Sub-plastidial localization of two different phage-type RNA polymerases in spinach chloroplasts

Abstract

Plant plastids contain a circular genome of approximately 150 kb organized into approximately 35 transcription units. The plastid genome is organized into nucleoids and attached to plastid membranes. This relatively small genome is transcribed by at least two different RNA polymerases, one being of the prokaryotic type and plastid-encoded (PEP), the other one being of the phage-type and nucleus-encoded (NEP). The presumed localization of a second phage-type RNA polymerase in plastids is still questionable. There is strong evidence for a sequential action of NEP and PEP enzymes during plant development attributing a prevailing role of NEP during early plant and plastid development, although NEP is present in mature chloroplasts. In the present paper, we have analysed two different NEP enzymes from spinach with respect to subcellular and intra-plastidial localization in mature chloroplasts with the help of specific antibodies. Results show the presence of the two different NEP enzymes in mature chloroplasts. Both enzymes are entirely membrane bound but, unlike previously thought, this membrane binding is not mediated via DNA. This finding indicates that NEP enzymes are not found as elongating transcription complexes on the template DNA in mature chloroplasts and raises the question of their function in mature chloroplasts.

Figure 1

Sequence analyses of the two spinach plastid phage-type RNA polymerases. (A) Phylogenetic analysis of the two spinach enzymes. The alignment that has been used for tree construction is shown in Supplementary Figure 1S. The parsimony tree is shown including bootstrap confidence values obtained from parsimony (upper values) and neighbour-joining (lower values) analyses. (B) Conservation of functionally important amino acids and motifs in the two spinach phage-type RNA polymerases. Numbering on the top of the conserved sequence blocks indicate the position of the corresponding amino acid and annotations below the sequence blocks indicate the name of the corresponding motif in the T7 RNA polymerase sequence. Abbreviations and accession nos are At (A.thaliana, Y08463, Y08137, AJ001037), Ca (Chenopodium album, Y08067), Hv (Hordeum vulgare, HVU507395, AJ586899), Ns (Nicotiana sylvestris, AJ302020, AJ302019, AJ416568), Pp (P.patens, AJ416854, AJ416855), So (Spinacia oleracea, Y18852, Y18853), Ta (Triticum aestivum, U34402, AF091838), T7 (T7 RNA polymerase, AAB28111) and Zm (Zea mays, AF127022, AJ005343). The Cm (C.merolae) sequence has been obtained from the entry of the Cyanidioschyzon project page (CMJ257C) and the sequence has not yet been experimentally verified.

Figure 2

Characterization of RPOTp and RPOTmp specific antibodies. (A) Alignment of the N-terminal parts of the Arabidopsis and spinach RPOTmp and RPOTp amino acid sequences. Predicted target sequence cleavage sites are indicated by closed (TargetP) or open (ChloroP) triangles. In the case of SoRPOTmp and SoRPOTp, Target P and ChloroP indicate the same cleavage sites. Peptides that have been used for production of specific antibodies are boxed. (B) The antisera (lane 2) and the pre-immune sera (lane 1) of the RPOTmp (left panel) and RPOTp enzymes (right panel) have been analysed by immunoblotting using 40 µg of total spinach protein extracts. (C) The specificity of the RPOTmp and RPOTp antibodies has been verified by dot blot analyses on all three peptides. 5, 50, 100, 150 and 200 ng (lanes 1–5, respectively) of peptides (indicated as RPOTp, RPOTmp2 and RPOTmp1 on the left panel of the figure) have been spotted onto nitrocellulose filter and the antibody cross-reactions have been tested using purified antibodies, i.e. peptide specific IgG fractions of the RPOTmp (left) and RPOTp (right) antibodies.

Figure 3

Both phage-type RNA polymerases, RPOTmp and RPOTp, are localized exclusively in plastids. Peptide specific IgG fractions have been obtained from the RPOTp and RPOTmp antisera as described in Materials and Methods. Equal amounts (40 µg) of total protein extracts (lanes 1 and 4), purified chloroplasts (lanes 2 and 5) and purified mitochondria (lanes 3 and 6) have been separated by PAGE. After transfer to nitrocellulose membranes the proteins have been stained by Ponceau S (lanes 1–3) or have been analysed by immunoblotting using antisera prepared against RPOTp, RPOTmp, ProteinT and KARI (lanes 4–6).

Figure 4

RPOTmp and RPOTp are bound to plastid thylakoid and envelope membranes. Aliquots (30 µg) of total chloroplast proteins (P), soluble chloroplast proteins (S), proteins obtained from purified thylakoid (Tk) and envelope (E) membranes have been separated by SDS–PAGE and transferred to nitrocellulose membranes. Membranes were analysed either (A) by staining with Coomassie (left panel) or by antibodies raised against plastid inner envelope protein IE37 (IE 37) plastid ribosomal protein L4 (L4) and plastid terminal oxidase (PTOX) (right panel) or (B) by purified anti-RPOTmp IgG (Figure 3B, left panel) and by purified anti-RPOTp IgG fractions (Figure 3B, right panel). (C) Aliquots of purified intact chloroplasts have been treated either with buffer (Co, lanes 1 and 2) or with different concentrations of thermolysin (lanes 3–12) in the absence (lanes 3–10) or in the presence (lanes 11 and 12) of EGTA (Materials and Methods). After purification of thylakoid and envelope fractions equal amounts of protein (30 and 15 µg, respectively) have been analysed by immunoblotting using antibodies made against RPOTp, RPOTmp and outer envelope protein 24 (OE24).

Figure 5

DNase treatment of purified plastid membrane fractions. (A) Schematic representation of the experiment. (B) Aliquots of fraction M1 corresponding to 60 µg of chlorophyll (A) have been re-suspended in low salt buffer. The solubilized membranes have been kept at room temperature for 1 h in the absence (−DNase, lanes 1 and 2) or presence (+DNase) of 7.5 µg (lanes 3 and 4), 15 µg (lanes 5 and 6) or 30 µg (lanes 7 and 8) of DNase before separating into membrane (M) and soluble (S) fractions by centrifugation. After separation of proteins by SDS–PAGE all fractions have been analysed using purified RPOTmp and RPOTp antibodies and antibodies made against the soluble plastid KARI and the membrane bound plastid terminal oxidase (PTOX). (C) DNase activity was verified by analysing the membrane fractions [M1, M3, M5 and M7, (A)] without (−, lane 1) and after DNase treatment (7.5, 15 and 30 µg of DNase, lanes 2–4, respectively) on agarose gels and staining the DNA by ethidium bromide.