Topological analysis of the Rhodobacter capsulatus PucC protein and effects of C-terminal deletions on light-harvesting complex II

Abstract

A theoretical model for the cytoplasmic membrane topology of the Rhodobacter capsulatus PucC protein was derived and tested experimentally with pucC::phoA gene fusions. The alkaline phosphatase (AP) activities of selected fusions were assayed, and the resultant pattern of high and low activity was compared with that of the theoretical model. High AP activity correlated well with fusion joints located in regions predicted to be periplasmic, and most fusions in predicted cytoplasmic loops yielded approximately 1/20th as much activity. Replacement of phoA with lacZ in nine of the fusions confirmed the topology, as ␤-galactosidase activities were generally reciprocal to the corresponding AP activity. On the basis of the theoretical analysis and the information provided by the activities of fusions, a model for PucC topology in which there are 12 membranespanning segments and both the N and C termini are located in the cytoplasm is proposed. Translationally out-of-frame pucC::phoA fusions were expressed in an R. capsulatus ⌬pucC strain. None of the fusions missing only one or two of the proposed C-terminal transmembrane segments restored the wild-type phenotype, suggesting that the C terminus of PucC is important for function.Such purple nonsulfur photosynthetic bacteria as Rhodobacter capsulatus are good model systems for investigations of membrane protein structure and function, especially in regard to proteins involved in photosynthesis. Crystal structures of the reaction center and light-harvesting (LH) integral membrane pigment-protein complexes from several species have been published (15,17,22). Although the structural proteins of these complexes are very hydrophobic and might be thought of as independently capable of complex assembly in the cytoplasmic membrane, several genes that do not encode crystal structural components and yet are required to obtain wild-type levels of the reaction center and LH complexes have been discovered (2,12,19,31). The results of preliminary experiments with one of these genes, F1696, indicate that it functions to enhance assembly of LH complex I (LHI) as opposed to inhibiting turnover (31a). The current evidence indicates that the protein components of pigment-protein complexes are capable of membrane association in the absence of bacteriochlorophyll, but complexes are not formed, and the proteins exhibit enhanced turnover rates (10).The crystal structure of the Rhodopseudomonas acidophila LHII complex was found to consist of a ring containing nine copies of a membrane-spanning ␣-␤ protein dimer, with bacteriochlorophyll and carotenoid pigments sandwiched between concentric transmembrane domains of the ␣ and ␤ proteins (22). The R. capsulatus LHII complex was purified and found to contain a third protein, designated ␥, which was removed from the complex by treatment with Na 2 CO 3 , with little effect on LHII light absorption properties (27). The LHII proteins are coded for by the pucBACDE operon, which has been cloned, sequenced, and studied by deletion-complementation analyses...

Structural Organization of the Protein-tyrosine Autokinase Wzc within Escherichia coli Cells

“…Strain BL21(pREP4-groESL), in which overproduction of the C-terminal domain of Wzc was obtained, was reported previously (24). Plasmids pSP72-lacZ and pUC19-phoA were obtained from J. T. Beatty and W. H. Bingle, respectively (25,26).…”

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

“…In contrast, due to improper folding of the PhoA moiety in the cytoplasm, in cytoplasmic fusion proteins the free PhoA moiety is rapidly degraded and is not or poorly detected by Western blotting. As a consequence, fusions with a periplasmic PhoA moiety show larger amounts of immunoreactive products on a Western blot than do cytoplasmic fusions (45,106,143,184). This systematic behavior can be used as an additional method to discriminate between fusions with a periplasmic or cytoplasmic PhoA moiety, but it is important that it is not used to normalize the activities.…”

“…Furthermore, in a few cases, the truncated membrane proteins adopt alternate conformations depending on which reporter protein is present. For unknown reasons, the correct folding in the membrane is more often achieved with PhoA as the reporter (57,106). In recent years, most topology studies have been done with just PhoA or ␤-lactamase.…”

“…Various topology models have been tested using a combination of PhoA and LacZ fusions (45,57,76,91,92,106,170,202,206). Random fusions to both reporter molecules are often constructed, and this is followed by screen- ing for positive clones on indicator plates.…”

Involvement of the C-Terminal Extension of the α Polypeptide and of the PucC Protein in LH2 Complex Biosynthesis inRubrivivax gelatinosus

“…PucC proteins contain 12 transmembrane segments (19,32), and the deletion in R. capsulatus of one or two C-terminal transmembrane helices abolished LH2 complex formation (17). This protein presents some similarities in sequence and function to the LhaA protein, which is essential for maximal LH1 formation.…”