of glycolipids. The increased con- mide, sulphatides, digalactosylceramide and globo- centrations of glycolipids within distinct membranes triaosylceramide (Ref. 38; see Fig. 2 for structures). appears to protect the vesicles from degradation by phospholipases, proteases and other hydrolases that Functional analysis of sphingolipid activator occur in high concentration in the lysosomes. In proteins general, glycoconjugates form a protective layer on The storage of GSLs can be demonstrated in culti- the anticytosolic face of biological membranes, pro- vated mutant cells by pulse-chase experiments3g. tecting them from enzymatic degradation. Specific Storage substances in cells from different patients can interaction may also play a role in protecting bio- be visualized by biosynthetic labelling of glycolipids logical membranes; for example, at a molar concen- with [lQZ]serine, followed by long chase periods. As tration of >25%, ganglioside GM1 protects phos- previously mentioned, the glycolipids that accumu- pholipid vesicles completely from the degradation by late reflect the types of GSLs synthesized in each cell phospholipase A2 (Ref. 41). The protective effect of type. In the case of fibroblasts, glucosylceramide GM1 on neuronal membranes may explain how accumulates in cells from patients deficient in treatment of patients who have Alzheimer’s disease SAP-C, ganglioside GM2 accumulates when there is type II with ganglioside GM1 can partially attenuate deficiency of the GM2 activator, and ceramide, glu- the disease progression4z. cosylceramide, lactosylceramide and ganglioside GM3 all accumulate when the SAP precursor is deficient. Future directions Cell-culture systems, such as these, are not only Future research will concentrate on elucidating suitable for diagnostic purposes, they also allow the both the fine details of the endocytic pathway for elucidation of the function of the different acti- plasma membrane fragments containing GSLs and vator proteins. Although the four SAPS are highly the mechanism of action of the SAPS.How does the homologous, their differences are revealed in cell decide whether membrane components join the these experiments. If purified SAP-Bis fed to the cells conventional endocytic pathway or become incor- deficient in SAP precursor, the storage of lactosyl- porated into an intralysosomal vesicle? How do the ceramide is abolished, but ceramide and glucosyl- activator proteins interact with the glycolipids, does ceramide still accumulate. By contrast, when SAP-D the activator enter the membrane or does the inter- is fed to the cells, ceramide accumulation is pre- action occur at the surface? Is activator action depen- vented. These experiments give the first hint to the dent on physical disturbance of biological membranes?

