A NOVEL METHOD FOR MEASURING PROTEIN EXPRESSION AT THE CELL-SURFACE

All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3'-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into suspension by proteinase K and EDTA treatment at 0 degrees C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin concanavalin A (ConA). The suspended cells were encapsulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After I-125-labelling the cell surface, protease-resistant I-125-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Surface I-125-labelled beta-Na+,K+-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, including endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected. Metabolically [S-35]methionine-labelled 'high-mannose' glycosylated beta Na+,K+-ATPase was not touched by DAB cytochemistry whereas complex-glycosylated surface-exposed S-35-beta-Na+,K+-ATPase was removed by the procedure. The results show that the method can be used to measure both endocytic uptake and biosynthetic arrival at the plasma membrane of membrane-associated proteins.