Characterization of FP21, a cytosolic glycoprotein from Dictyostelium.
FP21 is a glycoprotein which, when tracked by radioactivity in its fucosyl moiety, was previously detected in the cytosol of Dictyostelium cells after cell fractionation. This compartmentalization is confirmed by SDS-polyacrylamide gel electrophoresis/Western blotting of cell fractions using three different antibodies. Although a substantial fraction of FP21 is also detected in the particulate fraction using these new antibodies, particulate FP21 is released by disrupting protein-protein interactions, but not membrane disruption. Since purified FP21 is susceptible to aggregation, and purified nuclei do not contain FP21, particulate FP21 is also part of the cytosol. Additional compositional and structural information provides strong evidence that FP21 does not at any time traverse the rough endoplasmic reticulum. First, cDNAs spanning the entire coding region of the FP21 gene predict no hydrophobic motifs expected to promote membrane insertion, but do predict an NH2-terminal coiled coil domain which could explain aggregation. Second, monosaccharide composition analysis of the predominant glycoform of FP21 yields 2 mol of galactose, 1 mol of xylose, and 1 mol of fucose/mol of polypeptide; FP21 from a fucosylation-defective mutant contains 1 additional mol of xylose in place of fucose. Thus the N-glycosylation sequon present in FP21 is not utilized by oligosaccharyl transferase, which resides in the rough endoplasmic reticulum. These findings indicate that nascent FP21 remains in the cytosol after synthesis and is therefore glycosylated by unusual cytosolic xylosyl-, galactosyl-, and fucosyltransferases.
Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville 32610-0235.
E
E
Kozarov
H
H
van der Wel
M
M
Field
M
M
Gritzali
R D
RD
Brown
C M
CM
West
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D002241:Carbohydrates / Q000032:analysis; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D004023:Dictyostelium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D007106:Immune Sera; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D000596:Amino Acids; D002241:Carbohydrates; D017931:DNA Primers; D005656:Fungal Proteins; D006023:Glycoproteins; D007106:Immune Sera; D010446:Peptide Fragments; D010455:Peptides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C091987:glycoprotein FP21, Dictyostelium