The Puzzle of Metabolite Exchange and Identification of Putative Octotrico Peptide Repeat Expression Regulators in the Nascent Photosynthetic Organelles of Paulinella chromatophora

Abstract

The endosymbiotic acquisition of mitochondria and plastids more than one billion years ago was central for the evolution of eukaryotic life. However, owing to their ancient origin, these organelles provide only limited insights into the initial stages of organellogenesis. The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles—termed chromatophores—that evolved from a cyanobacterium ∼100 million years ago, independently from plastids in plants and algae. Despite the more recent origin of the chromatophore, it shows tight integration into the host cell. It imports hundreds of nucleus-encoded proteins, and diverse metabolites are continuously exchanged across the two chromatophore envelope membranes. However, the limited set of chromatophore-encoded solute transporters appears insufficient for supporting metabolic connectivity or protein import. Furthermore, chromatophore-localized biosynthetic pathways as well as multiprotein complexes include proteins of dual genetic origin, suggesting that mechanisms evolved that coordinate gene expression levels between chromatophore and nucleus. These findings imply that similar to the situation in mitochondria and plastids, also in P. chromatophora nuclear factors evolved that control metabolite exchange and gene expression in the chromatophore. Here we show by mass spectrometric analyses of enriched insoluble protein fractions that, unexpectedly, nucleus-encoded transporters are not inserted into the chromatophore inner envelope membrane. Thus, despite the apparent maintenance of its barrier function, canonical metabolite transporters are missing in this membrane. Instead we identified several expanded groups of short chromatophore-targeted orphan proteins. Members of one of these groups are characterized by a single transmembrane helix, and others contain amphipathic helices. We hypothesize that these proteins are involved in modulating membrane permeability. Thus, the mechanism generating metabolic connectivity of the chromatophore fundamentally differs from the one for mitochondria and plastids, but likely rather resembles the poorly understood mechanism in various bacterial endosymbionts in plants and insects. Furthermore, our mass spectrometric analysis revealed an expanded family of chromatophore-targeted helical repeat proteins. These proteins show similar domain architectures as known organelle-targeted expression regulators of the octotrico peptide repeat type in algae and plants. Apparently these chromatophore-targeted proteins evolved convergently to plastid-targeted expression regulators and are likely involved in gene expression control in the chromatophore.

Computational Methods and Tools in Antimicrobial Peptide Research

“…In the context of AMPs, n-grams are used to predict amino acid motifs with AMP activity. This allows AMPGram to not only check the activity of novel peptides but also scan for so-called “cryptic” AMPs, which are antimicrobial domains and segments of proteins that do not have antimicrobial activity and are obtained from proteolysis of the parent sequence. , For example, this approach has been used to find AMP-likely segments in the genome of chromatophores from the amoeba Paulinella chromotophora, in an effort to understand endosymbiosis and organellogenesis . Finally, XGBoost, which stands for eXtreme gradient boosting, is a relatively new ML algorithm.…”

“…146,147 For example, this approach has been used to find AMP-likely segments in the genome of chromatophores from the amoeba Paulinella chromotophora, in an effort to understand endosymbiosis and organellogenesis. 148 Finally, XGBoost, 145 which stands for eXtreme gradient boosting, is a relatively new ML algorithm. Gradient boosting, the parent model, iteratively constructs weak predictors which become stronger as the ensemble grows; XGBoost is a highly optimized version of this process that focuses on efficiency.…”

Why is primary endosymbiosis so rare?

“…Most solute transporters encoded on the chromatophore genome have been lost and, surprisingly, this has not been compensated by the retargeting of nuclear‐encoded transporters. Instead, the amoeba appears to have evolved small, single transmembrane helix containing proteins that are chromatophore‐targeted and may modulate membrane permeability (Oberleitner et al ., 2020).…”

Pas de Trois: An Overview of Penta-, Tetra-, and Octo-Tricopeptide Repeat Proteins From Chlamydomonas reinhardtii and Their Role in Chloroplast Gene Expression

“…Their structure is characterized by the presence of motifs of 38–40 amino acids repeated in tandem, in the same way as in PPR and TPR proteins, where each motif forms a pair of antiparallel α-helices ( Eberhard et al, 2011 ; Wang et al, 2015 ). Besides green algae, OPR proteins are present in some pathogenic bacteria ( Coxiella burnetii ; Rahire et al, 2012 ), symbiotic protozoa (e. g. Plasmodium falciparum , and Toxoplasma gondii ; Hillebrand et al, 2018 ; Hollin et al, 2021 ) and the cercozoan amoeba Paulinella chromatophora ( Oberleitner et al, 2020 ); but are rare in land plants. In A. thaliana , the presence of only one OPR protein has been reported, called RAP, which is involved in the processing of the 16S ribosomal RNA chloroplast gene ( Kleinknecht et al, 2014 ).…”