The lysosomal transport of prosaposin requires the conditional interaction of its highly conserved d domain with sphingomyelin.
Lysosomal prosaposin (65 kDa) is a nonenzymic protein that is transported to the lysosomes in a mannose 6-phosphate-independent manner. Selective deletion of the functional domains of prosaposin indicates that the D domain and the carboxyl-terminal region are necessary for its transport to the lysosomes. Inhibitors of sphingolipid biosynthesis, such as fumonisin B(1) (FB(1)) and tricyclodecan-9-yl xanthate potassium salt (D609), also interfere with the trafficking of prosaposin to lysosomes. In this study, we examine sphingomyelin as a direct candidate for the trafficking of prosaposin. Chinese hamster ovary and COS-7 cells overexpressing prosaposin or an albumin/prosaposin construct were incubated with these inhibitors, treated with sphingolipids, and then immunostained. Sphingomyelin restored the immunostaining in lysosomes in both FB(1)- and D609-treated cells and ceramide reestablished the immunostaining in FB(1)-treated cells only. D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which inhibits glycosphingolipids, had no effect on the immunostaining pattern. To determine whether sphingomyelin has the same effect on the transport of endogenous prosaposin, testicular explants were treated with FB(1) and D609. Sphingomyelin restored prosaposin immunogold labeling in the lysosomes of FB(1)- and D609-treated Sertoli cells, whereas ceramide restored the label in FB(1) treatment only. Albumin linked to the D and COOH-terminal domains of prosaposin was used as a dominant negative competitor. The construct blocked the targeting of prosaposin and induced accumulation of membrane in the lysosomes, demonstrating that the construct uses the same transport pathway as endogenous prosaposin. In conclusion, our results showed that sphingomyelin, the D domain, and its adjacent COOH-terminal region play a crucial role in the transport of prosaposin to lysosomes. Although the precise nature of this lipid-protein interaction is not well established, it is proposed that sphingomyelin microdomains (lipid rafts) are part of a mechanism ensuring correct intercellular trafficking of prosaposin.
Lefrancois
Stephane
S
Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B2, Canada.
May
Taymaa
T
Knight
Casey
C
Bourbeau
Danielle
D
Morales
Carlos R
CR
D000418:Albumins / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D019556:COS Cells; D002264:Carboxylic Acids / Q000494:pharmacology; D002401:Cathepsin B / Q000494:pharmacology; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D037341:Fumonisins; D006023:Glycoproteins / Q000378:metabolism*; D006028:Glycosphingolipids / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007150:Immunohistochemistry; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D049231:Saposins; D012671:Seminiferous Tubules / Q000187:drug effects; D013109:Sphingomyelins / Q000737:chemistry* / Q000502:physiology*; D014162:Transfection
D000418:Albumins; D002264:Carboxylic Acids; D018076:DNA, Complementary; D037341:Fumonisins; D006023:Glycoproteins; D006028:Glycosphingolipids; D009025:Morpholines; C483344:Psap protein, mouse; D049231:Saposins; D013109:Sphingomyelins; C056933:fumonisin B1; C033110:RV 538; D002401:Cathepsin B