Molecular Biology and Evolution 18:1455-1463 (2001)
© 2001 Society for Molecular Biology and Evolution
The Evolutionary Origins of Eukaryotic Protein Disulfide Isomerase Domains: New Evidence from the Amitochondriate Protist Giardia lamblia
Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, Massachusetts
Department of Pathology, Division of Infectious Diseases and the Center for Molecular Genetics, University of California at San Diego
Institute of Geophysics and Planetary Physics and Department of Microbiology and Immunology, University of California at Los Angeles
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Abstract |
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A phylogenetic analysis of protein disulfide isomerase (PDI) domain evolution was performed with the inclusion of recently reported PDIs from the amitochondriate protist Giardia lamblia, yeast PDIs that contain a single thioredoxin-like domain, and PDIs from a diverse selection of protists. We additionally report and include two new giardial PDIs, each with a single thioredoxin-like domain. Inclusion of protist PDIs in our analyses revealed that the evolutionary history of the endoplasmic reticulum may not be simple. Phylogenetic analyses support common ancestry of all eukaryotic PDIs from a thioredoxin ancestor and independent duplications of thioredoxin-like domains within PDIs throughout eukaryote evolution. This was particularly evident for Acanthamoeba PDI, Dictyostelium PDI, and mammalian erp5 domains. In contrast, gene duplication, instead of domain duplication, produces PDI diversity in G. lamblia. Based on our results and the known diversity of PDIs, we present a new hypothesis that the five single-domain PDIs of G. lamblia may reflect an ancestral mechanism of protein folding in the eukaryotic endoplasmic reticulum. The PDI complement of G. lamblia and yeast suggests that a combination of PDIs may be used as a redox chain analogous to that known for bacterial Dsb proteins.
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsAcknowledgementsReferences |
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The prokaryotic-eukaryotic divergence is a major evolutionary discontinuity whose details are not well understood. Unlike prokaryotes, eukaryotes segregate many functions into membrane-bounded compartments. For example, the complex endomembrane system for posttranslational modification of secretory proteins is highly conserved from yeast to humans. Because of their basal position in many molecular phylogenies, early-diverging amitochondriate protists like Giardia lamblia (Leipe et al. 1993
; Stiller and Hall 1997
; Roger et al. 1999
; but see Embley and Hirt 1998
; Stiller, Duffield, and Hall 1998
; Hirt et al. 1999
) may be highly informative for identification of the origins of the eukaryotic endomembrane system. Although Giardia has several prokaryotic-like features (e.g., Knodler et al. 1998
), the components of the giardial endomembrane system analyzed to date resemble their homologs in yeast and higher organisms at the sequence and/or functional levels (e.g., Murtagh et al. 1992
; Soltys, Falah, and Gupta 1996
; Svard et al. 1999
). One essential function of the eukaryotic endomembrane system is the formation of protein disulfide bonds, required for correct folding, transport, and function of secretory and membrane proteins. Considering the five protein disulfide isomerases of G. lamblia, we report an examination of the evolutionary origins of protein disulfide isomerase (PDI) to gain insights into the early organization of the eukaryotic endomembrane system.
