CHARACTERIZATION OF FP21, A CYTOSOLIC GLYCOPROTEIN FROM DICTYOSTELIUM

Citation
E. Kozarov et al., CHARACTERIZATION OF FP21, A CYTOSOLIC GLYCOPROTEIN FROM DICTYOSTELIUM, The Journal of biological chemistry, 270(7), 1995, pp. 3022-3030
Citations number
38
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
270
Issue
7
Year of publication
1995
Pages
3022 - 3030
Database
ISI
SICI code
0021-9258(1995)270:7<3022:COFACG>2.0.ZU;2-8
Abstract
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.