Identification of some plasma membrane proteins of cultured rat pigment epithelial cells by labeling with 125I

Abstract

Cultured rat retinal pigment epithelial (PE) cells were labeled with 125I by lactoperoxidase (LPO)-catalyzed radioiodination. Examination of 125I-labeled cells by electron microscopic autoradiography suggested that 125I was incorporated into proteins at both the apical and basal surfaces of the PE cells, and into the extracellular matrix (ECM). Analysis of labeled cells by SDS-polyacrylamide gel electrophoresis and autoradiography showed that 125I was incorporated into at least 15 polypeptides. In order to determine which of these labeled proteins were derived from the plasma membrane, 125I-labeled cells were subjected to differential detergent extraction. Triton X-100 (2% v/v), which removed the cell plasma membrane, solubilized only three of the labeled proteins (1 52 000, 1 38 000, 1 23 000 daltons). SDS (0·25 % w/v) completely removed cells from the tissue culture dish and solubilized all but four of the labeled proteins (225 000, 173 000, 87 000 and 72 100 daltons). When 125I-labeled PE cells were mechanically disrupted, and the resulting cell fractions separated by sucrose density gradient centrifugation, labeled proteins separated into two subcellular fractions. One fraction was especially enriched in the 1 52 000, 1 38 000 and 1 23 000 dalton labeled proteins, in addition to the plasma membrane marker enzymes Na +, K +-ATPase, and alkaline phosphodiesterase I. The second fraction was enriched in the 225 000, 1 96 000 and 1 73 000 dalton labeled proteins, the ECM proteins laminin and fibronectin, and the 43 000 actin band. It is proposed that 125I-labeled proteins in the former cell fraction are truly plasma membrane proteins, while those found in the latter cell fraction are a mixture of 125I-labeled ECM and basal plasma membrane proteins. The 1 52 000, 1 38 000 and 1 23 000 dalton labeled proteins of the plasma membrane fraction are glycoproteins and become firmly anchored to the Triton X-100 insoluble cytoskeleton when labeled cells are treated with concanavalin A.