In both prokaryotic and eukaryotic cells, the formation of disulfide bonds is sequestered within a specialized compartment and catalyzed by enzymes from the thioredoxin superfamily. In the endoplasmic reticulum (ER) of eukaryotes, PDIs catalyze the formation, isomerization, and reduction of disulfide bonds to ensure the correct folding of secretory proteins prior to their further modification and transport (Rietsch and Beckwith 1998
; Ferrari and Soling 1999
). Most, if not all, organisms studied to date have more than one PDI or PDI-related gene (Monnat et al. 1997
). PDIs are characterized by thioredoxin-like (Tx-like) domains, each containing a cysteine redox-active site (CGHC) responsible for disulfide bond formation and rearrangements via thiol-disulfide exchange reactions (fig. 1 ). Due to the high similarity between the active-site domains of PDI and thioredoxins (Tx), it is commonly assumed that eukaryote PDI domains and thioredoxins share common ancestry, a concept supported by sequence similarity and distribution of introns (Sahrawy et al. 1996
; Kanai et al. 1998
). The majority of known PDIs include two or more Tx-like domains, with the inference that the historical diversification of PDI proteins included both gene and domain duplication (Kanai et al. 1998
). In contrast, the disulfide-bond-forming enzymes of Gram-negative bacteria, known as Dsb proteins, have a single thioredoxin superfamily active site and are localized to the periplasmic space (Rietsch and Beckwith 1998
). Although eukaryotic PDIs are multifunctional, six bacterial Dsb proteins identified so far, each with a single, slightly different Tx-like CXXC active site, catalyze distinct major functions ranging from disulfide bond formation in secretory proteins to the regeneration of the active form of the Dsb enzymes.
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Recently, Knodler et al. (1999)
described the sequences and enzymatic activities of three PDI genes from G. lamblia. Subsequently, the Giardia lamblia Genome Sequencing Project (www.mbl.edu/Giardia) reported two additional PDI coding regions. All five predicted giardial PDI proteins are unusual in that each has only a single Tx-like domain and they differ greatly in size and sequence outside of the active-site region. The three giardial PDIs expressed and characterized by Knodler et al. (1999)
were capable of rearranging disulfide bonds and also had transglutaminase activity, as reported previously for filarial and human PDIs (Chandrashekar et al. 1998
). Thus, they were the first single active-site PDIs shown to be capable of forming both disulfide and isopeptide protein cross-links. In addition, Giardia PDI-3 and PDI-5 are very small compared with other eukaryotic PDIs and could represent "minimal" enzymes for disulfide bond formation (Knodler et al. 1999
). Knodler et al. (1999)
speculated that the giardial PDIs might reflect ancestral PDI organization but did not test this hypothesis.
PDIs are important in giardial biology, as the trophozoite form has a large repertoire of extremely cysteine-rich variable surface proteins which are likely important for avoidance of host defenses (Nash et al. 1990, 1997
). Recently, it was shown that surface localized PDIs can mediate cell adhesion (Lahav et al. 2000)
. This could potentially be involved in trophozoite attachment to host intestinal cell surfaces, a characteristic feature of giardiasis. Furthermore, during encystation, Giardia must secrete cyst wall proteins, which are cross-linked by disulfide bonds and are needed for survival in the external environment (Adam 1991
; Lujan et al. 1995
; Gillin, Reiner, and McCaffery 1996
). Giardia differs from most eukaryotes in using cysteine instead of glutathione as its ER thiol redox buffer (Brown, Upcroft, and Upcroft 1993
). The single Tx-like-domain PDIs from Giardia could reflect the ancestral characteristics of eukaryotic PDIs. However, Tachikawa et al. (1995, 1997)
, Bao et al. (2000)
, and Wang and Ward (2000)
have reported single Tx-domain PDIs from Fungi. In contrast to G. lamblia, Fungi belong to the eukaryotic "crown group" in molecular phylogenetic examinations, with a close relation to animals (Baldauf and Palmer 1993
; Wainright et al. 1993
). Most known fungal and crown group PDIs contain two Tx-like domains, indicating that single Tx-like-domain PDIs could alternately be the product of secondary domain loss. We included both fungal and giardial PDI sequences in our phylogenetic analysis with the aim of elucidating the early characteristics of eukaryotic thiol-disulfide reactions and the ER.
