Purification, primary structure, and homology relationships of a chloroplast ribosomal protein

Abstract

A chloroplast ribosomal protein that showed immunological homology to Escherichia coli ribosomal protein L12 was purified from spinach (Spinacia oleracea) leaves and its primary structure was determined by manual micro Edman degradation. The protein is composed of 130 amino acid residues and has Mr 13,576. It shows structural features characteristic of the L12 proteins of eubacterial 70S ribosomes (e.g., identical amino acid residues in about 50% of the sequence) but no apparent homology to the L12-type proteins of eukaryotic cytoplasmic 80S ribosomes. The homology to eubacterial proteins is highest in the COOH-terminal region (70%) and low in the NH2-terminal region (<20%).Plant cells contain three types of ribosomes-namely, an 80S in the free cytoplasm, and two distinct 70S types in the plastids (e.g., chloroplast) and in mitochondria (1-3). The organelle ribosomes synthesize a subset of organelle-specific proteins which are encoded in the organelle DNA. The products of synthesis of the chloroplast ribosomes (which includes the large subunit of the abundant leaf protein ribulosebisphosphate carboxylase) contributes up to halfthe total protein in green leaves (4).Previous studies have shown that chloroplast ribosomes are similar to bacterial ribosomes in functional properties (3,5,6) and in the structure and organization of their RNA components (7,8). For example, the 16S rRNA ofmaize chloroplast (8) shows about 70% homology to Escherichia coli 16S rRNA; it also carries at the 3' end the conserved oligonucleotide stretch important in prokaryotic initiation. The chloroplast rRNA genes are located (two copies each) in the chloroplast DNA (7).Like bacterial ribosomes, chloroplast ribosomes contain between 50 and 60 ribosomal proteins (rproteins) (3). The rprotein genes are divided (unlike the rRNA genes) between the nuclear and chloroplast genomes (9), and therefore the coordinate synthesis of chloroplast rproteins depends on mechanisms (not yet elucidated) of intergenomic coregulation. The system of chloroplast rproteins and their genes is important and interesting but little work has been done in characterizing the individual components.In the present paper we report the isolation, properties, and primary structure ofa chloroplast rprotein. In terms ofsequence homology, it corresponds to the functionally important and phylogenetically conserved rprotein L12 (10), which is present in E. coli ribosomes in four copies (11) and which, as a tetramer, forms a morphologically distinct "stalk" on the 50S subunit (12). MATERIALS AND METHODSPreparation of Chloroplast Ribosomes. Because of the need to process large quantities of leaves, we developed a simplified procedure (based on refs. 13 and 14). Spinach (Spinacia oleracea) leaves (purchased at the central market) were cleaned and deveined, homogenized (2 liters of buffer per kg of leaves) in a Waring blender (two 10-sec runs), and filtered through cheesecloth (all operations at 0-4OC). The filtrate was centrifuged (1,200 x g, 15 min) and the pellet was washed ...

The Plastid Ribosomal Proteins

“…Plastid L12 gave a yield of almost 200 pmol, four times the highest yield for the other proteins recovered in good yield. The result supports the existence of four copies of L12/ plastid 50 S ribosomal subunit, as has been previously deduced (17). Several of the plastid 50 S subunit proteins, e.g.…”

“…3), consistent with the presence of four copies/50 S subunit, as has been previously deduced for plastid 70 S ribosome (17). In E. coli, this protein exists in two forms: L12, with free N terminus and L7, ␣-N-acetylated form, the sum of the two forms constituting four copies/50 S ribosomal subunit (51), and the ratio of the two forms altering during the bacterial growth cycle (52).…”

“…Alwaro) ribosomes were prepared as described previously (17). Purification of 70 S ribosomes and the 50 S subunits were done by zonal centrifugation (5000 A 260 units of ribosome/zonal rotor for 70 S purification and 3000 A 260 units of purified 70 S/zonal rotor for 50 S isolation) as described previously (18).…”

The Plastid Ribosomal Proteins

“…Alwaro) chloroplast ribosomes and zonal purifications of 70 and 30 S subunits (5000 A 260 units of crude ribosome/zonal rotor for 70 S purification and 3000 A 260 units of purified 70 S/zonal rotor for 30 S isolation) were done as described (15,18). The approximate conversion factor, 1 A 260 unit of 30 S ϭ 20 g of protein, was used for protein estimation.…”

Chloroplast ribosomal protein L15, like L1, L13 and L21, is significantly larger than its E. coli homologue

“…The results together with the locations of other immunologically crossreacting proteins [7,8] and L21 [2] are presented in Fig. 5.…”