Oliver D. Caspari

Background: Leontynka is a non-photosynthetic lineage of the order Chlamydomonadales (Chlorophyta). Although many Chlamydomonadales members encode components of the anaerobic energy metabolism, studies focused on Chlamydomonadales algae thriving in hypoxia and not prospering in oxic conditions are missing. Using a combination of experimental approaches, comparative genomics, and advanced in silico protein localization analyses, we employed Leontynka as a model to investigate the evolution of anaerobiosis in Chlamydomonadales. Results: Leontynka spp. accumulate a wide range of storage forms, enabling them to cope with nutritional stresses. Their mitochondria contain well-developed cristae mediating a conventional aerobic energy metabolism. Moreover, colocalization of a Raman signal for cytochromes with the position of mitochondria in the cell indicates that oxidative phosphorylation is an important route of energy metabolism in the alga. Interestingly, Leontynka spp. concentrate enzymes potentially involved in oxygen-independent ATP synthesis within the plastid, which lost the ability to produce ATP using proton gradient generated by membrane complexes that exploit redox reactions. We analyzed the composition of prokaryotic communities co-isolated with Leontynka spp. and hypothesize that their preference for hypoxic/microoxic conditions is facilitated by metabolic interactions with certain microaerophilic and anaerobic bacteria. Conclusions: This study represents the first comprehensive analysis of microaerophilic Chlamydomonadales algae. Having retained several ancestral enzymes of the anaerobic energy metabolism, Leontynka represents a unique vantage point for understanding the evolution of the hydrogen production machinery and adaptations to low oxygen in Chlamydomonadales (and core chlorophytes in general). Our findings suggest that the plastid of non-photosynthetic Leontynka follows a similar evolutionary path as mitochondria when adapting to anaerobiosis and parallels the transition of a mitochondrion into a hydrogenosome.

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, abbreviated as SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets (e.g. nuclear proteins such as cbp1 and mis16, and the centromere-specific histone protein cnp1). The SExY protocol optimizations increasing protein yield could be beneficial for many studies, when targeting low abundance proteins, or for studies that rely on genetic labelling for various reasons (e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality).

SUMMARY: Chloroplast ATP synthase contains subunits of plastid and nuclear genetic origin. To investigate the coordinated biogenesis of this complex, we isolated novel ATP synthase mutants in the green alga Chlamydomonas reinhardtii by screening for high light sensitivity. We report here the characterization of mutants affecting the two peripheral stalk subunits b and b′, encoded respectively by the atpF and ATPG genes, and of three independent mutants which identify the nuclear factor MDE1, required to stabilize the chloroplast‐encoded atpE mRNA. Whole‐genome sequencing revealed a transposon insertion in the 3′UTR of ATPG while mass spectrometry shows a small accumulation of functional ATP synthase in this knock‐down ATPG mutant. In contrast, knock‐out ATPG mutants, obtained by CRISPR‐Cas9 gene editing, fully prevent ATP synthase function and accumulation, as also observed in an atpF frame‐shift mutant. Crossing ATP synthase mutants with the ftsh1‐1 mutant of the major thylakoid protease identifies AtpH as an FTSH substrate, and shows that FTSH significantly contributes to the concerted accumulation of ATP synthase subunits. In mde1 mutants, the absence of atpE transcript fully prevents ATP synthase biogenesis and photosynthesis. Using chimeric atpE genes to rescue atpE transcript accumulation, we demonstrate that MDE1, a novel octotricopeptide repeat (OPR) protein, genetically targets the atpE 5′UTR. In the perspective of the primary endosymbiosis (~1.5 Gy), the recruitment of MDE1 to its atpE target exemplifies a nucleus/chloroplast interplay that evolved rather recently, in the ancestor of the CS clade of Chlorophyceae, ~300 My ago.

