Analysis of Euglena gracilis Plastid-Targeted Proteins Reveals Different Classes of Transit Sequences

The plastid of Euglena gracilis was acquired secondarily throughan endosymbiotic event with a eukaryotic green alga, and asa result, it is surrounded by a third membrane. This membranecomplexity raises the question of how the plastid proteins aretargeted to and imported into the organelle. To further exploreplastid protein targeting in Euglena, we screened a total of9,461 expressed sequence tag (EST) clusters (derived from 19,013individual ESTs) for full-length proteins that are plastid localizedto characterize their targeting sequences and to infer potentialmodes of translocation. Of the 117 proteins identified as beingpotentially plastid localized whose N-terminal targeting sequencescould be inferred, 83 were unique and could be classified intotwo major groups. Class I proteins have tripartite targetingsequences, comprising (in order) an N-terminal signal sequence,a plastid transit peptide domain, and a predicted stop-transfersequence. Within this class of proteins are the lumen-targetedproteins (class IB), which have an additional hydrophobic domainsimilar to a signal sequence and required for further targetingacross the thylakoid membrane. Class II proteins lack the putativestop-transfer sequence and possess only a signal sequence atthe N terminus, followed by what, in amino acid composition,resembles a plastid transit peptide. Unexpectedly, a few unrelatedplastid-targeted proteins exhibit highly similar transit sequences,implying either a recent swapping of these domains or a conservedfunction. This work represents the most comprehensive descriptionto date of transit peptides in Euglena and hints at the complexroutes of plastid targeting that must exist in this organism.

INTRODUCTION

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Introduction

A fundamental problem in cell biology is the precise and efficienttargeting of proteins synthesized by cytoplasmic ribosomes totheir appropriate intracellular locations. Proteins destinedfor the endomembrane system, mitochondria, or the chloroplastusually have specific N-terminal targeting domains that arerequired for proper subcellular localization. These leader sequencesare often removed by specific proteases at the protein's destinationprior to it assuming its active conformation. For chloroplast-targetedproteins in plants and algae, an N-terminal transit peptide(TP) is both necessary and sufficient for correct plastid targeting(11). Transit peptides are not conserved in sequence but exhibitcharacteristic biochemical properties, such as an elevated contentof the hydroxylated amino acids serine and threonine as wellas a deficiency of acidic (aspartate and glutamate) amino acids(76). Within a typical chloroplast, there are six distinct locationsto which the constituent proteins must be sorted, and some proteinshave to cross up to three membranes (33). This complexity requiresadditional targeting information within the transit peptide,such as the signal sequence-like domain found in proteins targetedto the thylakoid membrane, or information contained within themature portion of the protein itself (62).

Plants, green algae, and red algae have plastids derived froman endosymbiotic cyanobacterium, with two membranes envelopingthe chloroplast (34). Protein targeting to these plastids isfairly well understood; generally, the transit peptides directnewly synthesized proteins to the outer envelope membrane, wherethey interact with receptors and other components of the translocationapparatus so that protein import and subsequent sorting cantake place (33). Many of the translocation components presentin the outer and inner envelopes have been identified in plants(31).

Many protists, however, possess secondary plastids that arebelieved to have arisen from endosymbiosis with a eukaryoticalga. These organisms have complex plastids with either threemembranes around the chloroplast, as occurs in the dinoflagellatesand Euglena spp., or four membranes, as in the stramenopilesand haptophytes (34). The presence of additional membranes surroundingthe plastid would seem to necessitate additional targeting information,complicating the process of translocation. We know, for example,that during the evolution of secondary plastids, genes fromthe endosymbiont were functionally transferred to the host'snuclear genome. These genes must then be expressed and theirprotein products targeted back to the organelle, and this processis undoubtedly more complicated than that in the case of primaryplastids. A significant hurdle in this pathway is the necessityto acquire appropriate targeting information that allows nucleus-encodedproteins to be directed to the plastid and to traverse additionalmembranes in the process. Understanding the mechanism of targetingand translocation in organisms with complex plastids has beenkey to understanding how the transition from algal symbiontto plastid occurred (12, 35, 47, 50, 63, 74).

In protists with four membranes around the plastid, the outermostmembrane often has ribosomes attached and is typically continuouswith the endoplasmic reticulum (ER) (23). Proteins directedto these plastids possess bipartite targeting sequences, withan N-terminal signal sequence (24) that directs them to thechloroplast ER, where they are cotranslationally imported acrossthe first membrane (4, 7, 30). The domain after the signal sequenceis the predicted transit peptide for transport across the innertwo membranes, in a process likely to resemble translocationacross plant chloroplast envelopes (43).

The euglenophytes and dinoflagellates have plastids with threemembranes, the outermost of which lacks bound ribosomes. Inboth cases, plastid proteins are targeted through the endomembranesystem (49, 53, 67, 70, 71). From studies of several complete,publicly available Euglena gracilis plastid protein sequences(13, 25, 27, 28, 38, 44, 52, 56, 61, 64, 66, 73), it was predictedthat the plastid proteins have an N-terminal signal sequence,an inference that was confirmed by both in vitro (38) and invivo (70, 71) experimental approaches. Following the signalsequence is the predicted transit peptide, which is sufficientfor translocation across plant chloroplast membranes (29), anda hydrophobic region that acts as a "stop-transfer" sequenceto prevent complete transport into the ER, such that the matureprotein remains in the cytoplasm (69). The protein is then targetedto the plastid, likely via a vesicular transport system (67).Also described for Euglena are tripartite transit sequencesthat possess an additional hydrophobic domain predicted to targetproteins to the thylakoid lumen (73).

