FIGURE 4.
Palmitoylation of GP Ibβ and GP IX through intracellular membrane-proximal cysteines is not essential for GEM localization in CHO cells. Intracellular membrane-proximal cysteines of both GP Ibβ (βC148S) and GP IX (IXC154S) were mutated to the amino acid serine. A, stable CHO cells expressing mutant GP IbβS or GP IXS or both with wild-type GP Ibα were generated (WM23 staining). B, when cells were labeled with [3H]palmitate and visualized using SDS-PAGE and fluorography, no sign of palmitic modification was shown, indicating that the intracellular membrane-proximal cysteines of GP Ibβ and GP IX are the sites for palmitoylation in the GP Ib-IX complex. C and D, further sucrose density fractionation analysis showed that the depalmitoylated GP Ibs in the double mutant CHO cells distributed to the same sucrose gradient as their palmitoylated counterparts in the wild-type cells (Figs. 2B and 3A).