Protease-stable integration of Lhcb1 into thylakoid membranes is dependenton chlorophyll b in allelic chlorina-f2 mutants of barley (Hordeum vulgareL.)

Allelic chlorina-f2 mutants of barley (Hordeum vulgare L.) growing under di fferent light and temperature conditions demonstrated that the chlorophyll b-free chlorina-f2(f2) and chlorina-f2(101) express a stable phenotype. Onl y 3 out of 10 light-harvesting chlorophyll alb-binding proteins, Lhca4 (pho tosystem I), and Lhcb1 and Lhcb6 (photosystem II), required chlorophyll b f or accumulation. The other light-harvesting proteins were found in all chlo rina-f2 alleles, indicating that the integration pathway of these proteins into mutant thylakoid membranes was not affected. Chlorina-f2 alleles with a thylakoid membrane capable of fullfilling photosynthesis and transport de mands, but with various amounts of chlorophyll b: chlorina-f2(101) (chlorop hyll b-free), chlorina-f2(123) (27% of chlorophyll b compared with the wild type) and chlorina-f2(122) (70% chlorophyll b), were chosen to investigate whether chlorophyll b is necessary for the protease-stable insertion of Lh cb 1 into mutant thylakoid membranes. The Lhcb1 was affected in almost all alleles and was most sensitive to chlorophyll b deficiency. The Lhcb1 antib ody confirmed the heterogeneity of the polypeptides of the light-harvesting chlorophyll alb-binding protein II (LHCII) and detected in wild-type membr anes, two protease-resistant, mature forms of Lhcb 1 with apparent molecula r masses of 28 and 29 kDa. Only one band reacting with the Lhcb1 antibody c ould be detected in chlorophyll b-free chlorinaf2(f2). It co-migrated with the 29-kDa band, but was completely digested after treatment of the isolate d mutant membranes with exogenous protease. This showed that in chlorina-f2 (f2) the Lhcb1 precursor was processed at one cleavage site only. The resul ting 29-kDa Lhcb1 was not provided with chlorophyll b, and, consequently, n ot properly folded and inserted into the membrane. It remained susceptible to protease and was inconvertable to a 28-kDa form.