Because relatively few Euglena plastid protein sequences arepublicly available, the study we report here more comprehensivelyexamines the characteristics of plastid-targeting sequences.Since many of the known Euglena proteins, including all of thosefor which biochemical analyses of targeting have been conducted,are encoded as polyproteins, we sought to determine whetherall plastid proteins are likely to proceed to the plastid viaa similar pathway in this organism. By examining the targetingsequences of a large number of plastid proteins, the majorityof which are not organized as polyproteins, we have been ableto define the characteristics that can be used to identify Euglenaplastid-targeted proteins with high confidence and to infermodes of transport to the plastid.

MATERIALS AND METHODS

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Materials and Methods

E. gracilis strain Z was cultured under several different conditions,and cDNA libraries were produced commercially in the PCDNA3.1(+)vector (DNA Technologies Inc.). Expressed sequence tag (EST)sequencing was performed at the Atlantic Genome Centre (Halifax,Nova Scotia, Canada) and the B.C. Cancer Agency (Vancouver,British Columbia, Canada). A total of 19,013 ESTs were retainedfollowing quality and vector trimming via the taxonomicallybroad EST database (TBestDB [http://tbestdb.bcm.umontreal.ca/searches/login.php]),under the auspices of the Protist EST Program. The ESTs wereclustered to form a total of 9,461 unique groups.

To search for plastid-targeted proteins, the 9,461 clusterswere translated in three reading frames (ORFs) (plus orientation),and the longest ORF of >19 amino acids starting with a methioninewas retained for further analyses (http://maven.smith.edu/ $~$ vvouille/sumCGI/translator.html).Screening for plastid-targeted proteins was carried out in severalrounds. First, all ORFs were screened for the presence of asignal sequence using the program SignalP3 (6, 51; http://www.cbs.dtu.dk/services/SignalP/).Any ORFs with a signal sequence predicted with the hidden Markovmodel (HMM) or the artificial neural network (NN) were retained.All selected ORFs were then rescreened, and those having a clearrole in plastid function and/or those whose top BLASTnr hitwas plant, algal, or cyanobacterial in origin were segregatedfor further consideration. Finally, the putative plastid-targetedproteins were screened further according to the following criteria:(i) the top BLAST hit (NCBI nonredundant database) was plant/algalor cyanobacterial and/or the protein has a clear role in plastidfunction, and (ii) the BLASTp E value was $≤$ 1e–05. The ORFwas considered to possess a complete transit sequence when (i)there was evidence for a spliced leader sequence (TTTTTTTCG)at the 5' end of the cDNA that would indicate that the cDNAwas full length (72), (ii) there was an extension of the ORFtoward the N terminus upstream of the first region of evidentamino acid sequence similarity following a BLASTp search, and(iii) the beginning of the mature protein was identified bycomparison with orthologous proteins.

Potential membrane-spanning regions were identified using thehidden Markov model-based program TMHMM (39; http://www.cbs.dtu.dk/services/TMHMM/).Hydrophobicity plots were generated using the Protscale programat the exPASy site (http://www.expasy.org/tools/protscale.html),using a Kyte-Doolittle scale with a sliding window length of7 or 19 nucleotides, as indicated. The amino acid content ofpeptides was calculated using the PEPSTATS program in the EMBOSSpackage, available at AnaBench (http://anabench.bcm.umontreal.ca/anabench/Anabench-Jsp/Welcome.jsp).Sequence logo displays were generated using the online programWebLogo (weblogo.berkeley.edu/logo.cgi).

Nucleotide sequence accession numbers. All individual EST sequences have been deposited in the NCBIdbEST database under accession numbers EG565093 [GenBank] to EG565263 [GenBank] .

RESULTS

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Results

From 9,461 individual Euglena EST clusters, a total of 117 full-lengthplastid proteins were identified. Eliminating nearly identicalisoforms from the data set left a total of 83 unique proteinsfor further analysis (Table 1). In addition to functioning inbasic photosynthetic reactions, the proteins identified hadpredicted roles in the biosynthesis of proteins, lipids, carotenoids,and chlorophyll. Proteins involved in signal transduction andplastid metabolism were also found. Through determination ofthe N-terminal-most regions of sequence similarity in BLASTpsearches, targeting domains were delineated and found to bevery long, with an average size (± standard deviation)of 152 ± 25 residues (Table 1). The shortest estimatedpresequence was 95 residues, for Rubisco activase, and the longestwas 211 (Albino3) (Table 1).

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TABLE 1. EST clusters

Of the few Euglena plastid proteins examined to date, all possessan N-terminal region similar to a eukaryotic signal sequence.Thus, the first strategy for identifying plastid-targeted proteinswas to search for the presence of such a sequence, using SignalP3.Of the final group of 83 plastid-targeted proteins examinedusing the SignalP hidden Markov model, 68% were predicted topossess a signal sequence. This value dropped to 56% when theartificial NN was employed. In cases where SignalP did not predicta signal peptide but other screens indicated a potential plastid-targetedprotein, there was nevertheless a clear hydrophobic region characteristicof a signal peptide. Based on the NN predictions for the signalsequence cleavage sites, the Euglena signal sequence was estimatedto be 33 ± 9 residues long (range, 18 to 59 residues).The predicted cleavage site was consistent with that in othereukaryotic signal sequences (Fig. 1C).

