Cerebroside sulfotransferase forms homodimers in living cells.
Cerebroside sulfotransferase (CST) catalyzes the 3'-sulfation of galactose residues in several glycolipids. Its major product in the mammalian brain is sulfatide, which is an essential myelin component. Using epitope-tagged variants, murine CST was found to localize to the Golgi apparatus, but in contrast to previous assumptions, not to the trans-Golgi network. An examination of enhanced green fluorescent protein (EGFP)-tagged CST suggests that CST forms homodimers and that dimerization is mediated by the lumenal domain of the enzyme, as shown by immunoprecipitation and density gradient centrifugation. In order to verify that dimerization of CST observed by biochemical methods reflects the behavior of the native protein within living cells, the mobility of CST-EGFP was examined using fluorescence correlation spectroscopy. These experiments confirmed the homodimerization of CST-EGFP fusion proteins in vivo. In contrast to full-length CST, a fusion protein of the amino-terminal 36 amino acids of CST fused to EGFP was exclusively found as a monomer but nevertheless showed Golgi localization.
Institut für Physiologische Chemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Germany.
Afshin
A
Yaghootfam
Thomas
T
Sorkalla
Hanns
H
Häberlein
Volkmar
V
Gieselmann
Joachim
J
Kappler
Matthias
M
Eckhardt
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D006056:Golgi Apparatus / Q000201:enzymology / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000737:chemistry / Q000201:enzymology
D008565:Membrane Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015238:Sulfotransferases; C020556:galactosylceramide sulfotransferase