102 trends in CELL BIOLOGY (Vol. 6) March 1996 These and other questions will be tackled by using 19 VAN DEURS, B. et al. (1993) Eur. 1. Cell Biol. 61, 208-224 cultured cells from human patients and also by 20 SUZUKI, K. et al. (1968) Path. Europ. 3, 389-408 studying the knockout mouse mutants that are cur- 21 HARZER, K. et al. (1989) Eur. 1. Pediatr. 149, 31-39 rently being developed. Finally, a better understand- 22 SCHNABEL, D. et al. (1992) 1. Biol. Chem. 267, 3312-3315 ing of the mode of action and other aspects of glyco- 23 SANDHOFF, K. et al. (1989) in The Metabolic and Molecular sphingolipid degradation will hopefully lead to Basis of Inherited Disease (Striver, C., Beaudet, A. L., Sly, W. S. therapeutic advances in the treatment of lipid and Valle, D., eds), pp. 1807-I 839, McGraw-Hill storage diseases. 24 GRAVEL, R. A. et al. (1995) in The Metabolic and Molecular Basis of Inherited Disease (Striver, C., Beaudet, A. L., Sly, W. S., References Valle, D., eds), pp. 2839-2879, McGraw-Hill 1 WIEGANDT, H. (1985) in New Comprehensive 25 SANDHOFF, K. et al. (1995) in The Metabolic and Molecular (Vol. 10) (Neuberger, A. and van Deenen, L. L. M., eds), Basis of Inherited Disease (Striver, C., Beaudet, A. L., Sly, W. S. pp. 199-260, Elsevier and Valle, D., eds), pp. 2427-2441, McGraw-Hill 2 IHAKOMORI, 5. (1981) Annu. Rev. Biochem. 50, 733-764 26 MEIER, E. M. et al. (1991) I. Biol. Chem. 266, 1879-1887 3 KARLSSON, K-A. (1989) Annu. Rev. Biochem. 58, 309-350 27 KYTZIA, H-J. and SANDHOFF, K. (1985) 1. Biol. Chem. 260, 4 HANNUN, Y. A. and OBEID, L. M. (1995) Trends Biochem. Sci. 7568-7572 20,73-77 28 KLIMA, H. et al. (1993) Biochem. 1. 292, 571-576 5 VAN ECHTEN, G. and SANDHOFF, K. (1993) I. Biol. Chem. 268, 29 MEHL, E. and JATZKEWITZ, H. (1964) Hoppe-Seyler’s Z. Physiol. 5341-5344 Chem. 339,260-276 6 COSTE, H. et al. (1986) Biochim. Biophys. Acta 858, 6-12 30 FURST, W. et al. (1990) Eur. 1. Biochem. 192, 709-714 7 SANDHOFF, K. and KOLTER, T. (1995) Naturwissenschaften 82, 31 LI, S-C. et al. (1988) 1. Biol. Chem. 263, 6588-6591 403-413 32 FUJIBAYASHI, 5. and WENGER, D. A. (1986) Biochim. Biophys. 8 CRIFFITHS, C. W. et al. (1988) Cell 52, 329-341 Acta 875,554-562 9 KOVAL, M. and PACANO, R. E. (1989) 1. Cell Biol. 108, 33 FORST, W. et al. (1988) Biol. Chem. Hoppe-Seyler 369, 317-328 2169-2181 34 O’BRIEN, ). S. et al. (1988) Science 241, 1098-l 101 10 KOVAL, M. and PACANO, R. E. (1990) 1. Celi Biol. 111,429-442 35 NAKANO, T. et al. (1989) 1. Biochem. 105, 152-l 54 11 WESSLINC-RESNICK, M. and BRAELL, W. A. (1990) 1. Biol. 36 CHRISTOMANOU, H. et al. (1986) Biol. Chem. Hoppe-Seyler Chem. 265, 690-699 367,879-890 12 KOK, 1. W. eta/. (1991)j. CellBiol. 114, 231-239 37 SCHNABEL, D. et al. (1991) RBS lett. 284, 57-59 13 CARLSSON, S. R. el al. (1988) 1. Biol. Chem. 263, 1891 l-l 8919 38 BRADOVA, V. et al. (1993) Hum. Cenet. 92, 143-I 52 Acknowledgement 14 FURST, W. and SANDHOFF, K. (1992) Biochim. Biophys. Acta 39 KLEIN, A. et al. (1994) Biochem. Biophys. Res. Commun. 200, 1126,1-16 1440-l 448 The work 15 ZACHGO, S. et al. (1992) 1. Cell Sci. 103, 81 l-822 40 HO, M. W. and O’BRIEN, 1. S. (1971) Proc. Nat/ Acad. Sci. USA performed in the 16 HOPKINS, C. R. et al. (1990) Nature 346, 335-339 68,2810-2813 authors’ laboratory 17 McKANNA, 1, A. et al. (I 979) Proc. Nat/ Acad. Sci. USA 76, 41 BIANCO, I. D. et al. (1989) Biochem. 1. 258, 95-99 was supported by 5689-5693 42 SVENNERHOLM, L. (1994) in Progress in Brain Research the Deutsche 18 RENFREW, C. A. and HUBBARD, A. L. (1991) 1. Biol. Chem. 266, (Vol. 101) (Svennerholm, L. et al., eds), pp. 391-404, Elsevier Forschungsgemein- 21265-21273 43 HOLTSCHMIDT, H. et a/. (1991) 1, Biol. Chem. 266, 7556-7560 schaft (SFB 284).

Tetracycline-responsive host cell lines

I Thank you to everyone who has provided information on the “3 availability of tetracycline-responsive ceil lines (Tet-&et: a call ” ! for ceils expressing the tetracycline-controllable transactivaQr;‘, :I

Sandra L. Schmid; trends in CELL BIOLOGY, July 1995). : ” ’

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