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FIG. 1. Characteristics of class I targeting sequences of Euglena. (A) Averaged TMHMM probabilities for 70 class I proteins identified in this study. Because the region upstream of the first TMH is of variable length (range, 2 to 32 amino acids; mean, 12.7 ± 6.7 amino acids), the data were normalized to a starting TMHMM probability of $≥$ 0.1, which corresponds to the beginning of a predicted membrane-spanning region, and then averaged. The error bars show 2 standard errors. Key features of a Euglena class I targeting sequence are depicted above the graph. (B) Overview (McClade) of amino acid categories of the targeting sequences of selected plastid-targeted proteins. Colors represent different amino acids, as follows: gray, hydrophobic and nonpolar (A, C, F, G, I, L, M, P, V, W, and Y); red, acidic (D and E); purple, basic (H, K, and R); yellow, hydroxylated (S and T); and blue, polar (Q and N). (C) Sequence logo plot showing occurrence of amino acids around the signal sequence cleavage site (arrow) predicted by SignalP (neural net). The y axis is displayed as bits, as described at weblogo.berkeley.edu/logo.cgi.

E. gracilis plastid-targeting sequences can be divided into two classes. Class I plastid-targeting sequences are designated by analogyto a similar type of targeting domain identified in dinoflagellates(55). This class encompassed 89% of the Euglena proteins examined,which were characterized by the presence of two hydrophobicregions that are predicted by the TMHMM program to be transmembranehelices (TMH) (Fig. 1A). Figure 1A shows the average TMHMM probabilityfor the class I plastid-targeting regions, with the first predictedtransmembrane helix (TMH1) corresponding to the hydrophobicdomain of a classic signal sequence (75). A basic amino acidprecedes the first TMH in all but six proteins, with the averagecharge of this N-terminal region being +1.6. In only one caseis the N-terminal region negatively charged (Table 1, cluster3881 [ATP/ADP transporter]).

The location of the second TMH is remarkably consistent, at60 ± 8 amino acids following the end of the first predictedTMH, with a range of 45 to 94 amino acids. We designate thislocalization the "60 ± 8 rule" (Fig. 1A). The propertiesof the amino acids within the targeting regions of selectedplastid-localized proteins are shown in Fig. 1B. In this figure,the hydrophobic regions (gray) are obvious. The presence ofthe two TMH motifs separated by 60 ± 8 amino acids hadexcellent discriminating power for identifying potential plastid-targetedproteins. For class I targeting sequences, the TMHMM programwas able to predict upwards of 95% of the plastid proteins simplyby searching for N-terminal regions with TMHs according to the60 ± 8 rule. If we combined the entire set of predictedplastid proteins (all classes), the TMHMM program would havean overall success rate of 82%. In cases where the TMHMM probabilitydid not meet the threshold for formal TMH prediction (Table1, underlined values), the probability of a TMH was usuallybetween 0.3 and 0.9, and the success rate would be very highif the threshold was reduced in subsequent rounds of screening.Rescreening the entire population of ORFs using the 60 ±8 rule detected all of the class I proteins listed in Table1, including isoforms, plus an additional 25 proteins classifiedas unknowns (data not shown). The TP domains of dinoflagellates,whose plastid leader sequences have a similar structure (49),are about half the size (25 ± 8 residues) (data not shown)of those of Euglena proteins.

Class IB proteins (Table 1) also possess two predicted TMHsseparated by 60 ± 8 amino acids, but they have a thirdhydrophobic domain with a mean distance of 17 residues (range,7 to 25 residues) downstream of the end of TMH2 (Fig. 2). Thisregion resembles a prokaryotic signal sequence and is postulatedto function in the targeting of proteins to the thylakoid lumen(73). We identified five proteins that are homologous to thylakoidlumen-localized proteins and for which biochemical evidencefor this location exists (four of these class IB proteins areshown in Fig. 2, along with a lumen-targeted class II protein[see below]). Two additional proteins are predicted to functionin the lumen, based on their annotation as well as their possessionof a putative lumen-targeting domain (LTD). Three of the sevenclass IB proteins (ascorbate peroxidase, HCF136, and OEE2) containa double Arg immediately preceding the third hydrophobic domain(data not shown); another two class IB proteins (PSI-III andcytochrome c₆) have the same motif within six amino acids ofthe start of the hydrophobic LTD, suggesting that the twin-argininetranslocation (Tat) pathway (58) is functional in Euglena.

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FIG. 2. Kyte-Doolittle hydropathy plots for class IB plastid-targeting sequences of Euglena. Hydrophobicity plots for five confirmed lumen-targeted proteins are shown. The analyses were conducted with a window size of 19, and the hydrophobic regions (positive scores) corresponding to the TMHs of the signal sequence (SS) and the stop-transfer sequence (ST) are indicated with black bars. The hydrophobic region corresponding to the LTD is indicated with gray bars. Oxygen-evolving enhancer 3 (OEE3) has a class II targeting sequence and thus lacks the typical ST region. TP, transit peptide; MP, mature protein.