Sahrawy et al. (1996)
previously performed a phylogenetic analysis of thioredoxins and Tx-like domains of eukaryotic PDIs and found strong support for monophyletic origins of PDIs relative to thioredoxin outgroups. They found additional support for common ancestry of PDIs in the distribution of thioredoxin and PDI introns. However, introns have not been identified in the giardial PDIs (or in other known giardial genes). The analyses of Sahrawy et al. (1996)
included PDIs with two or three (er72 protein) Tx-like domains and concluded that the ancestral PDI had two Tx-like domains and that er72 was derived via a duplication of the N-terminal domain (possibly independently for vertebrates and nematodes). Kanai et al. (1998)
supported this general conclusion with a broader sampling of PDI diversity (including the protist Acanthamoeba castellanii) and defined four classes of PDIs based on the position of the Tx-like domains (fig. 2
). Kanai et al. (1998)
focused their analyses on relationships between individual Tx-like domains and found numerous occurrences of domain loss and duplication during the evolution of the four PDI classes. Subsequent to Kanai et al.'s (1998)
analyses, an increasing number of PDIs with single active sites have been discovered in both crown group and lower eukaryotes (Tachikawa et al. 1995, 1997
; Knodler et al. 1999
; Bao et al. 2000
; Wang and Ward 2000
). The finding of PDIs with a single Tx-like domain in Fungi and Giardia challenges the concept that the ancestral PDI had two Tx-like domains. With the goal of examining the origins and diversification of eukaryotic PDI proteins, particularly newly discovered single-Tx-domain PDIs, we present here a phylogenetic analysis of PDI domain evolution. To increase phylogenetic resolution, we included sequences of animal and fungal PDIs, as well as PDIs from the alveolate Cryptosporidium parvum, the alga Chlamydomonas reinhardtii, and the slime mold Dictyostelium discoideum. This analysis expands that of Sahrawy et al. (1996)
and Kanai et al. (1998)
in that it examines the influence of Tx outgroups, three yeast single-Tx-domain PDIs, and the five single-Tx-domain PDIs of G. lamblia. It tests the hypothesis that the latter may represent ancestral PDI organization and function.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsAcknowledgementsReferences |
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Giardia lamblia Strain
The strain used by Knodler et al. (1999)
, the Giardia lamblia Genome Sequencing Project, and our sequencing of new G. lamblia PDI sequences has been deposited in ATCC (G. lamblia strain WB, clone C6, ATCC submission 50803).
Identification and Sequencing of Additional Giardia lamblia PDI Genes
In addition to the three giardial PDI sequences presented in Knodler et al. (1999)
, two additional giardial PDI genes were identified by the Giardia lamblia Genome Sequencing Project (www.mbl.edu/Giardia). PDI genes in the genome project sequences MD0702RA and MD0258RA were designated PDI-4 and PDI-5, respectively. The entire coding region of PDI-5 was contained within the random genomic clone MD0258, from which the sequence MD0285RA was generated. Overlapping fragments of the plasmid insert were individually subcloned into pBlueScript KS- vector (Strategene, La Jolla, Calif.). Subclone sequences were generated using a cycle-sequence protocol (Epicentre Technologies, Madison, Wis.) with IR-labeled vector primers (M13F and M13R) and resolved on a LI-COR 4200L sequencing apparatus (LI-COR Inc., Lincoln, Nebr.). Both strands were sequenced in their entirety. The final PDI-5 sequence was deposited in GenBank under accession number AAF89535.
PDI-4 was amplified by polymerase chain reaction (PCR) using 500 ng G. lamblia genomic DNA, Taq DNA polymerase (QIAGEN Inc., Valencia, Calif.), corresponding PCR buffer including 1.5 mM MgCl++, and a pair of primers designed to anneal outside of the predicted open reading frame: PDI4-5' (5'-CACAGCGATATGAACCAGCTTAG-3') and PDI4-3' (5'-CGTGACCTCGACATTACTAGTGTC-3'). PCR was performed on a Model PTC-200 thermal cycler (MJ Research, Watertown, Mass.) under the following conditions: initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 60 s, primer annealing at 55°C for 90 s, and extension at 72°C for 3 min, followed by a final extension at 72°C for 7 min. The 1,343-bp product was ligated into the pGEM-T Easy vector (Promega, Madison, Wis.). Clones were sequenced in both directions on an ABI 373A XL automated fluorescent DNA sequencer (Perkin Elmer, Foster City, Calif.) using Big Dye (Perkin Elmer) dye terminators. The final PDI-4 sequence was deposited in GenBank under accession number AF295634.
