Thylakoid Membrane Protein Topography LOCATION OF THE TERMINI OF THE CHLOROPLAST CYTOCHROME bs ON THE STROMAL SIDE OF THE MEMBRANE*

The orientation of cytochrome bs in the thylakoid membrane and the question of whether the number of membrane spanning helices is an even or odd number was tested through the relative trypsin susceptibility of epitopes (Asp-5 to Gln-14) and (Ile-205 to Leu-214) at the NH2 and COOH termini, respectively, of the 214residue cytochrome be polypeptide. A structure of the cytochrome with an even number of helices and the NH, and COOH termini on the stromal side of the membrane was inferred from the following: 1) cleavage of cytochrome be by trypsin added to thylakoids occurs by removal of both of the exposed NH2and COOH-terminal epitopes. The epitopes at the termini were more sensitive to trypsin after prior treatment of thylakoids with carboxypeptidase A, indicating that these epitopes are shielded on the stromal side of the membrane by the COOH termini of other proteins. 2) Both epitopes were more trypsin-sensitive in thylakoid membranes than was cytochrome f that is only sensitive to trypsin acting on the lumen side of the membrane. 3) The NH2and COOH-terminal epitopes of cytochrome be were also more sensitive to trypsin added to thylakoid membranes than were the oxygenevolving complex 16and 33-kDa proteins that are completely located on the lumen side. 4) The order of trypsin susceptibility was reversed in inside-out membranes, where the cytochrome NH2and COOH-terminal epitopes were less sensitive than the 16and 33kDa proteins. The decreased relative sensitivity of the cytochrome be epitopes occurs in spite of a greater absolute sensitivity of these epitopes to trypsin in inside-out membranes. 5) The greater absolute sensitivity can be explained by a 4-helix model that includes trypsin-sensitive sites on the lumen side.