Class II targeting sequences in Euglena represent a departurefrom the class I type in that they lack the second TMH regionupstream of the region specifying the mature protein, and hencedo not conform to the 60 ± 8 rule (Fig. 3). The TMHMMprobability scatter plot shows the presence of the hydrophobicregion associated with the signal sequence in all class II proteins.This class represents 14% of the identified population of plastid-targetedproteins. Of the 13 class II proteins delineated so far, 6 haveunambiguous functions in the plastid, while the others conceivablycould be targeted elsewhere. However, they all possess signalsequence-like N termini, and their predicted functions are expectedto occur within the plastid. Each of these proteins is alsorelated to homologs from photosynthetic taxa, as gauged by thetop BLASTp hit (Table 1), supporting a putative plastid localization.The OEE3 protein, which is located within the thylakoid lumen,exhibits a second hydrophobic region (Fig. 3, arrow) that representsan LTD analogous to that found in class IB targeting domains.The estimated presequence length is 100 amino acids. All ofthe class II sequences have a spliced leader sequence, indicatingthat they are not class I sequences that were artifactuallytruncated upstream of the stop-transfer domain.

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FIG. 3. Characteristics of class II targeting sequences of Euglena plastid proteins. (A) Scatter plot showing TMHMM probability for the first 100 amino acids. Because the region before the first TMH is of variable length, the data were normalized to a starting TMHMM probability of $≥$ 0.1. In all cases, a second TMH 60 ± 8 amino acids downstream from the first was absent. The hydrophobic region centered at position 45 is the LTD of OEE3. (B) Overview (McClade) of amino acid categories of the targeting sequences of class II plastid-targeted proteins. Colors represent defined categories of amino acids, as indicated in the legend to Fig. 1. The black arrowhead indicates the predicted signal sequence cleavage site.

Plastid transit peptides of class I and II proteins. In plants, targeting of proteins to the chloroplast is mediatedby a transit peptide (for a review, see reference 11). Althoughsequence conservation per se is lacking, there is a generalmaintenance of certain chemical properties, including enrichmentfor the hydroxylated amino acids serine and threonine and adeficiency in acidic residues (76). In Euglena class I proteins,the intervening region between the two TMHs likely functionsas a plastid TP (29). For class II proteins, we predicted thatthe region immediately following the signal sequence must havea role in targeting to the plastid. The exact length of theputative TP was difficult to assess, as we had little confidencein the ability of ChloroP to correctly predict the cleavagesite, and thus the values in Table 1 are only estimates. However,from the predicted signal sequence site to the first regionof clear sequence similarity to known proteins, the length rangedfrom 36 to 135 amino acids.

To test whether the class II TP region was similar to that ofclass I targeting sequences and to determine the chemical propertiesof both TP domains compared to the TPs of green algae and plants,we examined their amino acid compositions. We also comparedthese compositions to those of the mature region of proteinswith class I targeting domains as well as selected Chlamydomonasproteins. The amino acid composition was calculated from theentire intervening region between the TMH regions of class Iproteins (the predicted transit peptide), the estimated transitpeptide from class II proteins that was located after the signalsequence and before the predicted start of the mature protein,and the entire coding region from all proteins having classI targeting sequences.

The data for selected amino acids and amino acid categoriesare shown in the form of box-and-whisker plots (Fig. 4). Sinceplastid transit peptides are reportedly enriched in hydroxylatedamino acids and deficient in acidic amino acids (76), we analyzeda priori these amino acid categories in the putative TPs ofclass I and class II targeting sequences of Euglena in additionto a selection of 25 predicted TPs from Chlamydomonas proteins(Fig. 4). The region immediately downstream of the signal sequencein class I and II targeting sequences was significantly enrichedin Ser and Thr (22% and 17%, respectively) compared to the matureregions of proteins with class I targeting sequences (11%) (one-wayanalysis of variance [ANOVA] and Tukey's test [ ${alpha}$ $≤$ 0.05]). TheTPs of Chlamydomonas proteins were similarly enriched in Ser/Thr(17%) compared to the mature portions of the proteins (11%)(Fig. 4). The putative transit peptide regions of class I andII targeting sequences were also significantly depleted in acidicamino acids (Asp and Glu) compared to the mature regions ofthe same proteins (Fig. 4) (one-way ANOVA and Tukey's test [ ${alpha}$ $≤$ 0.05]). The predicted transit peptide regions were also foundto have a higher Ala and Pro content than the mature portionsof proteins (Fig. 4) (one-way ANOVA and Tukey's test [ ${alpha}$ $≤$ 0.05]).However, given that 20 tests were conducted and that the aminoacid composition is not truly independent, there is a possibilitythat some of these differences could be by chance. Althoughthe Chlamydomonas TP exhibited a clear elevation in Ala content,there was no difference in the amount of Pro compared to thatin the Euglena TPs. In terms of charged amino acids, the TPregion is deficient in acidic amino acids, yet there is littlesignificant change in the content of basic (His, Lys, and Arg)residues compared to the mature regions of the same proteins.However, examination of Lys and Arg separately reveals discriminationagainst Lys in the TP regions of class I and II targeting sequences(mean, 1.6% and 2.1%, respectively) compared to the mature proteins(mean, 5.8%; P < 0.001 [Kruskall-Wallis]) (Fig. 4). Therewere no significant differences in Arg content between the predictedtransit peptides and the mature portions of the same proteins.Chlamydomonas TPs discriminate strongly against acidic aminoacids (mean, 0.2%) and have an elevated content of Arg comparedto the mature regions of the same proteins. Unlike Euglena,Chlamydomonas shows no bias against Lys in the TP. Without exception,the amino acid compositions of the Euglena class I and II transitpeptides were the same, and both were significantly differentfrom the composition of the mature protein (Fig. 4).

