Inhibition of tyrosine sulfation in the trans-Golgi retards the transport of a constitutively secreted protein to the cell surface.
The effect of tyrosine sulfation on the transport of a constitutively secreted protein, yolk protein 2 (YP2) of Drosophila melanogaster, to the cell surface was investigated after expression of YP2 in mouse fibroblasts. Inhibition of YP2 sulfation was achieved by two distinct approaches. First, the single site of sulfation in YP2, tyrosine 172, was changed to phenylalanine by oligonucleotide-directed mutagenesis. Second, L cell clones stably expressing YP2 were treated with chlorate, a reversible inhibitor of sulfation. Pulse-chase experiments with transfected L cell clones showed that the half-time of transport from the rough endoplasmic reticulum to the cell surface of the unsulfated mutant YP2 and the unsulfated wild-type YP2 produced in the presence of chlorate was 15-18 min slower than that of the sulfated wild-type YP2. Control experiments indicated (a) that the tyrosine to phenylalanine change itself did not affect YP2 transport, (b) that the retardation of YP2 transport by chlorate occurred only with sulfatable but not with unsulfatable YP2, (c) that the transport difference between wild-type and mutant YP2 was not due to the level of YP2 expression, and (d) that transport of the endogenous secretory protein fibronectin was the same in L cell clones expressing wild-type and mutant YP2. Since the half-time of transport of wild-type YP2 from the intracellular site of sulfation, the trans-Golgi, to the cell surface was found to be 10 min, the 15-18-min retardation seen upon inhibition of tyrosine sulfation reflected a two- to threefold increase in the half-time of trans-Golgi to cell surface transport, which was most probably caused by an increased residence time of unsulfated YP2 in the trans-Golgi. The results demonstrate a role of tyrosine sulfation in the intracellular transport of a constitutively secreted protein.
Friederich
E
E
Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Fritz
H J
HJ
Huttner
W B
WB
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002704:Chlorates / Q000494:pharmacology; D004527:Egg Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007739:L Cells; D008565:Membrane Proteins / Q000378:metabolism*; D008715:Methionine; D009154:Mutation; D011233:Precipitin Tests; D013455:Sulfur / Q000378:metabolism / Q000502:physiology*; D013462:Sulfur Radioisotopes; D014443:Tyrosine / Q000378:metabolism / Q000502:physiology*
D002704:Chlorates; D004527:Egg Proteins; D005353:Fibronectins; D008565:Membrane Proteins; D013462:Sulfur Radioisotopes; D014443:Tyrosine; D013455:Sulfur; D008715:Methionine