Shi LX, Schroder WP
Compositional and topological studies of the PsbW protein in spinach thylakoid membrane
Photosynthesis Research: 1997 53:45-53
The lateral distribution and transversal orientation of the nuclear encoded PsbW protein (psbW gene product) has been investigated. The main part (80%) of the PsbW protein was found in the grana region of the thylakoid membrane, corroborating earlier observations that the PsbW protein was closely associated with Photosystem II (PS II). The localisation within the PS II complex was analysed by a comparative quantification of the PsbW content between PS II membrane fragments (BBY) and various isolated PS II reaction centres. Our results showed that the PsbW protein could be detected in all PS II reaction centre preparations, whereas the chlorophyll a proteins CP47 and CP43 were not detectable. However, a careful analysis based on the number of reaction centres, revealed that the amount of the PsbW protein found in the PS II reaction centre preparation (Nanba-Satoh type) was lower than that in a BBY preparation. These results suggested that the PsbW protein was located close to the D1/D2 heterodimer, but the PsbW protein could, at least partially, be removed from the PS II reaction centre during isolation. Quantification of the amounts of the PsbW protein in various reaction centre preparations indicated that the presence of Triton X-100 throughout the isolation procedure appeared to be a crucial point for obtaining low amounts of the PsbW protein in the PS LI reaction centre preparation. Trypsin digestion followed by SDS-PAGE, immunoblotting and Enzyme Linked Immunosorbent Assay (ELISA) revealed that the hydrophobic PsbW protein contained one transmembrane span with its C-terminus exposed on the stroma side while the N-terminus faced the lumen side of the thylakoid membrane. Thus, despite that the protein had a typical lumenal targeting presequence, it was an integral membrane protein. Moreover, it had its N-terminus on the opposite side of the membrane compared to other PS II reaction centre proteins.
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