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FIG. 4. Amino acid composition analyses of the predicted TPs of class I and II targeting sequences compared to the mature proteins (MP). The amino acid compositions of the intervening region between TMH1 and TMH2 of class I targeting sequences (TP, I; n = 70), the predicted transit peptide region for class II proteins (TP, II; n = 13), and the mature protein regions from class I proteins (MP, I; n = 70) were determined. Also shown are the amino acid compositions of Chlamydomonas reinhardti TPs (TP, Cr; n = 25) and mature proteins (MP, Cr; n = 25). Box-and-whisker plots were used to represent the data and are based on quartiles around the median value. The box encloses 50% of the data, with 25% above and below the median (solid line). Each whisker represents the data range of an additional 25% of the data. The existence of outliers beyond the 5% and 95% confidence ranges is indicated with a solid dot where applicable. Categories indicated with different letters on the plot are significantly different (one-way ANOVA and Tukey's test [ ${alpha}$ $≤$ 0.05]). All data were normal except for the Lys content in class II peptides, in which case nonparametric statistics were used to assess differences.

To examine the distribution of the acidic amino acids further,the class I transit peptide region was divided into thirds,and the acidic amino acid content was calculated (Fig. 5). Fromthis analysis, an asymmetric distribution of acidic amino acidswas apparent, such that the first third (TP1) lacked acidicresidues (1%) while the latter third (TP3) had the same acidiccontent as the mature protein (11%). The Ser/Thr compositionsof the putative TPs were not different among the three regions(TP1-3) (Fig. 5). The basic amino acid composition was the samewithin the three TP regions and the mature protein (Fig. 5).

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FIG. 5. Amino acid composition analysis of the plastid TP domain of class I targeting sequences. Each TP region was divided into three equal segments (TP1-3), and the basic (H, K, and R), acidic (D and E), and serine/threonine (Ser/Thr) contents were calculated. These values were compared to the averaged amino acid composition of the mature protein (MP).

Overall, the putative TP domains of the two classes of Euglenatargeting sequences have the same amino acid composition, andthis composition resembles that of plant chloroplast transitpeptides (11, 20, 76) in terms of an elevated content of Ser/Thr.These putative TP domains were also predicted to be transitpeptides by using ChloroP (18), with apparent success ratesof 83% and 67% for class I and II targeting sequences, respectively,when the signal sequence domain was removed. Surprisingly, thesuccess rates were still respectable when the signal sequencewas retained during the analysis (71% and 50%) but not whenthe entire targeting sequence was removed. One notable exceptionis the lumen-targeted protein OEE3, with a class II targetingsequence that has a mere seven amino acids between the end ofthe TMH (the signal sequence) and the putative hydrophobic LTD.Two of the seven residues are basic amino acids (no acidic residues),but the region immediately after the LTD is strongly acidic(data not shown). Euglena TPs, like others in the green algalineage, lack a requirement for a Phe at the N terminus thatis commonly observed in chromalveolates (15, 55, 57) and glaucophytes(68) and that is essential for plastid import in vivo in diatoms(37).

Stop-transfer sequences are a predicted feature of class I proteins. Stop-transfer sequences function to halt the cotranslationalimport of proteins into the ER and serve an important role indetermining the orientation of a protein in the membrane (8).For Euglena, it has been proposed that the second TMH acts asa stop-transfer sequence (69). From analysis of a large numberof proteins with class I targeting sequences, it is clear thata stop-transfer sequence is a common motif in Euglena plastid-targetedproteins. In a few cases (Table 1), the second TMH region wasnot predicted by the TMHMM program, and the probability of havinga TMH ranged from 0.1 to 0.9. Nevertheless, in these cases,subsequent hydropathy plots confirmed that these targeting domainsare still strongly hydrophobic (data not shown) and thereforelikely to have the same stop-transfer function. Immediatelyfollowing the second TMH and within six residues of its end,ca. 80% of proteins of this class have two or more basic aminoacids, and 97% of proteins have at least one. Only 2 of the71 class I proteins lack a positively charged residue immediatelyafter the TMH. The sharp change in polarity immediately afterthe second hydrophobic region, particularly towards positivelycharged residues, is apparent in the hydropathy plots encompassingthis region (Fig. 6A). Class I polypeptides display a sharpdecline in hydrophobicity immediately following the second TMH,a feature that presumably acts to block further insertion intothe membrane. In class IB proteins, an additional hydrophobicregion, the lumen-targeting domain, is located 25 to 30 aminoacids further downstream. In contrast, class II polypeptidesdo not exhibit this sharp increase in polarity immediately afterthe hydrophobic section of the predicted signal sequence. Thesequence logo illustrates the common occurrence of basic aminoacids immediately after the hydrophobic domain of the stop-transferdomain (Fig. 6B), which is not observed after the signal sequencesof class II proteins. These differences provide additional evidencethat the TMH of a class II protein is not simply the secondTMH of a 5'-truncated cDNA encoding a class I protein.

