A non-Golgi alpha 1,2-fucosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium.
Skp1 is a subunit of the SCF-E3 ubiquitin ligase that targets cell cycle and other regulatory factors for degradation. In Dictyostelium, Skp1 is modified by a pentasaccharide containing the type 1 blood group H trisaccharide at its core. To address how the third sugar, fucose alpha1,2-linked to galactose, is attached, a proteomics strategy was applied to determine the primary structure of FT85, previously shown to copurify with the GDP-Fuc:Skp1 alpha 1,2-fucosyltransferase. Tryptic-generated peptides of FT85 were sequenced de novo using Q-TOF tandem mass spectrometry. Degenerate primers were used to amplify FT85 genomic DNA, which was further extended by a novel linker polymerase chain reaction method to yield an intronless open reading frame of 768 amino acids. Disruption of the FT85 gene by homologous recombination resulted in viable cells, which had altered light scattering properties as revealed by flow cytometry. FT85 was necessary and sufficient for Skp1 fucosylation, based on biochemical analysis of FT85 mutant cells and Escherichia coli that express FT85 recombinantly. FT85 lacks sequence motifs that characterize all other known alpha 1,2-fucosyltransferases and lacks the signal-anchor sequence that targets them to the secretory pathway. The C-terminal region of FT85 harbors motifs found in inverting Family 2 glycosyltransferase domains, and its expression in FT85 mutant cells restores fucosyltransferase activity toward a simple disaccharide substrate. Whereas most prokaryote and eukaryote Family 2 glycosyltransferases are membrane-bound and oriented toward the cytoplasm where they glycosylate lipid-linked or polysaccharide precursors prior to membrane translocation, the soluble, eukaryotic Skp1-fucosyltransferase modifies a protein that resides in the cytoplasm and nucleus.
van Der Wel
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H
Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610-0235 and the Department of Biochemistry, Imperial College, London SW7 2AY United Kingdom.
Morris
H R
HR
Panico
M
M
Paxton
T
T
North
S J
SJ
Dell
A
A
Thomson
J M
JM
West
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000201:enzymology* / Q000378:metabolism; D004023:Dictyostelium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005647:Fucosyltransferases / Q000737:chemistry* / Q000235:genetics; D006056:Golgi Apparatus / Q000201:enzymology*; D007438:Introns; D008027:Light; D013058:Mass Spectrometry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D018797:Cell Cycle Proteins; D005647:Fucosyltransferases; D011994:Recombinant Proteins; D044786:S-Phase Kinase-Associated Proteins; D000097631:Galactoside 2-alpha-L-fucosyltransferase