Enzymes of the heme biosynthetic pathway in the nonphotosynthetic alga Polytomella sp

Abstract

Heme biosynthesis involves a number of enzymatic steps which in eukaryotes take place in different cell compartments. Enzyme compartmentalization differs between photosynthetic and nonphotosynthetic eukaryotes. Here we investigated the structures and subcellular localizations of three enzymes involved in the heme pathway in Polytomella sp., a colorless alga evolutionarily related to the green alga Chlamydomonas reinhardtii. Functional complementation of Escherichia coli mutant strains was used to isolate cDNAs encoding three heme biosynthetic enzymes, glutamate-1-semialdehyde aminotransferase, protoporphyrinogen IX oxidase, and ferrochelatase. All three proteins show highest similarity to their counterparts in photosynthetic organisms, including C. reinhardtii. All three proteins have N-terminal extensions suggestive of intracellular targeting, and immunoblot studies indicate their enrichment in a dense cell fraction that is enriched in amyloplasts. These results suggest that even though the plastids of Polytomella sp. are not photosynthetically active, they are the major site of heme biosynthesis. The presence of a gene for glutamate-1-semialdehyde aminotransferase suggests that Polytomella sp. uses the five-carbon pathway for synthesis of the heme precursor 5-aminolevulinic acid.

FIG. 1.

Sequence alignments of the N termini of predicted GSAT, PPO, and FeC of Polytomella sp. with representative prokaryotic and eukaryotic counterparts. The full-length, unprocessed eukaryotic precursors are shown. The alignments were generated using the CLUSTALW algorithm and refined manually. Residues that are similar or identical are shaded by gray or black, respectively; photosynthetic organisms are indicated in bold; and ↓ indicates the N termini of the mature proteins, predicted on the basis of the start of similarities with eukaryotic and prokaryotic sequences. (A) ARATH, Arabidopsis thaliana GSAT-II (GenBank accession number Q42522); CHLRE, C. reinhardtii (Q39566); CHLTE, Chlorobium tepidum TLS (NP_662973); ECOLI, E. coli (P23893); POLSP, Polytomella sp. (AY152854); THEEL, Thermosynechococcus elongatus BP-1 (Q8DLK8); TOBAC, N. tabacum (P31593). (B) ARATH-I, A. thaliana PPO-I (P55826); BACSU, Bacillus subtilis (P32397); CHLRE, C. reinhardtii (Q9ZTA7); HUMAN, Homo sapiens (P50336); POLSP, Polytomella sp. (AF332964); SOLTU, Solanum tuberosum (O64384); THEEL, T. elongatus BP-1 (Q8DLV2); TOBAC-I, N. tabacum PPO-I (O24163); TOBAC-II, N. tabacum PPO-II (O24164); YEAST, S. cerevisiae (P40012). (C) ARATH-I, A. thaliana FeC-I (P42043); ARATH-II, A. thaliana FeC-II (O04921); BACSU, B. subtilis (P32396); CHLRE, C. reinhardtii (Q9ATG8); ECOLI, E. coli (P23871); HUMAN, H. sapiens (P22830); POLSP, Polytomella sp. (AAK16729); SOLTU, S. tuberosum (O64391); THEEL, T. elongatus BP-1 (Q8DLV2); YEAST, S. cerevisiae (P16622).

FIG. 2.

Southern blot analysis of GSAT, PPO, and FeC in Polytomella sp. Total DNA (20 μg) was digested with restriction enzymes and subjected to DNA blot analysis. Hybridization was performed using Polytomella sp. predicted ORFs for GSAT, PPO, and FeC. Positions of size markers (kbp) are indicated at the left of each blot. GSAT cDNA contains one restriction site for AvaI, one for BamHI, and none for PstI; PPO cDNA contains three restriction sites for AvaI, none for NcoI, and two for SalI; and FeC cDNA contains one restriction site for BamHI, none for HindIII, and two for NcoI.

FIG. 3.

