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Secretion of the HasA hemophore is mediated by a C-terminal secretion signal as part of an ATP-binding cassette (ABC) pathway in the Gram-negative bacterium Serratia marcescens. We reconstituted the HasA secretion pathway in Escherichia coli. In E. coli, this pathway required three specific secretion functions and SecB, the general chaperone of the Sec pathway that recognizes HasA. The secretion of the isolated C-terminal secretion signal was not SecB-dependent. We have previously shown that intracellular folded HasA can no longer be secreted, and we proposed a step in the secretion process before the recognition of the secretion signal. Here we show that the secretion of a fully functional HasA variant, lacking the first 10 N-terminal amino acids, was less efficient than that of HasA and was SecB-independent. The N terminus of HasA was required, along with SecB, for the efficient secretion of the whole protein. We have also previously shown that HasA inhibits the secretion of metalloproteases from Erwinia chrysanthemiby their specific ABC transporter. Here we show that this abortive interaction between HasA and the E. chrysanthemimetalloprotease ABC transporter required both SecB and the N terminus of HasA. N-terminal fragments of HasA displayed this abortive interaction in vivoand also interacted specificallyin vitrowith the ABC protein of the Prt system. SecB also interacted specifically in vitrowith the ABC protein of the Prt system. Finally, the HasA variant, lacking the first 10 N-terminal amino acids did not display this abortive interaction with the Prt system. We suggest that the N-terminal domain of HasA specifically recognizes the ABC protein in a SecB-dependent fashion, facilitating functional interaction with the C-terminal secretion signal leading to efficient secretion.

Pulse‐labeling of wild‐type and a Photosystem II mutant strain of Chlamydomonas reinhardtiiwas carried out in the presence or absence of inhibitors of either cytoplasmic or chloroplast ribosomes, and their thylakoid membrane polypeptides were analyzed by polyacrylamide gel electrophoresis. A pulse‐chase study was also done on the wild‐type strain in the presence of anisomycin, an inhibitor of protein synthesis on cytoplasmic ribosomes. The following results were obtained: the Photosystem II reaction center is mainly composed of integral membrane proteins synthesized within the chloroplast. Several of the proteins of the Photosystem II reaction center are post‐translationally modified, after they have been inserted in the thylakoid membrane.

Pulse‐labeling of wild‐type and a Photosystem II mutant strain of Chlamydomonas reinhardtiiwas carried out in the presence or absence of inhibitors of either cytoplasmic or chloroplast ribosomes, and their thylakoid membrane polypeptides were analyzed by polyacrylamide gel electrophoresis. A pulse‐chase study was also done on the wild‐type strain in the presence of anisomycin, an inhibitor of protein synthesis on cytoplasmic ribosomes. The following results were obtained: the Photosystem II reaction center is mainly composed of integral membrane proteins synthesized within the chloroplast. Several of the proteins of the Photosystem II reaction center are post‐translationally modified, after they have been inserted in the thylakoid membrane.

We report the isolation and characterization of a uniparental mutant of Chlamydomonas reinhardtiithat is resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine). Such herbicides inhibit photosynthesis by preventing transfer of electrons in photosystem II from the primary stable electron acceptor Q to the secondary stable electron acceptor complex B, which is thought to contain a protein of 32 kDa and a bound quinone. It has been proposed that herbicide binding to the 32-kDa protein alters the B complex so that electron transfer from Q is prohibited. Both whole and broken-cell preparations of the mutant alga show a resistance to the effects of herbicide on electron transfer from Q to B, as measured by fluorescence-induction kinetics. In the absence of herbicide, mutant cells exhibit a slower rate of Q to B electron transfer than do wild-type cells. The 32-kDa protein from wild-type cells, but not mutant cells, binds azido[14C]atrazine at 0.1 μM. We have isolated psbA, the chloroplast gene for the 32-kDa protein, from both wild-type and herbicide-resistant algae and sequenced the coding regions of the gene that are contained in five exons. The only difference between the exon nucleotide sequences of the wild-type and mutant psbA is a single T-A to G-C transversion. This mutation results in a predicted amino acid change of serine in the wild-type protein to alanine in the mutant. We suggest that this alteration in the 32-kDa protein is the molecular basis for herbicide resistance in the C. reinhardtiimutant.

The crystal structure of the complex between the 50 kDa metallo-endo-proteinase fromSerratia marcescens(SMP), a member of the metzincin superfamily, and an inhibitor fromErwinia chrysanthemi (Inh)was solved by molecular replacement using the known structure of SMP, and refined at 2.30 Å resolution to a crystallographicR-factor of 0.195. TheE. chrysanthemiinhibitor folds into a compact eight-stranded antiparallel β-barrel of simple up-down topology such as is found for members of the retinol binding protein family. It mainly interacts with the proteaseviaits five N-terminal residues, which insert into the active site cleft, occupying the S′ sites. The first N-terminal residue, SerI1, is partially cleaved off by the protease, while SerI2 makes a hydrogen bond with the catalytically active glutamic acid, Glu177, of the protease. Further interactions are made between one face of the inhibitor formed by the strands s3, s4 and s5 and the protease segment 218 to 228, which is located immediately after the characteristic “Met-turn” of the metzincins.

Lithium dodecyl sulfate/polyacrylamide gel electrophoresis of Chlamydomonas reinhardtiithylakoid membranes at room temperature gave two chlorophyll-protein complexes, CP I and CP II, as had been reported previously. However, when the electrophoresis was performed at 4°C, there was an increase in the amount of chlorophyll associated with CP I and CP II, and in addition, three other chlorophyll-protein complexes appeared. Two of these complexes, designated CP III and CP IV, were characterized and found to be similar in their compositions. Each complex contains four to five molecules of chlorophyll a, one molecule of β-carotene, and one polypeptide chain. The apoprotein of CP III is polypeptide 5 (Mr50,000) and that of CP IV is polypeptide 6 (Mr47,000); the two polypeptides are structurally unrelated. Chlorophyll-protein complexes similar to C. reinhardtiiCP III and CP IV were also detected in higher plants (e.g., Pisum sativum). The apoproteins of the higher plant complexes are immunochemically related to those of the C. reinhardtiicomplexes, as shown by crossed immunoelectrophoresis. Absorption spectra of CP III and CP IV at -196°C revealed a component at 682 nm. This observation, together with the previous results on photosystem II mutants [Chua, N.-H. & Bennoun, P. (1975) Proc. Natl. Acad. Sci. USA72, 2175-2179], provides indirect evidence that CP III and CP IV may be involved in the primary photochemistry of photosystem II.