Density of newly synthesized plasma membrane proteins in intracellular membranes II. Biochemical studies

Abstract

Using two independent methods, incorporation of radioactive amino-acid and quantitative immunoblotting, we have determined that the rate of synthesis of each of the Semliki Forest virus (SFV) proteins in infected baby hamster kidney (BHK) cells is 1.2 x 105 copies/cell/min. Given the absolute surface areas of the endoplasmic reticulum and Golgi complex presented in the companion paper (Griffiths, G., G. Warren, P. Quinn, O. Mathieu Costello, and A. Hoppeler, 1984, J. Cell Biol. 98:2133-2141), and the approximate time spent in these organelles during their passage to the plasma membrane (Green J., G. Griffiths, D. Louvard, P. Quinn, and G. Warren 1981, J. Mol. Biol. 152:663-698), the mean density of each viral protein in these organelles can be calculated to be 90 and 750 molecules/μm2 membrane, respectively. In contrast, we have determined that the density of total endogenous integral membrane proteins in these organelles is ~ 30,000 molecules/μm2 so that the spike proteins constitute only 0.28 and 2.3% of total membrane protein in the endoplasmic reticulum and Golgi, respectively. Quantitative immunoblotting was used to give direct estimates of the concentrations of one of the viral membrane protein precursors (E1) in subcellular fractions; these agreed closely with the calculated values. The data are discussed with respect to the sorting of transported proteins from those endogenous to the intracellular membranes.