E. Kozarov et al., CHARACTERIZATION OF FP21, A CYTOSOLIC GLYCOPROTEIN FROM DICTYOSTELIUM, The Journal of biological chemistry, 270(7), 1995, pp. 3022-3030
FP21 is a glycoprotein which, when tracked by radio-activity in its fu
cosyl moiety, was previously detected in the cytosol, of Dictyostelium
cells after cell fractionation. This compartmentalization is confirme
d by SDS-polyacrylamide gel electrophoresis/Western blotting of cell f
ractions using three different antibodies. Although a substantial frac
tion of FP21 is also detected in the particulate fraction using these
new antibodies, particulate FP21 is released by disrupting protein-pro
tein 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 no
t at any time traverse the rough endoplasmic reticulum. First, cDNAs s
panning the entire coding region of the FP21 gene predict no hydrophob
ic motifs expected to promote membrane insertion, but do predict an NH
2-terminal coiled coil domain which could explain aggregation. Second,
monosaccharide composition analysis of the predominant glycoform of F
P21 yields 2 mol of galactose, 1 mol of xylose, and 1 mol of fucose/mo
l of polypep tide; FP21 from a fucosylation-defective mutant contains
1 additional mol of xylose in place of fucose. Thus the N-glycosylatio
n sequon present in FP21 is not utilized by oligosaccharyl transferase
, which resides in the rough endoplasmic reticulum. These findings ind
icate that nascent FP21 remains in the cytosol after synthesis and is
therefore glycosylated by unusual cytosolic xylosyl-, galactosyl-, and
fucosyltransferases.