Metamonada is a eukaryotic supergroup of free-living and parasitic anaerobic protists. Their characteristic feature is the presence of highly reduced mitochondria that have lost the ability to produce ATP by oxidative phosphorylation and in some cases even by substrate phosphorylation, with all ATP being imported from the cytosol. Given this striking difference in cellular ATP metabolism when compared to aerobic mitochondria, we studied the presence of mitochondrial carrier proteins (MCPs) mediating the transport of ATP across the inner mitochondrial membrane. Our bioinformatic analyses revealed remarkable reduction of MCP repertoire in Metamonada with striking loss of the major ADP/ATP carrier (AAC). Instead, nearly all species retained carriers orthologous to human SLC25A43 protein, a little-characterized MCP. Heterologous expression of metamonad SLC25A43 carriers confirmed their mitochondrial localization, and functional analysis revealed that SLC25A43 orthologues represent a distinct group of ATP transporters, which we designate as ATP-importing carriers (AIC). Together, our findings suggest that AIC facilitate the ATP import into highly reduced anaerobic mitochondria, compensating for their diminished or absent energy metabolism.

• Keywords
• ADP/ATP carrier, Metamonada, SLC25A43, mitochondrial carrier protein, mitochondrial evolution, mitochondrion-related organelle,
• Publication type
• Journal Article MeSH

Myeloid leukemia factor 1 (Mlf1) was identified as a proto-oncoprotein that affects hematopoietic differentiation in humans. However, its cellular function remains elusive, spanning roles from cell cycle regulation to modulation of protein aggregate formation and participation in ciliogenesis. Given that structurally conserved homologs of Mlf1 can be found across the eukaryotic tree of life, we decided to characterize its cellular role underlying this phenotypic pleiotropy. Using a model of the unicellular eukaryote Giardia intestinalis, we demonstrate that its Mlf1 homolog (GiMlf) mainly localizes to two types of cytosolic foci: microtubular structures, where it interacts with Hsp40, and ubiquitin-rich, membraneless compartments, found adjacent to mitochondrion-related organelles known as mitosomes, containing the 26S proteasome regulatory subunit 4. Upon cellular stress, GiMlf either relocates to the affected compartment or disperses across the cytoplasm, subsequently accumulating into enlarged foci during the recovery phase. In vitro assays suggest that GiMlf can be recruited to membranes through its affinity for signaling phospholipids. Importantly, cytosolic foci diminish in the gimlf knockout strain, which exhibits extensive proteomic changes indicative of compromised proteostasis. Consistent with data from other cellular systems, we propose that Mlf acts in the response to proteotoxic stress by mediating the formation of function-specific foci for protein folding and degradation.

• MeSH
• Giardia lamblia * metabolism   MeSH
• Humans MeSH
• Proteolysis * MeSH
• Protozoan Proteins * metabolism   genetics   MeSH
• Protein Folding * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Protozoan Proteins * MeSH

Mitochondrial metabolism is entirely dependent on the biosynthesis of the [4Fe-4S] clusters, which are part of the subunits of the respiratory chain. The mitochondrial late ISC pathway mediates the formation of these clusters from simpler [2Fe-2S] molecules and transfers them to client proteins. Here, we characterized the late ISC pathway in one of the simplest mitochondria, mitosomes, of the anaerobic protist Giardia intestinalis that lost the respiratory chain and other hallmarks of mitochondria. In addition to IscA2, Nfu1 and Grx5 we identified a novel BolA1 homologue in G. intestinalis mitosomes. It specifically interacts with Grx5 and according to the high-affinity pulldown also with other core mitosomal components. Using CRISPR/Cas9 we were able to establish full bolA1 knock out, the first cell line lacking a mitosomal protein. Despite the ISC pathway being the only metabolic role of the mitosome no significant changes in the mitosome biology could be observed as neither the number of the mitosomes or their capability to form [2Fe-2S] clusters in vitro was affected. We failed to identify natural client proteins that would require the [2Fe-2S] or [4Fe-4S] cluster within the mitosomes, with the exception of [2Fe-2S] ferredoxin, which is itself part of the ISC pathway. The overall uptake of iron into the cellular proteins remained unchanged as also observed for the grx5 knock out cell line. The pull-downs of all late ISC components were used to build the interactome of the pathway showing specific position of IscA2 due to its interaction with the outer mitosomal membrane proteins. Finally, the comparative analysis across Metamonada species suggested that the adaptation of the late ISC pathway identified in G. intestinalis occurred early in the evolution of this supergroup of eukaryotes.

• MeSH
• Anaerobiosis MeSH
• Giardia lamblia * genetics   metabolism   MeSH
• Humans MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria metabolism   MeSH
• Iron-Sulfur Proteins * genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Mitochondrial Proteins MeSH
• Iron-Sulfur Proteins * MeSH

Tail-anchored (TA) membrane proteins, accounting for roughly 2% of proteomes, are primarily targeted posttranslationally to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. For this complicated process, it remains unknown how the central targeting factor Get3 uses nucleotide to facilitate large conformational changes to recognize then bind clients while also preventing exposure of hydrophobic surfaces. Here, we identify the GET pathway in Giardia intestinalis and present the structure of the Get3-client complex in the critical postnucleotide-hydrolysis state, demonstrating that Get3 reorganizes the client-binding domain (CBD) to accommodate and shield the client transmembrane helix. Four additional structures of GiGet3, spanning the nucleotide-free (apo) open to closed transition and the ATP-bound state, reveal the details of nucleotide stabilization and occluded CBD. This work resolves key conundrums and allows for a complete model of the dramatic conformational landscape of Get3.

• MeSH
• Adenosine Triphosphatases metabolism   MeSH
• Humans MeSH
• Saccharomyces cerevisiae Proteins * metabolism   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Protein Transport MeSH
• Guanine Nucleotide Exchange Factors metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• Guanine Nucleotide Exchange Factors MeSH

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.

• Keywords
• CRISPR/Cas9, Giardia, gene knockout, multiploid,
• MeSH
• CRISPR-Cas Systems * MeSH
• Gene Editing methods   MeSH
• Giardia lamblia * genetics   MeSH
• Humans MeSH
• Tetraploidy MeSH
• RNA, Guide, CRISPR-Cas Systems MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Guide, CRISPR-Cas Systems MeSH

BACKGROUND: Eukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which is responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs; however, less is known about the role of these proteins across eukaryotic diversity. RESULTS: Here, we investigated the distribution and function of PufSF RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) 'classical' Pufs, which control the translation of mRNA; and (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors ('classical' Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of 'classical' Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization. CONCLUSIONS: Our data suggest that the last eukaryotic common ancestor (LECA) already contained all three types of PufSF proteins and that 'classical' Pufs then underwent lineage-specific expansions.

• Keywords
• Giardia intestinalis, LECA, Puf superfamily proteins, RNA processing, RNA-binding protein,
• MeSH
• Eukaryota genetics   metabolism   MeSH
• Phylogeny MeSH
• RNA, Messenger metabolism   MeSH
• RNA-Binding Proteins chemistry   genetics   metabolism   MeSH
• Protein Biosynthesis * MeSH
• RNA, Ribosomal, 18S metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Messenger MeSH
• RNA-Binding Proteins MeSH
• RNA, Ribosomal, 18S MeSH