Alignment Construction
With the goal of exploring the potential role of domain duplications in the evolution of PDI genes, individual PDI domains from the same protein were treated as distinct genes in our phylogenetic analyses. We included domain sequences of G. lamblia PDIs; protistan, animal, plant, and fungal PDIs; Escherichia coli thioredoxin; and eukaryote thioredoxins found in GenBank using the BlastX program (Altschul et al. 1997
) (table 1
). We also included within the alignment a PDI-like protein from the filarial worm Dirofilaria immitis, which was first characterized as a transglutaminase but has high similarity to PDI (Chandrashekar et al. 1998
). The alignment of PDI domains and the thioredoxins was produced using the computer program CLUSTAL W (Thompson, Higgins, and Gibson 1994
). Alignments were adjusted in the context of the PDI domain and thioredoxin superfamily alignments of PFAM (Bateman et al. 1999
) and Kanai et al. (1998)
. Only those alignment positions lacking gaps were included in phylogenetic analyses and all thioredoxin sequences were used as outgroups. A corresponding alignment of nucleotide sequences was constructed for nucleotide-based phylogenetic analyses. The computer programs TREE-PUZZLE (Strimmer and von Haesler 1996
) and PAUP* (Swofford 1999)
were used to test for differences in amino acid and nucleotide composition among sequences, respectively.
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Phylogenetic Analyses
Protein distance matrixes were calculated using the computer program TREE-PUZZLE (formerly PUZZLE; Strimmer and von Haesler 1996
) under the JTT substitution model (Jones, Taylor, and Thornton 1992
), with inclusion of observed amino acid frequencies, estimated proportion of invariant sites (I), and estimation of among-site rate variation for the remaining sites according to a gamma distribution (
). The JTT substitution model was chosen based on Akaike information criterion (AIC) statistics (see Hasegawa et al. 1993
) in preliminary analyses. Searches for the best tree under the Fitch-Margoliash optimality criterion were performed using the FITCH program of the PHYLIP package (Felsenstein 1993), with 100 random-addition replicates. Bootstrapping under parsimony optimality criteria was performed using the SEQBOOT, PROTDIST, and CONSENSE programs of the PHYLIP package and 100 bootstrap replicates with 10 random-addition replicates each. Bootstrapping under distance optimality criteria (TREE-PUZZLE/FITCH) was performed as above with the additional use of the PUZZLEBOOT program (Holder and Roger 1999) to produce TREE-PUZZLE-based distance matrices.
Additional nucleotide analyses were performed using the computer program PAUP* (Swofford 1999
). Searches for the best tree were performed under the minimum-evolution criterion using 100 random-addition replicates. Bootstrapping under parsimony and minimum-evolution optimality criteria were performed using 100 replicates. Substitution models for use in minimum-evolution searches were chosen using the likelihood ratio test (LRT), after Huelsenbeck and Crandall (1997)
and Sullivan, Markert, and Kilpatrick (1997)
. Models, their parameters, and likelihood scores were initially estimated from the most-parsimonious tree topologies. In case substitution dynamics severely misled parsimony, models and parameters were then reestimated from the best trees found under the initial minimum-evolution criteria and the minimum-evolution search was repeated. Reestimation of model parameters continued using the results of successive minimum-evolution searches until their values stabilized.
