Analysis of Euglena gracilis plastid-targeted proteins reveals different classes of transit sequences

Abstract

The plastid of Euglena gracilis was acquired secondarily through an endosymbiotic event with a eukaryotic green alga, and as a result, it is surrounded by a third membrane. This membrane complexity raises the question of how the plastid proteins are targeted to and imported into the organelle. To further explore plastid protein targeting in Euglena, we screened a total of 9,461 expressed sequence tag (EST) clusters (derived from 19,013 individual ESTs) for full-length proteins that are plastid localized to characterize their targeting sequences and to infer potential modes of translocation. Of the 117 proteins identified as being potentially plastid localized whose N-terminal targeting sequences could be inferred, 83 were unique and could be classified into two major groups. Class I proteins have tripartite targeting sequences, comprising (in order) an N-terminal signal sequence, a plastid transit peptide domain, and a predicted stop-transfer sequence. Within this class of proteins are the lumen-targeted proteins (class IB), which have an additional hydrophobic domain similar to a signal sequence and required for further targeting across the thylakoid membrane. Class II proteins lack the putative stop-transfer sequence and possess only a signal sequence at the N terminus, followed by what, in amino acid composition, resembles a plastid transit peptide. Unexpectedly, a few unrelated plastid-targeted proteins exhibit highly similar transit sequences, implying either a recent swapping of these domains or a conserved function. This work represents the most comprehensive description to date of transit peptides in Euglena and hints at the complex routes of plastid targeting that must exist in this organism.

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.

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.

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.

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 [α ≤ 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.

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).

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.

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.