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FIG. 6. (A) Kyte-Doolittle hydrophobicity profiles for the stop-transfer region of class I targeting sequences and the region immediately following the signal sequence of class II targeting domains. Plots begin 10 amino acid residues upstream of the start of the second TMH (for class I proteins) or the first TMH (for class II proteins), and the hydrophobicity profiles were calculated with a window size of 7 residues. The thick lines are the mean scores, and the thin lines on either side represent the 95% confidence intervals. The black bars above the hydrophobic regions indicate the location of the predicted TMH. (B) Sequence logo plot of class IA sequences when the second transmembrane helixes (TMH2) were aligned. Only the regions immediately before and after TMH2 are shown.

Some plastid transit sequences are conserved. When the plastid-targeting sequences of Euglena class I proteinswere used individually as tBLASTn queries against the Euglenadatabase, unexpected similarities in certain groups of unrelatedproteins were revealed. For instance, FNR and CP29 possess nearlyidentical targeting sequences despite having no functional relationship(Fig. 7A). There is also a group of targeting sequences thatshow various degrees of similarity, particularly within thesignal sequence and plastid-targeting domains of the transitpeptide. Within this group, the targeting sequences of rpL21and rpL3 (tBLASTn E value = 5e–59) are nearly identical,and these sequences share a high degree of similarity with thetargeting sequences of an acyl carrier protein (E = 2e–29)and two different light-harvesting complex (LHC) subunits (E= 2e–39 and 4e–21). Interestingly, the targetingdomain of the first LHCI-like sequence shares a greater degreeof similarity with those of the acyl carrier protein and ribosomalproteins than with the targeting domain of the other LHCI sequence(or of any other LHC sequence in the database). This similarityeven extends into the putative stop-transfer domain, a regionnot expected to be conserved. In other cases, a tBLASTn searchwith a specific targeting sequence allowed the detection, asexpected, of isoforms and members of a multigene family, a resultthat is attributable to gene duplication events. This searchapproach was able to recognize a variety of different plastid-targetingproteins, although the E values were generally >1e–20;thus, some of this similarity could simply be due to the constraintsplaced upon these regions by amino acid composition. In markedcontrast, many other targeting sequences produced no significanthits at all. With the exception of rpL3, which was representedby a single EST, the remainder of the EST clusters analyzedhere comprised multiple overlapping reads, with clear evidenceof a spliced leader sequence, eliminating clustering artifactsas an explanation for the observed similarity.

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FIG. 7. Alignment of targeting sequences from selected Euglena plastid-targeted proteins. (A) Comparison of FNR and CP29 targeting sequences. Identical amino acids are white on a black background. (B) Second group of proteins possessing similar targeting sequences. Identical amino acids compared to the top sequence are indicated by white letters on a black background. The hydrophobic regions of the signal sequence and stop-transfer domains are indicated by lines above the appropriate amino acids. The mature portions of the proteins, if shown, are indicated with double underlining.

DISCUSSION

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Discussion

The discovery of LHC precursors in Golgi dictyosomes of Euglena(53) and subsequent in vitro experiments demonstrated that theEuglena LHCII presequence does indeed possess a functional signalmotif (38), an inference that is strongly supported by the studyreported here. Although the presence of a signal sequence-likeregion was part of our selection criteria, we found no evidencein the entire database of a plastid-targeted protein that lackeda signal sequence, suggesting that in Euglena, all plastid proteinsproceed to the organelle via the endomembrane system. Althoughsome in vitro studies have suggested the potential direct importof proteins into Euglena plastids, thereby bypassing the ER(65), the bulk of relevant biochemical work indicates that transportvia the endomembrane system is required for plastid targeting(38, 67, 69-71). The endomembrane system is also important forplastid targeting in all protists with complex secondary plastids,including those with three (49, 55) and four (4, 7, 16, 19,37, 59, 78, 79) plastid membranes.

In Euglena, proteins targeted to the plastid do not fully insertinto the ER lumen or the membrane during translation due tothe presence of a stop-transfer domain, so the majority of theprotein remains exposed in the cytoplasm (69). Indeed, in classI proteins, the presequence has a second hydrophobic regionfollowed by positively charged amino acids, both of which arecharacteristics typical of stop-transfer sequences (14, 41).Although 2 of the 70 class I proteins lack positively chargedamino acids immediately after the second TMH, such residuesare not an absolute requirement for a stop-transfer function,with the effectiveness of targeting depending on a combinationof hydrophobicity, length, and charge (14, 41, 60). The presenceof a functioning stop-transfer motif in a plastid presequenceis unique to Euglena and dinoflagellates. Both groups have threeplastid membranes, leading Nassoury et al. (49) to suggest thatthe stop-transfer sequence arose from a mechanistic requirementdriven by the number of plastid membranes. It is generally agreedthat Euglena and dinoflagellates are phylogenetically distant;thus, the similarities between their targeting sequences, andpresumably the underlying transport mechanisms, would appearto be convergent as part of a necessary step in protein targeting.

