A mitochondrial-like chaperonin 60 gene in Giardia lamblia: evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria - PubMed (original) (raw)

A J Roger et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Diplomonads, parabasalids, as represented by trichomonads, and microsporidia are three protist lineages lacking mitochondria that branch earlier than all other eukaryotes in small subunit rRNA and elongation factor phylogenies. The absence of mitochondria and plastids in these organisms suggested that they diverged before the origin of these organelles. However, recent discoveries of mitochondrial-like heat shock protein 70 and/or chaperonin 60 (cpn60) genes in trichomonads and microsporidia imply that the ancestors of these two groups once harbored mitochondria or their endosymbiotic progenitors. In this report, we describe a mitochondrial-like cpn60 homolog from the diplomonad parasite Giardia lamblia. Northern and Western blots reveal that the expression of cpn60 is independent of cellular stress and, except during excystation, occurs throughout the G. lamblia life cycle. Phylogenetic analyses position the G. lamblia cpn60 in a clade that includes mitochondrial and hydrogenosomal cpn60 proteins. The most parsimonious interpretation of these data is that the cpn60 gene was transferred from the endosymbiotic ancestors of mitochondria to the nucleus early in eukaryotic evolution, before the divergence of the diplomonads and trichomonads from other extant eukaryotic lineages. A more complicated explanation requires that these genes originated from distinct alpha-proteobacterial endosymbioses that formed transiently within these protist lineages.

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Figures

Figure 1

Figure 1

Properties of the G. lamblia cpn60 gene and mRNA transcript. Upstream and downstream regions of the cpn60 gene are shown and numbered relative to the first base of the start codon (+1). The transcriptional start sites are shown in bold under vertical lines and rightward-pointing arrow (→). Two possible transcriptional signals are identified: an AT-rich transcription initiation signal (single underline), and an upstream promoter element (double underline) similar to the CAATTT signal reported for other G. lamblia genes (33). Possible sites of polyadenylation (↓), a putative polyadenylation signal (boxed), and the stop codon (∗) are also indicated.

Figure 2

Figure 2

Expression of cpn60 at different stages in the G. lamblia life cycle. (A) Northern blot analysis of total cellular RNA using a PCR fragment of the G lamblia cpn60 as a probe. Expression of the ≈1.8-kb cpn60 transcript was monitored in the vegetative trophozoite (T) stage, and after 5, 24, and 48 hr of encystation of G. lamblia. (B) Western blot analysis of total cellular protein isolated at different stages of encystation and excysation using a Synechococcus sp. anti-cpn60 antibody. Levels of the ≈60-kDa putative cpn60 protein were constant during the transition from the trophozoite stage (T) and throughout 0–66 hr of encystation. This protein was also present in the cyst phase (C) and after the first stage of induction of excystation (pI). A marked decrease in cpn60 protein was observed after the second stage of encystation (pII). Low levels of cpn60 persisted at 20 min, 90 min, 1 day, and 3 days after excystation, increasing after 6 days.

Figure 3

Figure 3

An alignment of the N termini of G. lamblia, E. histolytica, mitochondrial, hydrogenosomal, and eubacterial cpn60 homologs. The deduced N-terminal sequence of G. lamblia and E. histolytica cpn60s are aligned with homologs from L. tarentolae, T. vaginalis, and Caulobacter crescentus. The L. tarentolae and T. vaginalis targeting peptides (underlined) are removed during import into mitochondria and hydrogenosomes, respectively (9, 39). An N-terminal extension of the E. histolytica cpn60 homolog is suggestive of a targeting peptide for a cryptic organelle in this organism. G. lamblia cpn60 has a small, 2-aa N-terminal extension relative to C. crescentus. Amino acid identities of the G. lamblia cpn60 to other homologs are indicated by asterisks (∗) under the alignment.

Figure 4

Figure 4

Phylogenetic relationships of cpn60 homologs. Protein maximum likelihood analysis of 513 aligned amino acid positions yielded the tree shown (log likelihood = −18676.09). Optimal trees obtained by using protein distance and maximum parsimony methods differed from this topology in the branching order of the major eukaryotic groups and, to a lesser extent, within the α-, β-, and γ-proteobacterial clades. The branching order between these clades was identical for all methods. Bootstrap values obtained for the _G. lamblia/_mitochondrial-like cpn60 clade when using protein maximum likelihood (ML), protein distance matrix (DM), and maximum parsimony (MP) methods are shown in a box above the relevant node (indicated by an arrow). For all other nodes in the tree, protein ML bootstrap values (where >50%) are shown above each branch. The scale bar indicates estimated sequence divergence per unit branch length.

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