Diatom plastids depend on nucleotide import from the cytosol

Abstract

Diatoms are ecologically important algae that acquired their plastids by secondary endosymbiosis, resulting in a more complex cell structure and an altered distribution of metabolic pathways when compared with organisms with primary plastids. Diatom plastids are surrounded by 4 membranes; the outermost membrane is continuous with the endoplasmic reticulum. Genome analyses suggest that nucleotide biosynthesis is, in contrast to higher plants, not located in the plastid, but in the cytosol. As a consequence, nucleotides have to be imported into the organelle. However, the mechanism of nucleotide entry into the complex plastid is unknown. We identified a high number of putative nucleotide transporters (NTTs) in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum and characterized the first 2 isoforms (NTT1 and NTT2). GFP-based localization studies revealed that both investigated NTTs are targeted to the plastid membranes, and that NTT1 most likely enters the innermost plastid envelope via the stroma. Heterologously expressed NTT1 acts as a proton-dependent adenine nucleotide importer, whereas NTT2 facilitates the counter exchange of (deoxy-)nucleoside triphosphates. Therefore, these transporters functionally resemble NTTs from obligate intracellular bacteria with an impaired nucleotide metabolism rather than ATP/ADP exchanging NTTs from primary plastids. We suggest that diatoms harbor a specifically-adapted nucleotide transport system and that NTTs are the key players in nucleotide supply to the complex plastid.

Fig. 1.

Uptake of α³²P-labeled nucleotides into E. coli cells expressing diatom NTTs. Net import of [α³²P] nucleotides was calculated by subtraction of the corresponding control values (import into noninduced E. coli cells). The highest net value for each imported nucleotide was set to 100%, and the remaining uptake rates were calculated as percentage of this maximal transport. Data are the mean of at least 3 independent experiments. Standard errors are given. (A) Adenine nucleotide import by TpNTT1 (dark gray bars) and PtNTT1 (light gray bars). (B) Nucleotide and deoxynucleotide import by TpNTT2 (dark gray bars) and PtNTT2 (light gray bars).

Fig. 2.

Analysis of the proton dependency of diatom NTTs. Effect of the protonophore CCCP on [α³²P] ATP uptake by TpNTT1 (black bars), PtNTT1 (dark gray bars), TpNTT2 (gray bars), and PtNTT2 (light gray bars). Rates of nucleotide uptake are given as percentage of the control rates (nonaffected transport = 100%).

Fig. 3.

Analysis of the cellular localization of diatom NTTs. Cellular targeting of GFP fused to the NTT presequences or the full-length NTTs. Selected merged images (GFP fluorescence in green, chlorophyll autofluorescence in red, and Nomarski differential interference contrast in white) are displayed. See Fig. S4 for additional information. (Scale bars: 5 μm.)

Fig. 4.

Phylogenetic relationships of diatom NTTs with known bacterial and plastidial NTTs. An amino acid-based phylogenetic tree calculated by using the TREE-PUZZLE algorithm is shown. Black circles indicate nodes, which are supported by TREE-PUZZLE support, maximum parsimony, and ProML bootstrap values >80% (1,000 resamplings). Gray circles indicate nodes that are supported by 2 of the 3 above-mentioned treeing methods with support values >80%. TREE-PUZZLE support and bootstrap values <80% are not shown. Bar indicates 10% estimated evolutionary distance. Green algae and plant NTTs are marked in green, red algae are in red, heterokonts are in brown, diatoms are underlined, and C. paradoxa is in light gray. The rickettsial NTT group including NTT1 from L. intracellularis is highlighted by a gray box. A more detailed version of this figure and protein IDs are given in Fig. S6.