Determination of stoichiometry of the five multiprotein complexes in the thylakoid membranes
Takunori Matsumura, Shin-ichiro Ozawa and Yuichiro Takahashi
Graduate School of Natural Science and Technology, Okayama Univ., Okayama, Japan
Four multiprotein complexes, photosystem I (PSI), cytochrome b6f (Cyt. b6f), photosystem II (PSII), and light-harvesting complex II (LHCII), are involved in the light-harvesting and photosynthetic electron transfer while one complex, ATP synthase, in photophosphorylation. We have optimized the conditions for blue-native polyacrylamide gel electrophoresis (BN-PAGE) to separate the multiprotein complexes in the thylakoid membranes and for second SDS-PAGE to separate the constituent polypeptides. We then employed two dimensional (2D) PAGE to estimate relative abundance of the five multiprotein complexes and of the constituent polypeptides in the thylakoid membranes from Chlamydomonas wild-type cells grown in light (50 microE m-2 s-1). First we uniformly labeled total cell proteins with 14C-acetate and isolated thylakoid membranes. Subsequently the thylakoid membranes were solubilized with dodecyl-maltoside and the multiprotein complexes and the constituent polypeptides were separated by 2D-PAGE. The amount of the separated polypeptides was estimated with a luminoimage analyzer using imaging plate. It was estimated the ratio of PSI : PSII : Cytb6f : LHCII : ATP synthase to be 1.0 : 1.2 : 0.8 : 1.0 : 35 : 1.0. We will evaluate the present values by comparing them with those estimated by optically measuring the redox components (Nearle and Melis, 1986). We also report relative stoichiometry of subunits of ATP synthase (AtpA-I) and three types of LHCII polypeptides (Types I, III, and IV).
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