Trafficking of protein into the recently established photosynthetic organelles of Paulinella chromatophora

Abstract

Endosymbiotic acquisition of bacteria by a protist, with subsequent evolution of the bacteria into mitochondria and plastids, had a transformative impact on eukaryotic biology. Reconstructing events that created a stable association between endosymbiont and host during the process of organellogenesis--including establishment of regulated protein import into nascent organelles--is difficult because they date back more than 1 billion years. The amoeba Paulinella chromatophora contains nascent photosynthetic organelles of more recent evolutionary origin (∼60 Mya) termed chromatophores (CRs). After the initial endosymbiotic event, the CR genome was reduced to approximately 30% of its presumed original size and more than 30 expressed genes were transferred from the CR to the amoebal nuclear genome. Three transferred genes--psaE, psaK1, and psaK2--encode subunits of photosystem I. Here we report biochemical evidence that PsaE, PsaK1, and PsaK2 are synthesized in the amoeba cytoplasm and traffic into CRs, where they assemble with CR-encoded subunits into photosystem I complexes. Additionally, our data suggest that proteins routed to CRs pass through the Golgi apparatus. Whereas genome reduction and transfer of genes from bacterial to host genome have been reported to occur in other obligate bacterial endosymbioses, this report outlines the import of proteins encoded by such transferred genes into the compartment derived from the bacterial endosymbiont. Our study showcases P. chromatophora as an exceptional model in which to study early events in organellogenesis, and suggests that protein import into bacterial endosymbionts might be a phenomenon much more widespread than currently assumed.

Fig. 1.

Immunogold EM of sectioned P. chromatophora cells. (A) Cell cross-section. (B) Detailed cross-section of CR labeled with α-PsaE_pepC and α-rabbit-IgG-15 nm gold. (C) Close-up of area highlighted by the rectangle in B. Note dense decoration of thylakoids with gold particles (crisp black dots). (D) Cross-sectioned CR labeled with preimmune serum and α-rabbit-IgG-15 nm gold. Black arrows highlight gold particles. M, mitochondria; N, nucleus; PM, plasma membrane; SS, silica scales; T, theca.

Fig. 2.

Characterization of PSI isolated from P. chromatophora. (A) Fifteen micrograms of protein of PSI was resolved by SDS/PAGE on an 18% Schägger gel containing 7 M urea. Polypeptides were transferred to PVDF membranes for Western blot analysis using antibodies to various PSI subunits as indicated (lanes 1–5). Additionally, 140 μg protein of isolated PSI was resolved in the same gel system and stained with Sypro Ruby (lane 6); individual polypeptides were excised from the gels for MS analysis (PsaA/PsaB band) or transferred to PVDF membranes for N-terminal sequencing by Edman degradation (PsaE and PsaK1/PsaK2 band). (B) Edman degradation. Sequences shown represent the beginning of the full-length mRNA and their deduced translation products, with predicted initiator methionines encircled and stop codons designated by asterisks. Within the gray box are identities and quantities of the first 10 N-terminal amino acid residues, determined by Edman degradation. (C) Equal concentrations of protein (15 μg per lane) from isolated PSI complexes (left lanes) and from whole-cell extracts (right lanes) were resolved by SDS/PAGE, transferred to PVDF membranes, and subjected to immunoblot analyses using α-PsaC, α-PsaD, α-PsaE_pepN, α-PsaF, and α-PsaL. (D) Equal amounts of protein (15 μg per lane) from PSI complexes (lanes 1 and 4), thylakoid membranes (lanes 2 and 5), and whole-cell extracts (lanes 3 and 6) were resolved by SDS/PAGE, stained by CBB (lanes 1–3), or transferred to PVDF membranes and subjected to immunoblot analysis by using α-PsbA, α-PB, and α-PsaE_pepN. (lanes 4–6).

Fig. 3.

Translation of PsaE, PsaK1, and PsaK2 on 80S ribosomes. Equal amounts (45 μg protein per lane) of P. chromatophora PSI labeled in vivo with NaH¹⁴CO₃ without translation inhibitors (lane 1, A and B), in the presence of chloramphenicol (lane 2, A and B), cycloheximide (lane 3, A and B), or both (lane 4, A and B) were resolved by SDS/PAGE on an 18% polyacrylamide, 7 M urea Schägger gel. Resolved polypeptides were stained with CBB (A) and the ¹⁴C signal visualized using a PhosphorImager (B). Asterisks and arrowheads highlight presence or absence (respectively) of radiolabeled PsaE and PsaK1/PsaK2.

Fig. 4.

Immunogold EM of various compartments of P. chromatophora cells. (A) Labeling with α-PsaE_pepC and α-rabbit-IgG-15 nm gold. (B) Close up of Golgi in same section (area highlighted by black rectangle in A). Note the decoration of Golgi with gold particles (crisp black dots). (C) Statistical analysis of gold particle densities over CRs (Cr), Golgi (G), and other cell compartments (oC) in cells Immunogold-labeled with α-PsaE_pepC or with preimmune serum. Displayed are mean and SD; n = 6. One-way ANOVA with repeated measures for antibody treatment revealed differences for mean gold particle densities in CR, Golgi, and other cell compartments [F_(2,5) = 129.6; P < 0.001]; for preimmune treatment, no significant differences were found [F_(2,5) = 1.282; P = 0.32]. (*P < 0.001, Holm–Sidak test.) M, mitochondria; N, nucleus; PM, plasma membrane; T, theca.