New Role for Cdc14 Phosphatase: Localization to Basal Bodies in the Oomycete Phytophthora and Its Evolutionary Coinheritance with Eukaryotic Flagella

Cdc14 protein phosphatases are well known for regulating the eukaryotic cell cycle, particularly during mitosis. Here we reveal a distinctly new role for Cdc14 based on studies of the microbial eukaryote Phytophthora infestans, the Irish potato famine agent. While Cdc14 is transcribed constitutively in yeast and animal cells, the P. infestans ortholog is expressed exclusively in spore stages of the life cycle and not in vegetative hyphae where the bulk of mitosis takes place. PiCdc14 expression is first detected in nuclei at sporulation, and during zoospore formation the protein accumulates at the basal body, which is the site from which flagella develop. The association of PiCdc14 with basal bodies was supported by co-localization studies with the DIP13 basal body protein and flagellar β-tubulin, and by demonstrating the enrichment of PiCdc14 in purified flagella-basal body complexes. Overexpressing PiCdc14 did not cause defects in growth or mitosis in hyphae, but interfered with cytoplasmic partitioning during zoosporogenesis. This cytokinetic defect might relate to its ability to bind microtubules, which was shown using an in vitro cosedimentation assay. The use of gene silencing to reveal the precise function of PiCdc14 in flagella is not possible since we showed previously that silencing prevents the formation of the precursor stage, sporangia. Nevertheless, the association of Cdc14 with flagella and basal bodies is consistent with their phylogenetic distribution in eukaryotes, as species that lack the ability to produce flagella generally also lack Cdc14. An ancestral role of Cdc14 in the flagellar stage of eukaryotes is thereby proposed

Distribution of Cdc14 and flagella-associated structures among eukaryotes.

<p>Cdc14 sequences were identified from public databases and validated by the reciprocal best Blast strategy. Centrioles includes structures with either standard triple or singlet tubules <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone.0016725-CarvalhoSantos1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone.0016725-Woodland1" target="_blank">[43]</a>. Although <i>H. arabidopsidis</i> has not been examined for centrioles, their presence is inferred based on other downy mildews <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone.0016725-McKeen1" target="_blank">[44]</a>.</p

Complex formation by PiCdc14.

<p>Top panel: silver-stained gel from a microtubule binding assay, in which PiCdc14 fused to MBP and StrepTag (MBP/Cdc) or MBP alone from <i>E. coli</i> were incubated with or without microtubules (MT). After centrifugation, pellets (P) and supernatants (S) were resolved by SDS-PAGE and stained to detect the 95 kDa PiCdc14 fusion band. The strong 55 kDa band is tubulin, and the strong lower band in the left-most lane is MBP. Lanes S1/P1 and S2/P2 represent samples from independent experiments. A blank lane was deleted at the site marked by a vertical line. Lower panels: Western blots probed with anti-StrepTag. The lower left image shows samples from the upper gel, and confirms that PiCdc14 binds microtubules <i>in vitro</i>. The bottom right blot shows the partitioning of PiCdc14/StrepTag protein from <i>P. infestans</i> between supernatant (S) and pellet (P), and suggests that most PiCdc14 is insoluble <i>in vivo</i>.</p

Structures of Cdc14 proteins.