Although targeting in organisms with complex plastids firstrequires import of the protein into the ER, little is knownabout subsequent mechanisms of targeting to the plastid. Inorganisms with three plastid membranes, such as euglenophytesand dinoflagellates, targeting from the ER to the outer plastidmembrane involves vesicular transport via the Golgi system (49,53). The segregation of plastid-bound proteins into the propervesicles may involve receptors located in the endomembrane systemthat recognize the transit peptide and direct the protein toits appropriate destination. This pathway is analogous to thatin animal and fungal systems, where receptors within the endomembranesystem, such as the classic mannose-6-phosphate receptor systemfor targeting to the lysosome (22), are able to recognize featuresof the protein and ensure proper localization. Ultimately, cytoplasmicsorting factors, such as adaptins (9), may play a role in theaccumulation of plastid-targeted proteins and their segregationto vesicles destined for the plastid. Such cytosolic factorscould participate in the recognition of receptors that bindto plastid-targeted proteins and/or specific motifs just beyondthe stop-transfer domain of the targeted protein itself to facilitatetargeting. One potential series of residues includes the clusterof basic amino acids that immediately follows the stop-transferdomain. The importance of short, cytoplasm-exposed targetingmotifs for intracellular sorting is well known (9). For Euglena,Sláviková et al. (67) determined that this cytoplasm-exposedportion of the presequence is not required for plastid importin vitro, but they suggested that it may function in vesiclerouting.

Of particular interest here is our discovery of plastid-targetedproteins lacking the putative stop-transfer sequence (classII), implying that these proteins are inserted entirely intothe ER, leaving a soluble portion within the ER lumen and amembrane portion integrated within the ER membrane, once thesignal sequence is removed. Given that the Euglena class IIproteins comprise both soluble and membrane proteins, it isunlikely that other domains within the mature protein couldimpart a similar stop-transfer effect to compensate for thelack of such a region in the presequence. The targeting routefor class II proteins is conceptually similar to the targetingof proteins to the remnant plastid (apicoplast) in apicomplexans;apicoplast proteins lack the stop-transfer sequence and aretargeted to the plastid via the ER (19), presumably by vesiculartransport. Thus, for correct targeting, the putative transitpeptide, and possibly the mature protein, must contain featuresthat would be recognized by specific cofactors or receptorsthat are localized to the ER lumen, not the cytoplasm. Sinceclass II transit peptides are predicted to lack the stop-transfersequence and thus the cytoplasm-exposed region just beyond,redirection to the plastid must be facilitated solely by interactionwith targeting factors that bind to the TP and allow these precursorsto "hitchhike" in vesicles with the class I proteins. An alternative,albeit unlikely, mechanism is that the class II signal sequenceacts as a signal anchor, with the N terminus facing the ER lumen.However, in this orientation the transit peptide would be facingthe cytoplasm and presumably would be inaccessible to the targetingmachinery.

Even more surprising is the resemblance of this class of targetingsequence to those of dinoflagellates, whose plastid-targetedproteins also exhibit a similar proportion of presequences lackingstop-transfer domains (55), with the remainder resembling classI proteins. As possible explanations for the dinoflagellates,Patron et al. (55) ruled out the evolutionary history of thegene transfer or final destination of the protein, suggestinginstead that the "physical characteristics" of the plastid-targetedprotein may determine the nature of its presequence. In supportof the latter hypothesis, they found that the class I and IIdistinction was conserved between proteins in two dinoflagellatesexamined. If "physical characteristics" was the main factordetermining the mode of transport, then we would predict thatEuglena would exhibit a similar distribution of proteins havingclass I and II presequences. Some similarities are clearly evident,such as with phosphoribulose kinase and oxygen-evolving enhancer3 (PsbO), which lack a stop-transfer sequence in both dinoflagellatesand Euglena. However, other dinoflagellate proteins with classII (and III) targeting sequences are class I proteins in Euglena(acyl carrier protein, carbonic anhydrase, cytochrome c₆, andthe PSII 11-kDa protein). Although the sample size for comparisonis small, there do not appear to be any obvious inherent functionalor physical properties that would require a class I versus classII targeting sequence. In vitro import assays should help todefine the functional requirements of the different classesof presequence and determine whether either is essential forthe import of specific proteins.

With the exception of apicomplexans, complex plastids with fourplastid membranes often have ribosomes attached to the outermembrane (chloroplast ER [CER]). However, the primary plastid-sortingmechanism must still occur after cotranslational import across/intothe ER membrane, since in diatoms the signal sequences of ERand plastid-resident proteins are functionally equivalent (37),and in a raphidophyte, few ribosomes are bound to the CER (30).Thus, once inserted into the endomembrane system, the plastid-boundproteins still have to be targeted to and transverse at leastthree membranes, similar to the situation in Euglena and dinoflagellates.Though not involving the Golgi dictyosomes, a vesicular transferbetween the CER and the third membrane has been proposed (23),and a recent report supports such a mechanism (37). Apicomplexans,in particular, provide a valuable model system for dissectingthe targeting process in complex plastids with four membranes,with several studies indicating not only that there is partiallyredundant targeting information in the presequences of apicoplast-targetedproteins (26, 81, 82) but also that there is a distinction betweenthe information for targeting and that for import into the apicoplast(26). Recent work has even identified proteins that interactwith the TP and that may be involved in sorting from the ERto the apicoplast (82).

