The 5.6-Kilodalton Protein in Isolated Chlorosomes of Chloroflexus aurantiacus Strain 0k-70-fl is a Degradation Product Kai Griebenow, Alfred R. Holzwarth, and Kurt Schaffner Max-Planck-Institut für Strahlenchemie, D-4330 Mülheim a.d. Ruhr, Bundesrepublik Deutschland 7. Naturforsch. 45c, 823-828(1990); received March 19, 1990 Photosynthetic Antennae, Chloroflexus aurantiacus, Chlorosomes, 5.6-kDa Polypeptide Chlorosomes containing BChl a 790 have been isolated from Chloroflexus aurantiacus on sucrose density gradients using the detergents Miranol, Deriphat, N,N-dimethyldodecyl- amine-N-oxide, and dodecyl-ß-D-maltoside. All freshly prepared samples either lack the poly peptide of approximately 5 kDa, which appears identical with the 5.6-kDa protein previously assigned the role of BChl c-binding [R. G. Feick and R. C. Fuller, Biochemistry 23, 3693- 3700 (1984)], or they contain only a minor amount thereof. This polypeptide accumulates in the chlorosomes in vitro at room temperature within 24 h after isolation. The reaction cannot be prevented simply by addition of the protease inhibitors benzamidine, £-caproic acid, and phenylmethylsulfonyl fluoride. However, upon denaturation, as required tor gel electrophore sis, of the freshly isolated chlorosome sample the formation of the 5-kDa polypeptide is inhibit ed. We conclude that this species, viz. 5.6-kDa protein, is a degradation product of another - as yet unidentified - protein present in the chlorosome preparations. Despite the pronounced proteolytic activity which affords the 5-kDa fragment, the native absorption and fluorescence properties of BChl c and BChl a are essentially not changed in these chlorosome preparations. Introduction The Chlorobiaceae and Chloroflexaceae both contain so-called chlorosomes as the main light- harvesting antenna system [1]. Chlorosomes are vesicles with the dimensions of approx. 12 x 30 * 100 nm in Chloroflexus aurantiacus [2], They con tain bacteriochlorophyll (BChl) c as the main light-harvesting chromophore. BChl a is present in the chlorosomes in a small amount (about 4% of the total BChl [3]) when isolated by SDGC, but it is removed by GEF [4], and by treatment of the isolated chlorosomes with SDS [5, 6]. The organization of BChl c in chlorosomes is still a matter of debate. One of the models for the chlorosomes of C. aurantiacus [7] assumes binding of BChl c to a 5.6-kDa protein [3, 7, 8]. Six dimers of this protein, each with 10- 16 BChl c molecules Abbreviations: BChl, bacteriochlorophyll; DDM, dode- cyl-ß-D-maltoside; GEF, gel-electrophoretic filtration; LDAO, N,N-dimethyldodecylamine N-oxide; LDS, lithium dodecyl sulfate; Mr, relative molecular weight; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; SDGC, sucrose density gradient centrifugation; SDS, sodium dodecyl sulfate; Tris, tris(hydroxymethyl)aminomethane. Reprint requests to Dr. A. R. Holzwarth. Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen 0341-0382/90/0700-0823 $ 01.30/0 attached, were proposed to constitute one unit of the rod elements which had been detected by elec tron microscopy [2], However, Schmidt et al. [9] did not report the 5.6-kDa polypeptide in the chlo rosomes of C. aurantiacus strain 0k-70-fl. They rather found proteins with Mr values of 10, 15, 40, and 57 kDa only. An alternative organizational model is based on the finding that isolated BChl c molecules in vitro form aggregates whose spectroscopic properties in appropriate solvents match those of BChl c in vivo [10-14], Interestingly, Olson and coworkers [6] note that 3 proteins in the 10-20-kDa region are missing in SDS-treated BChl a-free chlorosomes of C. aurantiacus. We have recently found that the GEF procedure [4] either eliminates all proteins in the chlorosomes, or it reduces them to concentra tions which exclude their role as BChl obinding proteins [15, 16] as proposed by Wechsler et al. [7]. Estimates of the 5.6-kDa protein-to-BChl e ratio in our BChl a-free GEF chlorosomes indicate at least a 25-40-fold excess of BChl c [15, 16]. This constitutes the first biochemical evidence against relevant antenna functions by this protein and - together with other data [4, 17, 18] - provided evi dence in favour of chromophore-chromophore interactions as the main organizational principle in chlorosomes.