What might the plastid do? 1 General functions of plastids

The function o f the malarial plastid organelle remains a mystery, as it does for all apicomplexans. In most plants and algae, the chloroplast has a unique cellular role: photosynthesis. However, conversion from an autotrophic lifestyle to a parasitic one has occurred several times during plant evolution. Some o f these plants have totally lost their photosynthetic genes but have retained their plastids in a reduced form (Wolfe et a l, 1992). Although the highly derived malarial plastid genome of P. falciparum most resembles that of the non-photosynthetic parasitic flowering plant

The deletion o f genes coding for the RNA polymerase subunits, some o f the tRNA genes and ORF470 in Epifagus, which have been retained throughout apicomplexans, implies their plastids carry out different cellular functions (Preiser et a l, 1995). Two functional Epifagus plastid-encoded genes which are absent from the malarial plastid genome are accD and clpP. The former encodes the plastid homologue o f the p subunit o f the carboxy transferase component o f E. coli acetyl-CoA carboxylase (Li and Cronan, 1992), catalysing the first step in fatty acid synthesis. The other gene, clpP, encodes the plastid homologue o f the ATP-dependent protease (ClpP) of E. coli (Goldberg, 1992).

Plastids are the site for cellular biosynthesis o f porphyrin (needed for haem biosynthesis), essential amino acids, essential fatty acids (Boyer et a l, 1989; Howe and Smith, 1991; Wallsgrove, 1991), tetrapyrroles, isoprenoids, pyrimidine nucleotides, vitamin B l, the reduction of nitrite, starch metabolism and glycolysis. At least some o f these functions make plastids indispensable to plants and algae, so perhaps this is the role o f the apicoplast too (Wilson et a l, 1996; Kohler et a l, 1997; McFadden et a l, 1997; McFadden and Waller, 1997). Although essential haem and amino acid biosynthesis in the apicoplast cannot be discounted, available evidence suggests these functions are fulfilled by other cytosolic pathways (Wilson et a l, 1996; Kohler et a l, 1997; Mcfadden et a l, 1997; Wilson and Williamson, 1997). In the case of haem synthesis for example, an alternative pathway is already used in the mitochondrion o f the malarial parasite (Surolia and Padmanaban, 1992). The cases for and against the apicoplast as a site of fatty acid and amino acid biosynthesis are given below.

1.5.2.2 Fatty acid biosynthesis?

As discussed in section 1.4.3.2, three candidate genes involved in fatty acid biosynthesis (acpP, fa b H and fabT) have been identified in the nuclear genome of both T. gondii and P. falciparum and carry characteristic plastid import N-terminal signal sequences. The product o f one o f these genes, acpP, has also been shown to localize to the apicoplast (Waller et a l, 1998). These genes are members o f the type II fatty acid synthase multi-enzyme complex (Cronan and Rock, 1996). This type of biosynthesis is widespread in bacteria but otherwise is restricted to the plastids of plants and algae. Within fatty acid biosynthesis, ACP (encoded by acpP) plays a

central role by holding the forming acyl chains. FabH and FabZ on the other hand are involved in the condensation (FabH) and dehydration (FabZ) steps o f acetyl addition during acyl chain elongation. Apicoplast fatty acid biosynthesis has been implicated further by the discovery o f another fatty acid synthase component in P. falciparum, fabF. This gene encodes P-ketoacyl-ACP synthase II (Cronan and Rock, 1996).

Lipid synthesis within the apicoplast seems probable following the identification o f these nuclear encoded, apicoplast-targeted genes (Waller et a l, 1998). However, it could merely be a housekeeping function for the organelle. Conversely, it could be a compartmentalized site o f fatty acid biosynthesis (Waller et a l, 1997). Hopkins et a l (1999), also suggested that the apicomplexan plastid could be the membrane lipid “factory” o f the parasite. It was hypothesized that the lipids synthesized in the apicoplast were budded off in vesicular form, between the inner and outer plastid membranes and transported to adjacent organelles via extensions of its surface.

1.5.2.3 Amino acid biosynthesis?

It has been speculated that the apicoplast harbours a shikimate pathway for essential amino acid biosynthesis (Roberts et a l, 1998). The shikimate pathway is essential in higher plants, bacteria and fungi, but is absent from mammals (Kishore and Shah, 1988). Roberts et a l (1998) found that in vitro growth o f T. gondii, P. falciparum and Cryptosporidium parvum was inhibited by the herbicide glyphospate,

which inhibits the shikimate pathway enzyme 5-endopyruvyl shikimate 3-phosphate synthase (Kishore and Shah, 1988). In T. gondii and P. falciparum the inhibition of growth following treatment with this herbicide was reversed by the addition of p- aminobenzoate. This suggested the shikimate pathway supplies folate precursors for parasite growth. In the shikimate pathway, eiythrose 4-phosphate and phosphophenol pyruvate are converted to chorismate in seven enzymatic steps. Chorismate is essential for the synthesis o f /?-aminobenzoate and folate and is also required for the synthesis o f ubiquinone and aromatic amino acids.

