Organization of Photosystem I Polypeptides (Identification of PsaB Domains That May Interact with PsaD)

Abstract

-PsaA and PsaB are homologous integral membrane-proteins that form the heterodimeric core of photosystem I (PSI). We used subunit-deficient PSI complexes from the mutant strains of the cyanobacterium Synechocysfis sp. PCC 6803 t o examine interactions between PsaB and other PSI subunits. lncubation of the wild-type PSI with thermolysin yielded 22-kD C-terminal fragments of PsaB that were resistant t o further proteolysis. Modification of the wildtype PSI with Khydroxysuccinimidobiotin and subsequent cleavage by thermolysin showed that the lysyl residues i n the 22-kD Cterminal domain were inaccessible to modification by Khydroxysuccinimidobiotin. The absence of PsaE, PsaF, Psal, PsaJ, or PsaL facilitated accumulation of the 22-kD C-terminal fragments of PsaB but did not alter their resistance t o further proteolysis. When the PsaD-less PSI was treated with thermolysin, the 22-kD C-terminal fragments of PsaB were rapidly cleaved, with concomitant accumulation of a 16-kD fragment and then a 3.4-kD one. We mapped the N termini of these fragments by N-terminal amino acid sequencing and the C termini from their positive reaction with an antibody against the C-terminal peptide of PsaB. The cleavage sites were proposed to be in the extramembrane loops on the cytoplasmic side. Western blot analyses showed resistance of PsaC and Psal to proteolysis prior to cleavage of the 22-kD fragments. Therefore, we propose that PsaD shields two extramembrane loops of PsaB and protects the C-terminal domain of PsaB from in vitro proteolysis.

Topography of the Photosystem I Core Proteins of the Cyanobacterium Synechocystis sp. PCC 6803

“…Previous work has indicated that a 16-kDa proteolytic fragment detected by anti-PsaB718 in thermolysin-treated ADC4 PS I complex resulted from a cleavage at Leu-531 in the I loop of PsaB (28). Similar proteolytic fragments, such as ChB, ClB, LyA, LyB, ThA in different mutant PS I complexes, were detected.…”

“…4). Additionally, Xu and Chitnis (28) identified that PsaD may shield the I and K loops of PsaB. When the PS I complexes from the AFK6 and AIC9 strains were treated by proteases, no additional fragments from the N-terminal domains of PsaA and PsaB were observed.…”

“…Isolation of the Photosynthetic Membranes and Purification of PS I Complexes-Photosynthetic membranes were isolated after cell breakage with a bead beater (Biospec Products) (28). To isolate PS I, the membranes were solubilized with Triton X-100 and subjected to DEAEcellulose chromatography and sucrose gradient centrifugation (31).…”

“…The Lys-C and trypsin treatments of the osmotically shocked cells in this paper suggested that the N terminus of PsaA is located on the n side of the thylakoid membrane. Xu and Chitnis (28) showed that the K loop interacts with PsaD which is located on the n side of the thylakoid membrane. Vallon and Bogorad (29) located the G loop on the n side and the F and H loops on the p side by using immunogold labeling.…”

“…During the past few years, major advances in x-ray crystallography (25,26), electron microscopy (27), molecular genetics (10,22), and biochemical studies (28,29) have provided a framework for understanding the overall architecture of PS I. The PS I complex has an elongated shape with a local pseudo-2-fold symmetry.…”