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Results and Discussion |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsAcknowledgementsReferences |
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Figure 3 presents an alignment of the five G. lamblia PDI proteins. Like the previously published G. lamblia PDI-1 to PDI-3 genes, PDI-4 and PDI-5 each had a single classical PDI active site, F(Y/F)APWCGHCK. Unlike the multiple Dsb proteins of bacteria, the five giardial PDIs had identical active sites. The predicted lengths and molecular masses of PDI-4 and PDI-5 were 354 amino acids/40,342 Da and 116 amino acids/12,528 Da, respectively. Potential N-glycosylation sites (NxS/T) were found in PDI-1 (two) and PDI-4 (one). In the alignable Tx-like domain, giardial PDIs were 36%–81% similar to each other, 26%–51% similar to other PDIs, and 21%–32% similar to thioredoxins.
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Giardia PDI-3 and PDI-5 are the smallest eukaryotic proteins containing the PDI active site found to date. In size, overall sequence, Tx-like-domain sequence, and predicted N-terminal signal peptides, PDI-5 was strikingly similar to PDI-3, suggestive of recent gene duplication. In contrast to PDI-3, PDI-5 does not have a likely C-terminal ER retention signal. Mechanisms for ER localization are unknown for Giardia, but the ER chaperone Bip has a classical KDEL ER retention signal (Gupta et al. 1994
). However, in other organisms, it is becoming more evident that not all ER-resident proteins have a KDEL or KKXX ER-targeting signal. For example, localization of Dictyostelium PDI, which lacks a KDEL-type retrieval signal, is dictated by the 57 C-terminal amino acid residues of this protein (Monnat et al. 2000)
.
PDI-4 was similar to PDI-2 except that PDI-4 had a shorter C-terminal domain lacking the potential hydrophobic transmembrane domain of PDI-2, as predicted by the TMHMM software (Sonnhammer, von Heijne, and Krogh 1998
). Very few PDIs have a predicted transmembrane domain, but cell-surface-associated PDIs are known from other organisms (Jiang et al. 1999
; Xiao et al. 1999
; Zai et al. 1999
). While PDI-2 has a C-terminal ER retention signal (KRKK; Vincent, Martin, and Compans 1998
), it is uncertain if the C-terminal DKEL of PDI-4 serves the same function. Interestingly, the cytoplasmic tail of PDI-2 is very basic: 17 R/K residues out of 42 amino acids. Functionally, PDI-1, PDI-2, and PDI-3 have been localized to the giardial ER with specific antibodies (Knodler et al. 1999
).
A total of 49 PDI domains (from 31 individual PDIs) were aligned with three thioredoxin outgroup sequences. Attempts to include prokaryote Dsb proteins in the alignment failed due to insignificant similarity to thioredoxin and PDI amino acid sequences. The sister genes to eukaryotic PDIs appear to be eukaryotic and prokaryotic thioredoxins, which have only one active-site domain (Sahrawy et al. 1996
). Dsb proteins appear to be more distant relatives in our alignment attempts. Preliminary phylogenetic analyses (not shown) revealed an excessively long branch associated with the Saccharomyces cerevisiae MPD2 PDI protein (a single-domain PDI; Tachikawa et al. 1997
). This sequence was excluded from final phylogenetic analyses, as exceptionally long branches only contribute noise to phylogenetic reconstruction (Swofford et al. 1996
). In addition, preliminary phylogenetic analyses confirmed the problematic behavior of alfalfa (Medicago sativa) PDI domains in phylogenetic analyses, as encountered by Kanai et al. (1998)
, and they were also excluded from final phylogenetic analyses. After exclusion of sites with alignment gaps, the final amino acid alignment contained 86 sites, 77 of which were informative under parsimony. After exclusion of the third positions of all codons and sites with alignment gaps, the final nucleotide alignment contained 172 sites, 144 of which were informative under parsimony. Neither the amino acid nor the nucleotide alignment exhibited significant differences in amino acid and nucleotide composition among sequences, respectively.