Immunoblots identifying the enzymes involved in the heme biosynthetic pathway in Polytomella sp. Protein samples were separated by 12% (wt/vol) SDS-PAGE and transferred to nitrocellulose membranes. Immunodetection was performed with specific polyclonal antibodies produced in this work. (A) Coomassie blue-stained SDS-PAGE loaded with cell extracts (40 μg each) from Polytomella sp. (Ps) and C. reinhardtii (Cr). (B) Immunodetection of GSAT in cell extracts (40 μg each). (C) Immunodetection of PPO in cell extracts (40 μg). (D) Immunodetection of FeC in Polytomella sp. cell extracts (100 μg) in the presence of 1 mM β-mercaptoethanol (βme) or 50 mM dithiothreitol (DTT). Positions of molecular mass markers (kDa) are indicated at the left of the gel and blots.

FIG. 4.

Distribution of GSAT, PPO, and FeC between membrane and soluble fractions of Polytomella sp. Whole Polytomella sp. cells grown on acetate as the sole carbon source were sonicated and fractionated into their membrane and soluble components. Proteins in cell fractions were electrophoresed on a 12% (wt/vol) SDS-PAGE gel and transferred to nitrocellulose membranes. (A) SDS-PAGE gel stained with Coomassie blue. (B) Immunoblot analysis with the indicated antibody probes. Ce, whole cells; Sol, soluble fraction; Mb, membrane fraction. Positions of molecular mass markers (kDa) are indicated at the left of the gel and blots.

FIG. 5.

Subcellular localization of GSAT, PPO, and FeC in Polytomella sp. Proteins in cell fractions were electrophoresed on a 12% (wt/vol) SDS-PAGE gel and transferred to nitrocellulose membranes. (A) SDS-PAGE gel stained with Coomassie blue. (B) Immunoblot analysis with the indicated antibody probes. Samples were as follows: extract of whole cells (Ce) (50 μg), a fraction enriched in amyloplasts (P1, 50 μg), and fractions enriched in mitochondria (P2, 50 μg, and P3, 75 μg). Positions of molecular mass markers (kDa) are indicated at the left of the gel and blots.

FIG. 6.

Bootstrapped neighbor-joined rootless trees showing the relatedness of GSATs from various sources. For the eukaryotic enzymes, full-length preproteins were used in the analyses. Bootstrap values above 50 are shown at the nodes. The bar labeled “0.1” is the branch length representing a mean difference of 0.1 per residue along each branch. Photosynthetic organisms are shaded as follows: dark gray, eukaryotes; medium gray, cyanobacteria; light gray, chlorobia. ARATH1, Arabidopsis thaliana GSAT-I (GenBank accession number P42799); ARATH2, A. thaliana GSAT-II (Q42522); ARCGL, Archaeoglobus fulgidus DSM 4304 (AAB90001); BACSU, Bacillus subtilis (NP_390690); BIGNA, Bigelowiella natans (Q7XYK0); BRANA, Brassica napus (Q85WB7); CHLRE, C. reinhardtii (Q39566); CHLTE, Chlorobium tepidum TLS (NP_662973); CORDI, Corynebacterium diphtheriae (Q6NJJ2); DESHA, Desulfitobacterium hafniense DCB-2 (ZP_00098839); DESPS, Desulfotalea psychrophila LSv54 (YP_064548); ECOLI, E. coli (P23893); GEOSU, Geobacter sulfurreducens PCA (NP_951397); HELPY, Helicobacter pylori (P56115); METBA, Methanosarcina barkeri (ZP_00297199); METJA, Methanocaldococcus jannaschii (C64375); NOSPU, Nostoc punctiforme PCC 73102 (ZP_00111760); OCEIH, Oceanobacillus iheyensis HTE831 (NP_692986); POLSP, Polytomella sp. Pringsheim 198.80 (AAN74531); PROAE, Prosthecochloris aestuarii DSM 271 (ZP_00591188); PROMA, Prochlorococcus marinus (NP_874875); RALME, Ralstonia metallidurans CH34 (ZP_00275477); SALTY, Salmonella enterica (NP_804085); SOYBN, Glycine max (P45621); STRCO, Streptomyces coelicolor (Q9F2S0); SULSO, Sulfolobus solfataricus (Q980U5); THEEL, Thermosynechococcus elongatus BP-1 (Q8DLK8); THIDE, Thiobacillus denitrificans ATCC 25259 (ZP_00334465); TOBAC, N. tabacum (P31593); VIBVU, Vibrio vulnificus YJ016 (Q7MHY9); WOLSU, Wolinella succinogenes (Q7M847).