When a partial chorismate synthase sequence was identified from the T. gondii sequence database it had an amino acid identity of between 44 and 51% with other chorismate synthases. However, no N-terminal extensions were seen on the T. gondii or P. falciparum homologues (Roberts et a l, 1998) suggesting the product has a cytoplasmic location.

As the genomic databases for apicomplexan parasites increase, other nuclear encoded genes whose products are targeted to the plastid will be identified which could indicate still further functions.

1.5.2.4 Plastid encoded open reading frames

Sequence analysis o f the plastid genome o f Plasmodium sp. has not indicated a cellular role for the extrachromosomal plDNA. As mentioned previously, most o f its genes have been linked to gene expression (Wilson et a l, 1996). This system might be required for expression of one o f the open reading frames (ORFs) o f unknown function also present on the circle (Fig. 1.2). Mostly these are small, and it has been suggested they may code for additional very divergent ribosomal proteins (Wilson et a l, 1996). In P. falciparum, on the other hand, ORF470 codes for a large, reasonably well conserved protein (45-50% identity with other orthologues at the amino acid level) corresponding to the chloroplast geneyc/'24 (Wilson and Williamson, 1997).

1.6 Open reading fram e O RF 470 iycf24) 1.6.1 D istribution

y o f 24, specifies the last major gene to be designated a function on the vestigial plastid genome of P. falciparum. This ORF (1414bp; 471 amino acids) is encoded by other apicomplexan pathogens, for example Toxoplasma and Eimeria (Denny et a l, 1998) and lies downstream o f an LSU rRNA gene (Williamson et a l, 1994), (Fig. 1.2).

Although yc/"24 has a limited distribution in known plastids, orthologues (45- 55% amino acid identity) are found in a diverse array o f organisms. Some examples are shown in the sequence alignment in Figure 1.9. This shows the predicted amino acid sequences o f plastid encoded genes from two apicomplexan parasites {P. falciparum and T. gondii) alongside those fi*om the red alga, P. purpurea, the Gram

negative bacterium, E. coli, the cyanobacterium, Synechocystis PCC6803, and Mycobacterium leprae. The most marked conservation is observed towards the C- terminus.

y c flA is also found in the plastid genomes of two diatoms, Odontella sinensis and Skeletonema costatum, the cryptomonad Guillardia theta and other red algae including Cyanophora paradoxa and Cyanidium caldarium. Furthermore, it is widely distributed on the nuclear genome o f prokaryotes. These include the archaebacterium

Figure 1.9

An alignment of the hypothetical translation products of y c f 24,

Shown are the predicted amino acid sequences of plastid encoded genes from two apicomplexan parasites [Plasmodium falciparum^ Toxplasma gondii (partial)] and the red alga, Porphyra purpurea. Also included are sequences from the Gram negative bacterium {E. coli), the cyanobacterium {Synechocystis PCC6803) and Mycobacterium leprae (ppsl).

y c f 2 4 . e c o l i p p s l s y n e c h o c y s t i p o r p h y r a p la sm o d iu m t o x o . o r f y c f 2 4 . e c o l i p p s l s y n e c h o c y s t i p o r p h y r a p la sm o d iu m t o x o . o r f y c f 2 4 . e c o l i p p s l s y n e c h o c y s t i p o r p h y r a p la sm o d iu m t o x o . o r f y c f 2 4 . e c o l i 151 p p s l 151 VA |g b s y n e c h o c y s t i 151 p o r p h y r a 151 p la sm o d iu m 151 sisi; t o x o . o r f 151 LIL! y c f 2 4 . e c o l i p p s l s y n e c h o c y s t i p o r p h y r a p la sm o d iu m t o x o . o r f y c f 2 4 . e c o l i p p s l s y n e c h o c y s t i p o r p h y r a p la sm o d iu m t o x o . o r f y c f 2 4 . e c o l i p p s l s y n e c h o c y s t i p o r p h y r a p la sm o d iu m t o x o . o r f y c f 2 4 . e c o l i p p s l s y n e c h o c y s t i p o r p h y r a p la sm o d iu m t o x o . o r f y c f 2 4 . e c o l i 401 p p s l 401 s y n e c h o c y s t i 401 p o r p h y r a 401 p la sm o d iu m 401 FgB t o x o . o r f 401 y c f 2 4 . e c o l i 451 p p s l 451 s y n e c h o c y s t i 451 p o r p h y r a 451 p la sm o d iu m 451 t o x o . o r f 451 20 30 TDDVKTWTGG P

AP13L, LTQQQAIDSL GKYGYGWADS