Localization of polypeptides to the cytosolic side of the outer envelope membrane of spinach chloroplasts

Abstract

Nonpenetrating proteolytic enzymes (such as thermolysin) were used to probe the cytosolic surface of the outer envelope membrane from spinach chloroplasts. Up to 20 different envelope polypeptides were susceptible to a mild digestion of isolated intact chloroplasts by thermolysin. Most of the thermolysin-sensitive envelope polypeptides were not extracted by a mixture of chloroform/methanol (2:1, v/v). A clear exception was E10 which is hydrophobic and, in addition, is an integral membrane polypeptide. Using antibodies to envelope polypeptides sensitive (E10 and E24) and insensitive (E30 and E37) to thermolysin, we demonstrated that only antibodies to E10 and E24, but not antibodies to E30 and E37, induced agglutination of intact chloroplasts. In addition, immunofluorescence experiments demonstrated that only antibodies to E10 and E24, but not antibodies to E30 and E37, gave a green fluorescence at the outer surface of intact chloroplasts. These experiments demonstrate that E10 and E24, and probably all the thermolysin-sensitive envelope polypeptides, are accessible from the cytosolic side of the outer membrane of the chloroplast envelope.