Incorporation of light-harvesting complex I alpha and beta polypeptides into the intracytoplasmic membrane of Rhodobacter capsulatus

Abstract

The light-harvesting complex I (LHI) of Rhodobacter capsulatus is an oligomer of basic subunits each consisting of the two different pigment-binding polypeptides LHI a and LHI 0, encoded by the pufA (LHI a) and puJB (LHI j1) genes. Pulse-labeling experiments showed that in the presence of the LHI a polypeptide, the LHI , polypeptide was inserted earlier into the intracytoplasmic membrane than was the LHI a polypeptide.Each of the pufA and puJB genes was deleted to test whether the LHI a and polypeptides, respectively, are inserted into the intracytoplasmic membrane independently of the LHI partner polypeptide. Neither deletion mutant strain formed the LHI antenna, but a functional reaction center complex was present. Pulse-labeling experiments indicated that the LHI , polypeptide was inserted into the intracytoplasmic membrane with the same kinetics and in the same amounts regardless of whether the LHI a polypeptide was present. However, the LHI , polypeptide did not accumulate in the membrane in the absence of the LHI a protein but was degraded linearly within about 12 min. In contrast to the LHI ,1 protein, only trace amounts of the LHI a polypeptide were inserted into or attached to the membrane if the LHI , polypeptide was not synthesized.The facultative phototrophic bacterium Rhodobacter capsulatus has the ability to transduce light energy into metabolic energy. Two different light-harvesting (LH) complexes are involved in absorption of light quanta and transfer of excitation energy to the photosynthetic reaction center (RC). LH complex I (LHI) or B870 is associated with the RC forming the core complex (13, 21). LHII (B800-850) surrounds the core complex and is synthesized in variable amounts, depending on growth conditions (7, 13).The pigment-protein complexes are located in the intracytoplasmic membrane (ICM). Each complex is composed of two pigment-binding integral membrane proteins: LHI a and ,B, LHII a and 3, and RC-L and RC-M. A non-pigmentbinding protein is present in the LHII antenna (LHII -y polypeptide) and in the RC (RC-H subunit) (for reviews, see references 13 and 14). The pigments bacteriochlorophyll and carotenoids are bound noncovalently in stoichiometric ratios to the antenna and RC polypeptides (for a review, see reference 13).Pigments and pigment-binding proteins are coordinately assembled in distinct membrane areas (9) whose relative phospholipid concentration has an influence on the insertion (3,21,22,31,43 The LHI a and LHI fi polypeptides are encoded by the puf operon (1). Synthesis of these polypeptides is controlled at the mRNA level in response to changes of oxygen partial pressure or light intensity (1,5,22,50 hydrophobic a-helical central segment (39,41,51). The N-terminal segments of LHI a and LHI P polypeptides are oppositely charged (40, 42) and located on the cytoplasmic site of the membrane (41). The C termini of both LHI polypeptides extend into the periplasmic space (40). It was proposed that the LHI complex is stabilized by ionic interaction between the oppositely char...

Characterization of LHI- and LHI+ Rhodobacter capsulatus pufA mutants

“…The LHI 1 protein, however, has a negatively charged NH2 terminus and is able to integrate temporarily into the membrane in the absence of the LHI a protein (43). The LHI 13 protein was observed to be inserted earlier into the ICM than was the LHI a protein (43).…”

“…1) synthesized at the University of Bielefeld were used for introduction of mutations in the pufA gene coding for the LHI ao polypeptide. The mutations were carried out in a manner analogous to that described for deletion of the pufA and pufB genes (43). The mutations were confirmed by sequencing with appropriate primers (42)(43)(44).…”

“…The NH2 terminus of the LHI a polypeptide is located in the cytoplasm and has a short hydrophobic central part of amino acid residues flanked by positively charged residues which are next to a conserved proline (51,52). The LHI a protein is inserted into the ICM in wild-type amounts only in the presence of the LHI 1 protein (43). The LHI 1 protein, however, has a negatively charged NH2 terminus and is able to integrate temporarily into the membrane in the absence of the LHI a protein (43).…”

“…The mutations were carried out in a manner analogous to that described for deletion of the pufA and pufB genes (43). The mutations were confirmed by sequencing with appropriate primers (42)(43)(44). The plasmid constructions containing mutated puf operons were designated serially as pTPR13 to pTPR18 (Table 1).…”

“…All bacterial strains and plasmids used in this study are listed in Table 1. Growth conditions for Eschenchia coli strains are described elsewhere (39,43). Mutant strains of R. capsulatus were grown in malate mineral medium (18) Analytical methods.…”