Importance of Cys, Gln, and Tyr from the transmembrane domain of human alpha 3/4 fucosyltransferase III for its localization and sorting in the Golgi of baby hamster kidney cells.
Human fucosyltransferase III (EC ) (FT3wt) is localized in the Golgi of baby hamster kidney cells and synthesizes Lewis determinants associated with cell adhesion events. Replacement of the amino acid residues from the transmembrane domain (TM) Cys-16, Gln-23, Cys-29, and Tyr-33 by Leu (FT3np) caused a shift in enzyme localization to the plasma membrane. The mislocalization caused a dramatic decrease in the amount of biosynthetic products of FT3wt, the Lewis determinants. Determination of the expression levels on the surface with mutants of the enzyme, where one, two, or three of these residues were replaced by Leu, suggested that Cys from the TM was required for the localization of FT3 in the Golgi. Furthermore, Cys-23 and Cys-29 mediated the formation of disulfide-bonded dimers but not higher molecular weight oligomers. In vitro reconstitution of intra-Golgi transport showed that FT3wt was incorporated into coatomer protein (COP) I vesicles, contrary to FT3np. These data suggested that Cys, Gln, and Tyr residues are important for FT3wt sorting into the transport vesicles possibly due to interactions with other membrane proteins.
Sousa
Victor L
VL
Laboratory of Glycobiology, Instituto de Tecnologia Quimica e Biológica, Apartado 127, 2780 Oeiras, Portugal.
Brito
Catarina
C
Costa
Teresa
T
Lanoix
Joel
J
Nilsson
Tommy
T
Costa
Julia
J
D020816:Amino Acid Motifs; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry*; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D005647:Fucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry*; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry*
D004220:Disulfides; D005973:Glutamine; D014443:Tyrosine; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase; D003545:Cysteine