Import and assembly of the alpha and beta-polypeptides of the light-harvesting complex I (B870) in the membrane system of Rhodobacter capsulatus investigated in an in vitro translation system

Transcripts of the genes pufBA, pufB or pufA from Rhodobacter capsulatus were translated in a cell-free system of R. capsulatus. The incorporation of the nascent polypeptides LHI alpha and beta in various types of membranes and the assembly of the light-harvesting (LH) complex I (B870) were investigated. The highest rate of stable incorporation of LHI alpha and beta into the membrane was observed with membranes from the wild type strain grown under chemotrophic conditions. Addition of membranes from cells defective in biosynthesis of pigment-binding proteins resulted in a less efficient or less stable incorporation of LHI alpha beta. The single polypeptides LHI alpha or beta were synthesized and inserted into the membrane but were extractable to a higher percentage by 6 M urea than the pairwise inserted LHI polypeptides. If the ribosomes and the S135 extract were depleted of DnaK the rate of synthesis of both polypeptides, LHI alpha and beta, was strongly reduced. Removal of GroEL from the cell-free system did not impair the synthesis and membrane association of both proteins, but affected the stable insertion. A high percentage of the LHI alpha beta polypeptides became extractable by 6 M urea if the cell-free system was depleted of GroEL. Addition of GroEL to the cell-free system restored the capacity of stable insertion of both proteins into the membrane. GroEL interacted with LHI alpha and beta before membrane targeting as shown by immunological means. A protein fraction, which can be removed from the membrane with a low-salt buffer, supported the effective and stable incorporation of LHI alpha beta into the membrane. It is concluded that the assembly of the LHI complex in the membrane system of R. capsulatus is a multistep process guided and supported by polypeptides located in the cytoplasm and in the membrane. In the cell-free in vitro system not only the correct insertion of the LHI polypeptides but also an assembly with bacteriochlorophyll was observed. BChl was synthesized from delta-amino levulinate in the cell free system.