Cloning and sequencing of the genes encoding the light-harvesting B806-866 polypeptides and initial studies on the transcriptional organization of puf2B, puf2A and puf2C in Chloroflexus aurantiacus.

The genes encoding the alpha- and beta-polypeptide subunits of the B806-866 membrane-bound light-harvesting complex of Chloroflexus aurantiacus have been cloned and the nucleotide sequences determined. The gene puf2A, which encodes the B806-866 alpha-polypeptide, began 28 bases downstream of the stop codon of puf2B, which encodes the B806-866 beta gene. The gene-encoding cytochrome c-554, puf2C, was found about 250 bp downstream of puf2A. puf2A encoded a 13 amino acid extension at the C-terminus of the B806-866 alpha-polypeptide that was not present in the mature protein. These genes, unlike those of purple nonsulfur bacteria, did not form a contiguous operon with puf1L or puf1M, the genes encoding the L and M subunits of the photochemical reaction center. The occurrence of the two latter genes and of puf2B and puf2A in two separate operons has not been observed in purple bacteria. Under photoheterotrophic growth conditions, puf2B and puf2A were encoded on an abundant mRNA that was 0.5 kb long. Two monocistronic transcripts for puf2C were observed that had different 5'-ends. One transcript encoding all three genes was also detected. Nucleotide sequences very similar to the consensus promoter sequence of the Escherichia coli RNA polymerase sigma 70 subunit were found seven and eight bases upstream of the 5'-end of mRNA encoding puf2B and for one of the monocistronic mRNA encoding puf2C, respectively.

The primary structure of two chlorosome proteins from Chloroflexus aurantiacus.

The complete nucleotide sequence of two chlorosome proteins with apparent molecular weights of M(r) 18,000 and M(r) 11,000 from Chloroflexus aurantiacus have been determined. The two polypeptides were 145 and 97 amino acids long and possessed true molecular masses of 15,545 and 10,820 Da, respectively. Protein chemical sequencing was done in parallel to confirm the primary structure deduced from nucleotide sequencing. By Northern blot analysis of RNA isolated from phototrophically grown cells a transcript of 0.95 kb was detected which is the expected length for a mRNA encoding both genes.

A high-yield method for the isolation of hydrophobic proteins and peptides from polyacrylamide gels for protein sequencing.

A methodological approach is described which allows the isolation of hydrophobic and hydrophilic proteins and peptides in high yield. The technique consists of (1) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) protein elution from polyacrylamide gels with an organic solvent mixture composed of formic acid/acetonitrile/isopropanol/H2O (50/25/15/10, v/v/v/v), and (3) purification of eluted proteins by size exclusion chromatography on a Superose 12 column using this organic solvent mixture as eluant. The efficiency of this technique was tested with radioactively labeled polypeptides. These proteins were reaction center from Chloroflexus aurantiacus, bacteriorhodopsin, halorhodopsin from Halobacterium halobium, bovine serum albumin, ovalbumin, alpha-chymotrypsinogen A, and cytochrome c. The elution recoveries from polyacrylamide gels were 77-95%; the final yield after chromatographic purification was still 67-76% (with one exception). Subsequent amino acid sequencing was possible without further sample treatment. The sensitivity of the method described was found to be at least 20-30 micrograms protein.

Binding of plasma membrane glycoproteins to the cytoskeleton during patching and capping is consistent with an entropy-enhancement model.

Concentrations of concanavalin A that induced patching and capping of cell surface receptors on Dictyostelium discoideum also induce binding of the receptors to the cortical cytoskeleton, which was isolated by density-gradient centrifugation. The receptors were solubilized by deoxycholate, purified by affinity chromatography, and used to determine whether the receptors bound directly to the cytoskeletal protein, actin. As the concentration of actin was increased, many of the receptors became bound to purified filamentous rabbit muscle actin, even in the absence of concanavalin A. As in the ligation-induced binding of receptors to the cortical cytoskeleton in cells, concanavalin A induced much stronger binding of the purified receptors to filamentous actin. The results were consistent with a previously stated hypothesis that induction of receptor binding to the cytoskeleton during their patching and capping is driven by clustering the receptors, which reduces their translational entropy and by doing so enhances their avidity for the cytoskeleton.

The primary structure of the Chloroflexus aurantiacus reaction-center polypeptides.

The complete nucleotide sequence of two Chloroflexus aurantiacus reaction-center genes has been obtained. The amino acid sequence deduced from the first gene showed 40% similarity to the L subunit of the Rhodobacter sphaeroides reaction center. This L subunit was 310 amino acids long and had an approximate molecular mass of 35 kDa. The second gene began 17 bases downstream from the first gene. The amino acid sequence deduced from it (307 amino acids; 34950 Da) was 42% similar to the M subunit of the Rhodobacter sphaeroides reaction center. 20% of the deduced primary structure were confirmed through automated Edman degradation of cyanogen bromide peptide fragments or N-chlorosuccinimide peptide fragments isolated from the purified reaction-center complex or from the individual subunits. The peptides were isolated by preparative gel electrophoresis combined with molecular sieve chromatography in the presence of a mixture of formic acid, acetonitrile, 2-propanol and water. This method appeared to be applicable to the isolation of other hydrophobic proteins and their peptides.

