New insights in the topology of the biosynthesis of 5-aminolevulinic acid

Abstract

The formation of 5-aminolevulinic acid (ALA) at the beginning of the pathway is the rate limiting step of tetrapyrrole biosynthesis and target of multiple timely and spatially organized control mechanisms. Recent discovery of a glutamyl-tRNA reductase-binding protein (GluTRBP), reveals a new insight in the topology of regulation of plant ALA biosynthesis.

Shortly after the first reports on the cellular organelles, spatial substructures of these classical compartments have been detected consisting of membranes, granules, globules, and soluble or more hydrophobic areas. In recent years, the advantages of spatial structuring inside each cellular compartment have been approved. Proteins acting in expression control, metabolism or signaling are not randomly distributed inside their hydrophilic or hydrophobic intracellular environment, but are subjected to additional organizing principles and are localized in defined subcellular compartments, so called subcompartments. There are well known examples of intracellular topology, including the nucleolus inside the nucleus or asymmetric accumulation of mRNA in the cytoplasm in close proximity to the organelles, in which the synthesized proteins are imported,^1-3 or the spatial enrichment of glycolytic enzymes in cytoplasmatic zones nearby mitochondrial membranes for preferential repartitioning of substrates into the mitochondrial respiratory pathway.⁴

The identification of a novel protein binding glutamyl-tRNA reductase (GluTR) opened the view on the spatial organization of the tetrapyrrole biosynthetic pathway that enables metabolic channeling within this pathway.⁵ Plant tetrapyrrole biosynthesis is located in plastids and starting with tRNA^Glu up to 20 subsequent enzymatic steps are passed through the branched pathway before end products chlorophyll (Chl), heme, phytochromobiline and siroheme are finally assembled with apoproteins. It is generally accepted that all enzymatic steps of the tetrapyrrole metabolism leading to protoporphyrinogen IX are located in the stroma of plastids, while the successive proteins are located in the plastid membranes. This view was substantiated by proteomic approaches which identified proteins of tetrapyrrole biosynthesis in both, the thylakoid and envelope membranes.⁶

The GluTR-binding protein (GluTRBP) was recently introduced as a new component of 5-aminolevulinic acid (ALA) synthesis. GluTR is the first committed enzyme in tetrapyrrole biosynthesis reducing the activated tRNA-bound glutamate to glutamate-1-semialdehyde, which is subsequently transaminated by glutamate-1-semialdehyde aminotransferase (GSAT) to form ALA. As previously proposed ALA formation is the rate limiting step of tetrapyrrole biosynthesis and temporally controlled by GluTR expression. GluTRBP was identified by a yeast-two-hybrid screen for GluTR-interacting proteins and the physical interaction of GluTR and GluTRBP was confirmed by multiple techniques, such as the bimolecular fluorescence complementation assays in transgenic tobacco plants, the surface plasmon resonance technique and protein pull-down experiments. GluTR was known to be localized in the stroma, but GluTRBP is a thylakoid membrane-associated protein and binds a minor portion of GluTR at the membrane fraction of chloroplasts in order to separate a small amount of GluTR from bulk soluble GluTR.⁵ The confirmation of the GluTR-GluTRBP interaction was important as, to the displeasure of the authors, the proof of a putative function of GluTRBP proved to be rather complex. Enzyme assays using recombinant Chlamydomonas proteins revealed that the presence or absence of GluTRBP did not affect catalytic activities of GluTR or GSAT. Attempts to analyze the role of GluTRBP in transgenic tobacco and Arabidopsis knockout or RNAi lines failed and were hampered by an embryo/seedling-lethal phenotype. However, Arabidopsis GluTRBP-antisense lines showing a weak reduction in GluTRBP levels were available to analyze their tetrapyrrole metabolism.

Fortunately, an Arabidopsis pgr7 mutant was published, which was compromised in photosynthesis.⁷ The pgr7 mutant has a frame shift mutation at the 3′ end of the GluTRBP coding sequence, which likely results in the synthesis of a truncated protein. The most obvious tetrapyrrole biosynthesis-related phenotypes of both, pgr7 and GluTRBP antisense lines were a consistently reduced heme content, most likely as a result of partially impaired heme synthesis, and in course of seedling development a slightly lower Chl content.⁵ It was concluded that the strong binding capacity of GluTR to its membrane-bound binding protein ensures its tight association to thylakoid membranes enabling a spatial separation of ALA formation with potential implications for simultaneously discrete metabolic controls.

The spatial arrangement of certain amounts of GluTR in the thylakoid membranes via GluTRBP is interpreted as a principle of structuring ALA synthesis in a soluble stroma and, to a smaller extent, in a membrane-associated compartment. It was proposed that the partitioning of ALA in two pools allows a parallel and independent control of ALA synthesis dedicated to either Chl or heme formation.