The N-terminal sequence stretch that defines subcellular targeting for most nuclear encoded chloroplast proteins is usually considered identical to the sequence that is cleaved upon import. Yet here this study shows that for eight out of ten tested Chlamydomonas chloroplast transit peptides, significant additional sequence stretches past the cleavage site are required to enable efficient chloroplast import of heterologous cargo proteins. Analysis of Chlamydomonas cTPs with known cleavage sites and replacements of native post-cleavage residues with alternative sequences points to a role for unstructured sequence at mature protein N-termini.

Summary: We asked what peptide features govern targeting to the mitochondria versus the chloroplast using antimicrobial peptides as a starting point. This approach was inspired by the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell, to which organelle progenitors became resistant. To explore the molecular changes required to convert antimicrobial into targeting peptides, we expressed a set of 13 antimicrobial peptides in Chlamydomonas reinhardtii . Peptides were systematically modified to test distinctive features of mitochondrial and chloroplast targeting peptides, and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as cargo protein. Mitochondrial targeting can be achieved by some unmodified antimicrobial peptide sequences. Targeting to both organelles is improved by replacing Lysines with Arginines. Chloroplast targeting is enabled by the presence of flanking unstructured sequences, additional constraints consistent with chloroplast endosymbiosis having occurred in a cell that already contained mitochondria. If indeed targeting peptides evolved from antimicrobial peptides, required modifications imply a temporal evolutionary scenario with an early exchange of cationic residues, and a late acquisition of chloroplast specific motifs.

Expression of proteins in the chloroplast or mitochondria of the model green alga Chlamydomonas reinhardtii can be achieved by directly inserting transgenes into organellar genomes, or through nuclear expression and post-translational import. A number of tools have been developed in the literature for achieving high expression levels from the nuclear genome despite messy genomic integration and widespread silencing of transgenes. Here, recent advances in the field are combined and two systems of bicistronic expression, based on ribosome reinitiation or ribosomal skip induced by a viral 2A sequence, are compared side-by-side. Further, the small subunit of Rubisco (RBCS) was developed as a functional nuclear reporter for successful chloroplast import and restoration of photosynthesis: To be able to combine RBCS with a Venus fluorescent reporter without compromising photosynthetic activity, a leaky stop codon is introduced as a novel molecular tool that allows the simultaneous expression of functional and fluorescently tagged versions of the protein from a single construct.

Mitochondria and chloroplasts emerged from primary endosymbiosis. Most proteins of the endosymbiont were subsequently expressed in the nucleo-cytosol of the host and organelle-targeted via the acquisition of N-terminal presequences, whose evolutionary origin remains enigmatic. Using a quantitative assessment of their physico-chemical properties, we show that organelle targeting peptides, which are distinct from signal peptides targeting other subcellular compartments, group with a subset of antimicrobial peptides. We demonstrate that extant antimicrobial peptides target a fluorescent reporter to either the mitochondria or the chloroplast in the green alga Chlamydomonas reinhardtii and, conversely, that extant targeting peptides still display antimicrobial activity. Thus, we provide strong computational and functional evidence for an evolutionary link between organelle-targeting and antimicrobial peptides. Our results support the view that resistance of bacterial progenitors of organelles to the attack of host antimicrobial peptides has been instrumental in eukaryogenesis and in the emergence of photosynthetic eukaryotes.

Expression of proteins in the chloroplast or mitochondria of the model green alga Chlamydomonas reinhardtii can be achieved by directly inserting transgenes into organellar genomes, or through nuclear expression and post-translational import. A number of tools have been developed in the literature for achieving high expression levels from the nuclear genome despite messy genomic integration and widespread silencing of transgenes. Here, recent advances in the field are combined and two systems of bicistronic expression, based on ribosome reinitiation or ribosomal skip induced by a viral 2A sequence, are compared side-by-side. Further, the small subunit of Rubisco (RBCS) was developed as a functional nuclear reporter for successful chloroplast import and restoration of photosynthesis: To be able to combine RBCS with a Venus fluorescent reporter without compromising photosynthetic activity, a leaky stop codon is introduced as a novel molecular tool that allows the simultaneous expression of functional and fluorescently tagged versions of the protein from a single construct.