The photochemical reaction center of Chloroflexus aurantiacus is composed of two structurally similar polypeptides.

A method has been devised which allowed the isolation of highly purified reaction center from the thermophilic green bacterium, Chloroflexus aurantiacus. The procedure consisted of three chromatography steps. The final step was fast protein liquid chromatography on Mono Q in the presence of nonanoyl-N-methylglucamide (Mega-9). The purified reaction center complex was photochemically active and had an A280/A813 of 1.4 or less. Under non-denaturing conditions, a pigmented protein band having a Mr of 52,000-55,000 was observed in sodium dodecyl sulfate gels. When the isolated complex was heat-dissociated in the presence of sodium dodecyl sulfate, just two polypeptides having very similar Mr (24,000 and 24,500) were observed. Two protein bands were also observed in two-dimensional isoelectric focusing/sodium-dodecyl-sulfate polyacrylamide gel electrophoresis; the PI values of the two polypeptides were 6.5 and 6.7. Partial peptide mapping of the two isolated subunits, using both enzymatic and chemical cleavage techniques, yielded almost identical patterns which indicated a high degree of sequence homology between the two polypeptides. The N-terminal amino acid sequences of the two polypeptides were identical and did not exhibit any homology to reaction center subunits of purple sulfur bacteria. The Chloroflexus reaction center is believed to be composed of one molecule of each polypeptide, the photoactive bacteriochlorophyll a dimer and, as accessory pigments, an additional bacteriochlorophyll a and three bacteriopheophytins. Hence, it appears to be the smallest photochemically active reaction center isolated to date.

Topography of the Dictyostelium discoideum plasma membrane: analysis of membrane asymmetry and intermolecular disulfide bonds.

Through the application of a unique method for isolating plasma membranes, it was possible to specifically iodinate cytoplasm-exposed plasma membrane proteins in vegetative cells of the cellular slime mold Dictyostelium discoideum. The original procedure [Chaney, L. K., & Jacobson, B. S. (1983) J. Biol. Chem. 258, 10062] which involved coating cells with colloidal silica has been modified to yield a more pure preparation. The presence of the continuous and dense silica pellicle on the outside surface of the isolated plasma membrane permitted the specific labeling of cytoplasm-exposed membrane proteins. Lactoperoxidase-catalyzed iodination was employed to label cell-surface and cytoplasm-exposed membrane proteins. The isolated and radioiodinated membranes were then compared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cell-surface and cytoplasmic face labeling patterns were distinct. A total of 65 proteins were found to be accessible to at least one surface of the membrane. Sixteen intermolecular disulfide bond complexes were observed in the plasma membrane of Dictyostelium; most of these complexes involved glycoproteins and, hence, were exposed to the cell surface.

Linear and circular dichroism of membranes from Rhodopseudomonas capsulata.

Absorption, linear dichroism and circular dichroism spectra of Rhodopseudomonas capsulata (wild-type-St. Louis strain, mutant Y5 and mutant Ala+) are particularly sensitive to the nature of the light-harvesting bacteriochlorophyll-carotenoid-protein complexes. Evidence for exciton-type interactions is seen near 855 nm in the membranes from the wild-type and from mutant Y5, as well as in an isolated B-800 + 850 light-harvesting complex from mutant Y5. The strong circular dichroism that reflects these interactions is attenuated more than 10-fold in membranes from the Ala+ mutant, which lacks both B-800 + 850 and colored carotenoids and contains only the B-875 light-harvesting complex. These results lead to the conclusion that these two light-harvesting complexes have significantly different chromophore arrangements or local environments.

Isolation and characterization of the polypeptide components from light-harvesting pigment-protein complex B800--850 of Rhodopseudomonas capsulata.

The light-harvesting bacteriochlorophyll-carotenoid-protein complex B800--850 has been isolated from membranes of the phototroph-negative mutant strain Y5 of Rhodopseudomonas capsulata. The three polypeptides of the complex have been found to be soluble in chloroform-methanol (1:1, v/v) in the presence of 0.1 M ammonium acetate. They were extracted from the complex and separated by gel filtration on Sephadex LH-60 in the same solvent mixture. Minimum molecular weights based on amino acid composition are 12 000, 9300, and 5100. Values previously determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis are 14 000, 10 000 and 8000. The two smaller polypeptides (polarities 31% and 39%) are completely soluble in chloroform/methanol/ammonium acetate while the largest and most polar (41%) polypeptide is only partially soluble. The largest polypeptide contains no tryptophan. The middle polypeptide contains no cysteine and arginine, while the small polypeptide lacks cysteine. Methionine is shown to be the amino terminus for the small and middle polypeptides by two independent methods (Edman degradation and dansylation). Both methods also indicated that the N terminus of the 14 000 polypeptide seems to be blocked. Partial N-terminal amino acid sequences were obtained for the two smaller polypeptides. No homology between the two proteins was observed.