There are good reasons for two synchronous control mechanisms of ALA synthesis in plants. In angiosperms, ALA biosynthesis is repressed during darkness, to prevent excessive accumulation of the Chl precursor protochlorophyllide (Pchlide). Pchlide oxidoreductase can reduce Pchlide only by a light-driven catalytic mechanism using its own substrate to trigger the enzymatic reaction.⁸ However, ALA molecules are still required as precursors for heme synthesis during dark periods.

FLU, another GluTR binding protein, was introduced as a negative regulator of ALA synthesis, which was discovered in an Arabidopsis mutant screen for Pchlide accumulating etiolated seedlings.⁹ Light-dark grown flu seedlings sustain irreversible photooxidative damages caused by Pchlide that accumulated in darkness. FLU is also attached to thylakoid membranes, but both proteins, FLU and GluTRBP do not interact in planta (data not shown) and seem to be spatially separated within the plastid membranes. It is currently postulated that binding of GluTR to GluTRBP prevents a complete dark-repression of ALA synthesis operated by FLU (Fig. 1). Parallel actions of both binding proteins lead to inactivation of most GluTR proteins by interacting with FLU (in darkness), while minor GluTR amounts interact with GluTRBP and are protected from FLU-inactivation (Fig. 1). The two sites of ALA synthesis, in the stroma and in close proximity to GluTRBP, respectively, do not require additional spatial separation of the consecutive enzymes of porphyrin synthesis, as ALA once synthesized is directed toward the subsequent enzymes. At the branch point of Chl and heme synthesis, additional regulatory mechanisms distribute the metabolites to both branches of tetrapyrrole biosynthesis depending of demands during any time of the day.

Figure 1. Spatial organization of 5-aminolevulinic acid (ALA) formation in chloroplasts. Majority of a glutamyl-tRNA reductase (GluTR) and glutamate-1 semialdehyde aminotransferase (GSAT) protein complex is located in the stroma and forms ALA starting with glutamyl-tRNA^Glu, while a minor part of the active protein complex is attached to the thylakoid membrane via a GluTR-binding protein (GluTRBP). At night the FLU protein binds the soluble GluTR fraction to the thylakoid membrane and thereby inactivates ALA formation. Only the GluTRBP bound fraction of GluTR can continue to synthesize ALA during dark periods, preventing both a lack of heme during darkness and excessive accumulation of phototoxic intermediates of chlorophyll biosynthesis.

GluTRBP is tightly bound to chloroplast membranes, but an obvious transmembrane domain is missing in its structure.⁵ We propose an additional anchor protein stabilizing the association of GluTRBP to the thylakoid membranes. Then, GluTRBP acts as a localization protein that conveys GluTR away from the FLU protein.

Apart from the details in guidance and localization in different substructures of thylakoid membranes, a number of other questions remain open:

• What is the mechanism of GluTRBP to scaffold GluTR? Are additional proteins assembled?

• What is the function of GluTRBP in gymnosperms and green algae, where a strict suppression of Chl biosynthesis in darkness is not destined as a light independent Pchlide reduction facilitates chlorophyll synthesis in darkness?

• Are there additional indications for spatial organizations of the tetrapyrrole biosynthetic pathway?

• Are there additional indications in living organisms for spatial arrangements and organization of metabolic pathways in substructures?

Several other protein-protein interactions among enzymes of the tetrapyrrole biosynthetic pathway have been reported or seem likely.^8,10-13 Recently, the LIL3 protein, a member of the light-harvesting protein family, has been reported to interact with and to stabilize geranylgeranyl reductase.¹⁴ Additionally, LIL3 containing protein complexes accumulate Pchlide in plastid membranes of de-etiolated barley leaves.¹⁵ Future work will clarify, whether LIL3 is involved in association of different enzymatic steps of late Chl synthesis, including the synthesis of the long hydrophobic phytyl chain or Pchlide reduction, and integration of Chl synthesis with the assembly of pigments and chlorophyll-binding proteins of the photosynthetic complexes.

Metabolic activities in cells benefit from evolved spatial organization, including multiple enzyme complexes, membrane engulfed vesicles or subcompartments. These quaternary structures facilitate substrate channeling, prevent diffusion and loss of metabolites to competing pathways, and protect labile intermediates. Here, a first structural component for the organization of ALA synthesis is reviewed that contributes to controlled activity of ALA biosynthesis. Future experiments will explore the effectiveness of these structuring principles and the contribution of scaffold, anchor or escort proteins involved in the organization of the metabolic flow.

Glossary

Abbreviations:

ALA

5-aminolevulinic acid

Chl

chlorophyll

GluTR

glutamyl-tRNA reductase

GluTRBP

GluTR binding protein

GSAT

glutamate-1-semialdehyde aminotransferase

Pchlide

protochlorophyllide

Czarnecki O, Hedtke B, Melzer M, Rothbart M, Richter A, Schröter Y, et al. An Arabidopsis GluTR binding protein mediates spatial separation of 5-aminolevulinic acid synthesis in chloroplasts. Plant Cell. 2011;23:4476–91. doi: 10.1105/tpc.111.086421.