Abstract
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.
Highlights
Glycosyltransferases (GTs)1 mediate the transfer of monosaccharide residues from the corresponding nucleotide sugar donor onto glycoproteins or glycolipids during their transport along the secretory pathway
Cys-16, Gln-23, Cys-29, and Tyr-33 from the Transmembrane Domain of FT3wt Are Required for Its Localization in the Golgi—The full-length membrane-bound form of fucosyltransferase III has been stably expressed in Baby Hamster Kidney (BHK) cells (BHK-FT3wt) (Fig. 2, A–C) [4]
After examining the transmembrane domain (TM) of FT3wt we have identified amino acid residues more likely to participate in protein-protein interactions within the hydrophobic environment of the membrane
Summary
Forward mutagenesis primers of the transmembrane fucosyltransferase III mutant forms Sequence. 5Ј-CC ATG GAT CCC CTG GGT GCA GCC AAG CCA CAA TGG CCA TGG CGC CGC CTG CTG GCC GCA CTG CTA TTT CTG CTG CTG GTG GCT GTG CTG TTC TTC TCC CTC CTG CGT GTG TC-3Ј. 5Ј-CC ATG GAT CCC CTG GGT GCA GCC AAG CCA CAA TGG CCA TGG CGC CGC TGT CTG GCC GCA CTG CTA TTT CAG CTG CTG GTG GCT GTG TGT TTC TTC TCC CTC CTG CGT GTG TC-3Ј. 5Ј-CC ATG GAT CCC CTG GGT GCA GCC AAG CCA CAA TGG CCA TGG CGC CGC TGT CTG GCC GCA CTG CTA TTT CAG CTG CTG GTG GCT GTG CTG TTC TTC TCC TA-3Ј. 5Ј-CC ATG GAT CCC CTG GGT GCA GCC AAG CCA CAA TGG CCA TGG CGC CGC TGT CTG GCC GCA CTG CTA TTT CTG CTG CTG GTG GC-3Ј. FT3wt was concentrated into COPI vesicles contrary to the mutant FT3np, indicating that the Cys, Gln, and Tyr residues are important for the sorting of FT3wt into the transport vesicles
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