Plasma membrane isolated with a defined orientation used to investigate protein topography

Abstract

An investigation into the protein topography of tomato plasma membrane proteins was undertaken. Plasma membrane was isolated by phase partitioning to expose the extracellular leaflet, and by coating the protoplasts with silica microbeads to expose the cytosolic surface. Marker enzyme analysis indicated that both methods yielded relatively pure plasma membrane. Orientation of these plasma membrane fractions was established by investigating the latency of H +-ATPase activity. Triton X-100 stimulated H +-ATPase activity by 6-fold in the phase-partitioned plasma membrane fraction but did not stimulate this enzyme in the silica microbead-isolated plasma membrane. The impermeant photoactivable probes, 3-azido-(2,7)-naphthalene disulfonate and 5-azido-1-naphthalene monosulfonate, were used to probe the hydrophilic and hydrophobic regions of the plasma membrane, respectively. Using 5-azido-1-naphthalene monosulfonate, six proteins were labeled from the cytosolic leaflet of the plasma membrane and five proteins were labeled from the extracellular leaflet. Only two proteins were labeled by 3-azido-(2,7)-naphthalene disulfonate, and these were from the cytosolic-facing leaflet. The results indicate that these photoactive probes can be used in conjunction with aqueous two-phase partitioning and silica microbeads for transmembrane mapping of plasma membrane proteins.