Phylogenetic analyses based on protein (fig. 4
) and nucleotide (not shown) alignments produced very similar results. Overall, there was only marginal resolution of PDI domain phylogeny. Bootstrap support was high for monophyly of the PDI domains relative to the thioredoxin outgroups. Bootstrap support was additionally high for paraphyly of the class 1 C-terminal domains, as the clade included the most C-terminal Tx-like domain of er72 PDIs. As suggested by Kanai et al. (1998)
, the two most N-terminal Tx-like domains of er72 PDIs appear to be the result of an N-terminal domain duplication in a class 1 ancestral PDI. Overall, our results support the conclusions of Sahrawy et al. (1996)
and Kanai et al. (1998)
that domain duplication has occurred multiple times during the evolution of PDIs. Bootstrapping supported independent domain duplications leading to the origination of the erp5 proteins, Dictyostelium class 2 PDI, and (weakly) Acanthamoeba class 2 PDI. Given the lack of overall bootstrap support, it is uncertain if these duplications were preceded by domain loss (i.e., ancestral state of two domains) or if domain duplication was from an ancestral PDI containing one domain. The phylogenetic trees, albeit without strong bootstrap support, similarly favor domain triplication leading to the formation of human PDIR from an uncertain ancestor.
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The bootstrap results do not robustly support a basal position for the single-domain PDIs of Giardia and yeast or their common ancestry. Instead, the trees support common ancestry of Giardia PDI-2 and PDI-4, common ancestry of Giardia PDI-3 and PDI-5, and common ancestry of Giardia PDI-1 and yeast single-domain PDIs. The placement of the yeast-Giardia PDI-1 clade within a clade of multidomain PDIs is suggestive of secondary domain loss, but bootstrap support is negligible. Since the phylogenetic trees and bootstrap results do not favor recent common ancestry of all Giardia PDI genes, we can infer that multiple lineages of PDI existed early in eukaryotic history and were subsequently inherited by the diplomonads. The consistent monophyly of PDIs (Sahrawy et al. 1996
; our analyses) suggests that these lineages were the product of gene duplication from a single PDI ancestor. The PDI genes of Giardia suggest that gene duplication, and not domain duplication, was predominant in early eukaryotic history. The high similarity of Giardia PDI-3 and PDI-5 suggests that gene duplication may still be important in the evolution of the giardial ER.
The evolution of protein disulfide isomerases presents a difficult but important phylogenetic question. Phylogenetic resolution with molecular sequences is generally improved by reducing random error associated with the data by obtaining longer alignments, a solution not possible for the alignment of PDI domains. When examining PDI domain evolution, we are thus restricted to examining a deep phylogenetic question with a small amount of data. The lack of overall bootstrap support in both the amino acid and the nucleotide trees is a function of limited data and a long timescale. While we have attempted to reduce any systematic bias in the data through the use of appropriate substitution models that include among-sites rate variation and corrections for unequal amino acid and nucleotide frequencies, little can be done to increase the signal-to-random-noise ratio. Thus, we are left with an important question: why does G. lamblia have five PDIs, each with a single Tx-like domain? The quartet analyses of Kanai et al. (1998)
aside, we find that the poor basal resolution of PDI domain phylogeny is insufficient evidence for domain loss. One hypothesis is that in vivo, the PDI proteins of G. lamblia interact to form a redox chain analogous to that known for bacterial Dsb proteins. In this way, the earliest eukaryotes may have achieved protein folding in a manner similar to that of prokaryotes (in the absence of glutathione). The single-domain PDIs of G. lamblia thus would represent an ancestral mechanism of protein folding in the eukaryotic ER. We suspect that although the five giardial PDIs are identical in their active sites (unlike the redox chain of bacterial Dsb proteins), their greatly differing molecular masses and sequences outside of the active-site region may represent a subdivision of function in protein folding. The finding that filarial, human two-domain, and giardial single-domain PDIs all have transglutaminase activity and catalyze isopeptide bond formation suggests that this distinct protein cross-linking activity may be an inherent property of all PDIs.