In Euglena, the region between the signal sequence and the stop-transfersequence in class I proteins functions as a TP (67). This regionand the TPs of class II proteins possess characteristics typicalof most TPs. These similarities include enrichment in Ser/Thr(S/T bias) and Ala. S/T bias is a common feature of most transitpeptides of plants and algae (2, 4, 11, 15, 20, 49, 54, 55,59, 76). Some notable exceptions to this rule include apicomplexans(77, 78) and nucleomorph-encoded plastid proteins from the cryptomonadalga Guillardia theta (57). Replacement of all Ser/Thr residuesin the TP of Plasmodium had no effect on plastid targeting,demonstrating a lack of a requirement for such residues (78).Although an elevated Ser/Thr content is evidently dispensablein apicomplexans, it remains one of the more consistent featuresof most TPs, which may reflect a requirement for phosphorylation-dependentbinding of 14-3-3 proteins as part of a preinsertion guidancecomplex (46).

Euglena TPs also have an overall positive charge, an apparentlyuniversal feature of TPs (57), that is primarily due to a reductionin the content of acidic amino acids. Of particular interestis the asymmetric distribution of acidic amino acids in theTP, with the first two-thirds being deficient in such residues,whereas the remaining third has a composition resembling thatof the mature protein. This asymmetry may reflect a distinctionbetween functional TPs (with a bias against acidic residues)and regions having a different function. The importance of aTP depleted in acidic residues was demonstrated in Plasmodium,where the replacement of basic with acidic amino acids eliminatedapicoplast targeting (19). Interestingly, Euglena TPs are alsodeficient in Lys (but not Arg) and have biases in favor of Alaand Pro compared to mature proteins, which are also featuresof the TPs of the chlorarachniophyte Bigelowiella natans (59).Some of the shared features of TPs, such as a bias against acidicamino acids and a bias in favor of some hydrophobic residues,may be due to a requirement for binding of import factors, suchas molecular chaperones (Hsp70) (83). Although the biologicalsignificance of the biased amino acid composition in TPs isnot entirely understood, and despite any differences in primarystructure, TPs from diverse plastid types are functionally sufficientin heterologous import assays (3, 32, 42, 49, 67, 79).

The striking amino acid similarity between certain plastid-targetingsequences is surprising. In general, transit peptides lack evidentsequence similarity, even among paralogs of the same gene family,so the detection of clusters of related targeting sequencesmay shed light on how targeting sequences were acquired followingtransfer of the endosymbiont's genes to the host nucleus duringplastid evolution. Reports of highly similar plastid and mitochondrialTPs are relatively rare, but the examples can be separated intotwo categories. In the first case, homologs from different speciesexhibit a greater-than-expected similarity within the TP regioncompared to that of the mature proteins, which is attributedto a conserved functional role (80). The second category includesunrelated proteins that possess highly similar TPs (1, 5, 40,45), which is what we observe in Euglena. This similarity isoften attributed to exon shuffling, as introns commonly separatethe transit peptide from the mature protein (36, 45). Thereare also reports of transit peptide acquisition through insertioninto preexisting genes for plastid (5)- and mitochondrion-targeted(1) proteins. Thus, the newly transferred genes would acquirenot only the targeting mechanism but also the regulatory sequencesrequired for expression, in the so-called "lucky insertion scenario"(21). Although we lack the appropriate genomic information fromEuglena to be able to completely assess the mechanism of TPacquisition, a genomic sequence for an LHCII gene of this organismdoes have an intron that roughly separates the predicted targetingdomain from the mature protein (48), suggesting exon shufflingas a potential mode of TP acquisition. However, the similarityof the rpL3 and rpL21 presequences to a small portion of theLHCI mature protein (GFDPLGL) (Fig. 7) suggests that TP acquisitionby insertion into a preexisting copy of the LHCI gene is alsoa strong possibility. The maintenance of a continued high degreeof conservation between rpL21-rpL3 and CP29-FNR could also implyrecent recombination, or perhaps alternative splicing, as describedfor rice mitochondrion-targeted rpS14 and SDHB proteins (40).The pronounced sequence conservation within these regions alsoraises the possibility that these targeting sequences have anadditional function(s) in the cell, either before or after cleavage,as proposed for some mammalian signal sequences (10, 17).

In summary, we have characterized two distinct classes of Euglenaplastid presequences, i.e., classes I and II, that differ bythe presence and absence of a predicted stop-transfer sequence,respectively, revealing an additional level of complexity inthe protein transport mechanism. In addition to enhancing ourability to predict Euglena presequences, we expect that thecharacteristics of these TPs will stimulate further import studies,both in vitro and in vivo, seeking to dissect the processesof targeting and import into the complex plastids of Euglena.

ACKNOWLEDGMENTS

This work was carried out under the auspices of a Genome Canadalarge-scale genomics project, the Protist EST Program, withfunding provided through Genome Atlantic and the Atlantic InnovationFund. M.W.G. gratefully acknowledges salary support from theCanada Research Chairs Program and the Canadian Institute forAdvanced Research (Program in Evolutionary Biology). D.G.D.also thanks the Natural Sciences and Engineering Research Council(NSERC) for ongoing support.

We are grateful to Patrick Keeling for sharing a paper on dinoflagellatetargeting sequences prior to publication. We also thank SteveHeard and Penny Humby for helpful discussions on statistics.The technical assistance of H. Rissler, who isolated RNAs fromEuglena for the construction of two of the five cDNA librariessequenced for this study, is acknowledged.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada E3B 5A3. Phone: (506) 452-6207. Fax: (453) 453-3583. E-mail: durnford{at}unb.ca.

Published ahead of print on 22 September 2006.

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