FIG. 7.

Bootstrapped neighbor-joined rootless trees showing the relatedness of PPO (A) and FeC (B) enzymes from various sources. For the eukaryotic enzymes, full-length preproteins were used in the analyses. Bootstrap values above 50 are shown at the nodes. The bar labeled “0.1” is the branch length representing a mean difference of 0.1 per residue along each branch. Photosynthetic organisms are shaded as described in the legend for Fig. 6. (A) ARATH-I, Arabidopsis thaliana (GenBank accession number P55826); BACSU, Bacillus subtilis (P32397); CHLRE, C. reinhardtii (Q9ZTA7); CHLTE, Chlorobium tepidum (Q8KB91); CORDI, Corynebacterium diphtheriae (Q6NJJ3); CRYNE, Cryptococcus neoformans (Q5KDI9); DICDI, Dictyostelium discoideum (Q54DT8); ECOLI, E. coli (P27863); HELPY, Helicobacter pylori (O25143); HUMAN, Homo sapiens (P50336); MOUSE, Mus musculus (P51175); OCEIH, Oceanobacillus iheyensis HTE831 (NP_692090); PLAFA, P. falciparum isolate 3D7 (Q8IJC3); POLSP, Polytomella sp. (Q9ATG6); PROAE, Prosthecochloris aestuarii DSM 271 (ZP_00591000); PROFR, Propionibacterium freudenreichii (O32434); RICPR, Rickettsia prowazekii strain Madrid E (NP_221195); SALTY, Salmonella enterica subsp. enterica serovar Typhi (CAD07906); SCHPO, Schizosaccharomyces pombe (P40012); SOLTU, Solanum tuberosum (O64384); SPIOL, Spinacia oleracea (Q94IG7); STRCO, Streptomyces coelicolor (Q8CJP6); THEEL, Thermosynechococcus elongatus (Q8DLV2); TOBAC-I, N. tabacum PPOI (O24163); TOBAC-II, N. tabacum PPOII (O24164); TRIER, Trichodesmium erythraeum IMS101 (ZP_00325289); VIBVU, Vibrio vulnificus YJ016 (NP_932826); WOLSU, Wolinella succinogenes (Q7MAI5); YEAST, S. cerevisiae (P40012). (B) ARATH-I, A. thaliana HEMH1 (P42043); ARATH-II, A. thaliana HEMH2 (O04921); BACSU, B. subtilis (P32396); CHLRE, C. reinhardtii (Q9ATG8); CHLTE, C. tepidum (Q8KEC6); CORDI, C. diphtheriae (Q6NH66); CUCSA-I, Cucumis sativus (Q9FEK8); CUCSA-II, C. sativus (P42044); DESHA, Desulfitobacterium hafniense DCB-2 (ZP_00102659); DICDI, D. discoideum (Q54IA8); ECOLI, (P23871); HELPY, H. pylori (P56107); HUMAN, H. sapiens (P22830); MOUSE, M. musculus (P22315); NEUCR, Neurospora crassa (Q7SA94); OCEIH, O. iheyensis (Q8ERX9); ORYZA-I, Oryza sativa (Q69TB1); PLAFA, P. falciparum (Q8IFR0); POLSP, Polytomella sp. (AAK16729); PROAE, P. aestuarii DSM 271 (ZP_00591682); PROFR, P. freudenreichii subsp. shermanii (P72183); PROMA, Prochlorococcus marinus (Q7VD58); RICPR, R. prowazekii (Q9ZC84); SALTY, S. enterica serovar Typhimurium (P37408); SCHPO, S. pombe (O59786); SOLTU, S. tuberosum (O64391); STRCO, S. coelicolor (O50533); SYNPX, Synechococcus sp. WH8102 (Q7U5G0); THEEL, T. elongatus (Q8DGU6); VIBVU, V. vulnificus (Q8DFM2); WOLSU, W. succinogenes (Q7M7P9); YEAST, S. cerevisiae (P16622).