Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex.
Although glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.
Sprong
H
H
Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, 1100 DE, Amsterdam, Netherlands.
Degroote
S
S
Claessens
T
T
van Drunen
J
J
Oorschot
V
V
Westerink
B H
BH
Hirabayashi
Y
Y
Klumperman
J
J
van der Sluijs
P
P
van Meer
G
G
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004789:Enzyme Activation; D005964:Glucosyltransferases / Q000235:genetics; D006028:Glycosphingolipids / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D007980:Levodopa / Q000096:biosynthesis; D008247:Lysosomes / Q000378:metabolism; D008543:Melanins / Q000378:metabolism; D020460:Melanosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins; D051379:Mice; D014442:Monophenol Monooxygenase / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism*; D010088:Oxidoreductases; D010858:Pigmentation; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism; D011609:Psychosine / Q000031:analogs & derivatives; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014617:Vacuoles / Q000378:metabolism
D006028:Glycosphingolipids; D008543:Melanins; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D011506:Proteins; D011609:Psychosine; D007980:Levodopa; C035742:sphingosyl beta-glucoside; D010088:Oxidoreductases; C079621:Tyrp1 protein, mouse; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D013110:Sphingosine