Molecular basis for semidominance of missense mutations in the XANTHA-H (42-kDa) subunit of magnesium chelatase

Abstract

During biosynthesis of bacteriochlorophyll or chlorophyll, three protein subunits of 140, 70, and 42 kDa interact to insert Mg2+ into protoporphyrin IX. The semidominant Chlorina-125, -157, and -161 mutants in barley are deficient in this step and accumulate protoporphyrin IX after feeding on 5-aminolevulinate. Chlorina-125, -157, and -161 are allelic to the recessive xantha-h mutants and contain G559A, G806A, and C271T mutations, respectively. These mutations cause single amino acid substitutions in residues that are conserved in all known primary structures of the 42-kDa subunit. In vitro complementation and reconstitution of Mg-chelatase activity show that the 42-kDa subunits are defective in the semidominant Chlorina mutants. A mutated protein is maintained in the Chlorina plastids, unlike in the xantha-h plastids. Heterozygous Chlorina seedlings have 25-50% of the Mg-chelatase activity of wild-type seedlings. Codominant expression of active and inactive 42-kDa subunits in heterozygous Chlorina seedlings is likely to produce two types of heterodimers between the strongly interacting 42-kDa and 70-kDa subunits. Reduced Mg-chelatase activity is explained by the capacity of heterodimers consisting of mutated 42-kDa and wild-type 70-kDa protein to bind to the 140-kDa subunit. The 42-kDa subunit is similar to chaperones that refold denatured polypeptides with respect to its ATP-to-ADP exchange activity and its ability to generate ATPase activity with the 70-kDa subunit. We hypothesize that the association of the 42-kDa subunit with the 70-kDa subunit allows them to form a specific complex with the 140-kDa subunit and that this complex inserts Mg2+ into protoporphyrin IX.

Figure 1

Leaves (6 days old) obtained from the seeds of a heterozygous barley Chlorina mutant. Because of the semidominant behavior of the Chlorina mutation, the leaves segregate into a phenotypic ratio of one normal green wild-type to two light-green heterozygotes to one yellow, chlorophyll-lacking homozygote. In this spike, the ratio is 3 normal green to 13 light-green to 5 yellow.

Figure 2

Polypeptide sequence alignment of 42-kDa subunits of Mg-chelatase. The barley sequence and 5 of the 14 sequences available in the GenBank Database: Hvu (Hordeum vulgare; barley; GenBank accession no. U26545), Gma (Glycine max; soybean; D45857), Nta (N. tabacum; tobacco; AF014053), Ath (Arabidopsis thaliana; thale cress; X91411), Rca (Rhodobacter capsulatus; Z11165), and Rsp [R. sphaeroides; AF017642 (AJ001690)]. The N-terminal sequence of the soybean, tobacco, and thale cress polypeptides is the same as that of the unprocessed proteins. Conserved amino acid residues in all 14 sequences are marked by asterisks. The species of origin of the 42-kDa subunits (not displayed) are Porphyra purpurea (U38804), Odontella sinensis (Z67753), Cryptomonas phi (Z21976), Anabaena variabilis (D49426), Synechocystis PCC6803 (U35144), Olisthodiscus luteus (Z21959), Chlorobium vibrioforme (Z83933), and Heliobacillus mobilis (AF080002). Arrows indicate the amino acid residues changed in mutants: L91F, D187N, and R269K. The tobacco Sulfur mutation is N269I.

Figure 3

Western blot analysis of the barley Mg-chelatase subunits XANTHA-H (Top), XANTHA-F (Middle), and XANTHA-G (Bottom). Lanes: 1, Chlorina-125; 2, Chlorina-157; 3, Chlorina-161; 4, xantha-h⁵⁷; 5, xantha-g²⁸; 6, xantha-f¹⁰; 7, wild-type chloroplast high-speed supernatant; and 8, wild-type chloroplast high-speed pellet. The molecular masses of the standard bands labeled in lanes 7 and 8 are 42 kDa for H, 150 kDa for F, and 76 kDa for G.