<p>(<b>A</b>) Proteins from the species in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone-0016725-t001" target="_blank">Table 1</a>. The sequences are taken from their respective genome databases, except for the <i>Naegleria</i>, <i>Selaginella</i>, <i>Trypanosoma</i>, and <i>Thalassiosira</i> proteins which are based on manually curated gene models. The predicted proteins range from 341 to 822-aa as marked to the right of each model. Following a N-terminal region that shows little similarity between the proteins (yellow), each protein contains a fairly conserved stretch of about 300 aa (red). The latter includes the phosphatase domain which is marked as pfam00782, with the catalytic residue indicated. The C-terminal portions of the proteins (blue) show little conservation except for a roughly 85 aa region that is fairly conserved between <i>C. elegans</i>, human, and <i>X. laevis</i> (light blue). This includes the nuclear exit sequence (NES) and one or two QGD repeats. Nuclear localization signals (NLS) are also marked as detected by PSORTII; these include an experimentally validated NLS near the C-terminus of the <i>S. cerevisiae</i> protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone.0016725-Mohl1" target="_blank">[45]</a>, NLSs in the N-terminal regions of the human and <i>X. laevis</i> proteins which appear to have functions based on mutagenesis studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone.0016725-Kaiser1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone.0016725-Wu1" target="_blank">[46]</a>, and a NLS predicted in the C-terminal region of the <i>C. merolae</i> protein. (<b>B</b>) Similarity between Cdc14 of <i>P. infestans</i>, <i>S. cerevisiae</i>, and human Cdc14A. The program SSEARCH was used to calculate the percent amino acid identity in the region upstream, upstream, and C-terminal to the pfam00782 phosphatase domain. <i>E</i>-values for each match are also provided, which indicate that the similarity at the C-terminus is insignificant.</p

Colocalization of PiCdc14 and DIP13.

<p>Shown are the locations of the two proteins in transformants expressing Cdc14 and DIP3 fused to GFP or mCherry, respectively, in a cleaving sporangium (top row) and zoospores (bottom rows). Indicated are the basal bodies (arrowheads) and flagella (F). Bars represent 4 µm.</p

PiCdc14 association with flagellar basal body complexes.

<p>(<b>A</b>) FBBC from strain expressing PiCdc14/GFP, showing the protein in basal bodies (b), flagella (f), and nuclei (n). (<b>B</b>) Detection of PiCdc14/StrepTag in purified FBBCs and whole zoospores, using equal amounts of protein per lane and anti-StrepTag. (<b>C, D</b>) Colocalization in zoospores of PiCdc14/GFP (green) with flagella (pink, stained with anti-β-tubulin). Basal bodies and selected flagella are indicated. Bars represent 2 µm.</p

Rethinking the evolution of eukaryotic metabolism: novel cellular partitioning of enzymes in stramenopiles links serine biosynthesis to glycolysis in mitochondria

Abstract Background An important feature of eukaryotic evolution is metabolic compartmentalization, in which certain pathways are restricted to the cytosol or specific organelles. Glycolysis in eukaryotes is described as a cytosolic process. The universality of this canon has been challenged by recent genome data that suggest that some glycolytic enzymes made by stramenopiles bear mitochondrial targeting peptides. Results Mining of oomycete, diatom, and brown algal genomes indicates that stramenopiles encode two forms of enzymes for the second half of glycolysis, one with and the other without mitochondrial targeting peptides. The predicted mitochondrial targeting was confirmed by using fluorescent tags to localize phosphoglycerate kinase, phosphoglycerate mutase, and pyruvate kinase in Phytophthora infestans, the oomycete that causes potato blight. A genome-wide search for other enzymes with atypical mitochondrial locations identified phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase, which form a pathway for generating serine from the glycolytic intermediate 3-phosphoglycerate. Fluorescent tags confirmed the delivery of these serine biosynthetic enzymes to P. infestans mitochondria. A cytosolic form of this serine biosynthetic pathway, which occurs in most eukaryotes, is missing from oomycetes and most other stramenopiles. The glycolysis and serine metabolism pathways of oomycetes appear to be mosaics of enzymes with different ancestries. While some of the noncanonical oomycete mitochondrial enzymes have the closest affinity in phylogenetic analyses with proteins from other stramenopiles, others cluster with bacterial, plant, or animal proteins. The genes encoding the mitochondrial phosphoglycerate kinase and serine-forming enzymes are physically linked on oomycete chromosomes, which suggests a shared origin. Conclusions Stramenopile metabolism appears to have been shaped through the acquisition of genes by descent and lateral or endosymbiotic gene transfer, along with the targeting of the proteins to locations that are novel compared to other eukaryotes. Colocalization of the glycolytic and serine biosynthesis enzymes in mitochondria is apparently necessary since they share a common intermediate. The results indicate that descriptions of metabolism in textbooks do not cover the full diversity of eukaryotic biology