The single-domain PDIs of Fungi present a very interesting discovery. If fungal single-domain PDIs are secondarily derived products of domain loss, then the phylogenetic placement of Giardia PDI-1 suggests the existence of a two-domain PDI gene in Giardia's evolutionary history. Despite fourfold coverage of the G. lamblia genome, no additional Giardia PDI genes have been identified and there is no suggestion of a two- or three-domain gene (unpublished data; www.mbl.edu/Giardia). Discovery of such a gene among other basal eukaryotes, particularly other diplomonads and also trichomonads, would provide support for two-domain PDIs appearing early in eukaryotic history. The alternative, predicted by our hypothesis of early PDI evolution, is that lineages of single-domain PDI have existed since the origin of eukaryotes and persist throughout eukaryotic diversity. Notably, a single-domain PDI has been discovered in the trypanosomatid Leishmania (A. Debrabant, personal communication), suggesting that further investigation of protist PDI diversity could help elucidate the ancestral design of eukaryotic PDIs.
Knodler et al. (1999)
illustrated that the Giardia proteins are the first single active-site PDIs capable of forming both disulfide and isopeptide protein cross-links. Could this also be the case for the yeast single-domain PDI proteins? If so, the phylogenetic placement of Giardia PDI-1 in our analyses may actually reflect convergent function, not a shared history. Giardia and yeast may both use a diversity of PDI proteins to form a redox chain (see Frand, Cuozzo, and Kaiser 2000)
, adding an additional twist to our hypothesis that Giardia's ER reflects ancestral design. Recombinant PDI-2 had higher activity in insulin reduction than PDI-3, while PDI-1 was inactive (Knodler et al. 1999
). In addition, only PDI-2 complemented an E. coli dsbA mutant (Knodler et al. 1999
), suggesting that the main role of PDI-2 may be in disulfide bond reduction. In contrast, PDI-2 had lower transglutaminase activity than PDI-1 or PDI-3. However, these observations could simply be due to differences in folding of the expressed enzymes. An examination of functional similarities and differences between the single-domain PDIs of yeast and the five Giardia PDIs may prove very fruitful for understanding the multifunctional capabilities of PDIs.
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Conclusions |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsAcknowledgementsReferences |
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Inclusion of protist PDIs in our analyses, particularly those from G. lamblia, has revealed that the evolutionary history of the ER may not be simple. For example, we now know that single Tx-like-domain PDIs exist in diplomonads, trypanosomatids, and Fungi and that domain duplications have occurred independently throughout eukaryote diversification. The PDI complement of Giardia and yeast suggests that a diversity of PDIs may be used as a redox chain analogous to that known for bacterial Dsb proteins. Further investigation of eukaryote PDI diversity, coupled with investigation of giardial and yeast PDI function, could transform our concepts of the origins and mechanism of protein folding within the ER.
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsAcknowledgementsReferences |
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We thank H. Morrison, N. Passamaneck, J. Nixon, and U. Kim for their considerable contribution. S. Kanai provided a useful electronic copy of Kanai et al.'s (1998)
PDI domain alignment and reviewed early versions of the manuscript. A. Roger provided important insight into protein phylogenetics and use of the computer program TREE-PUZZLE. D. Reiner, P. Wheeler, and M. Holder provided logistical assistance. This work was supported by National Institute of Health grant AI43273 to M.L.S. ("Giardia—A Model for Ancient Eukaryotic Genome Analysis"), National Institute of Health grants AI42488, GM53835, and DK35108 to F.D.G., the G. Unger Vetlesen Foundation, and LI-COR Biotechnology. |
Footnotes |
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Mark Ragan, Reviewing Editor
1 Present address: Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, Canada. 
1 Keywords: protein disulfide isomerase
thioredoxin
Giardia
phylogeny.
2 Address for correspondence and reprints: Mitchell L. Sogin, Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts 02543-1015. sogin{at}mbl.edu
.
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsAcknowledgementsReferences |
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