N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

Abstract

bMitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PP i -dependent phosphofructokinase (TvPP i -PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPP i -PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to "short" bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.T he transition of the mitochondrion into an ATP-producing organelle was the crucial event at the eukaryote origin (1). ATP synthesis in eukaryotes is typically compartmentalized, with glycolysis in the cytosol and pyruvate oxidation in the mitochondria, which is linked to highly efficient oxidative phosphorylation (1, 2). In protists, however, there are notable exceptions to the usual scheme regarding both glycolysis and pyruvate oxidation. In Trichomonas vaginalis and other eukaryotes that possess an anaerobic form of mitochonria called hydrogenosomes, pyruvate is oxidized within the organelle via less efficient anaerobic fermentation (3). Giardia intestinalis, Entamoeba histolytica, and other eukaryotes possess a reduced form of mitochondria called mitosomes that do not produce ATP at all (4). In these organisms, pyruvate oxidation takes place exclusively in the cytosol (1). In kinetoplastids, glycolysis is compartmentalized in specialized microbodies called glycosomes (5). In some green algae, the first half of the glycolytic pathway is localized in the chloroplast (6, 7), while in the diatom Phaeodactylum tricornutum and other stramenopiles, several glycolytic enzymes are targeted to multiple compartments, such...

Investigation of the Secretory Pathway in Trichomonas vaginalis Argues against a Moonlighting Function of Hydrogenosomal Enzymes

“…Moreover, recombinant PDI‐BirA‐V5 was expressed under the control of a weak promoter to avoid any possible mistargeting due to the strong expression of recombinant proteins (Rada et al. ). In transfected cells, PDI‐BirA‐V5 appeared in the typical ER ring around the nucleus and tubulovesicular structures (Fig.…”

“…Therefore, we transformed the cell line expressing BirA-V5 in the cytosol with vectors that allowed the expression of the complete aor b-SCS with a C-terminal ACP and HA tag. Immunofluorescence microscopy of double-transfected trichomonads revealed that biotinylated aand b-SCS-ACP-HA localized exclusively to spherical vesicles exhibiting a distribution, number, and shape corresponding to hydrogenosomes (Dolezal et al 2005;Hrd y et al 2004;Rada et al 2011Rada et al , 2015Wexler-Cohen et al 2014). No labeling of structures associated with the secretory pathway or plasma membrane was observed.…”

“…Then, the proteins are processed and further exported via the Golgi apparatus (Trombetta and Parodi 2003), although some membrane proteins can reach the cell surface independently on the Golgi (Nickel and Rabouille 2009;Nickel and Seedorf 2008;Rabouille 2017). Therefore, it is not clear how the hydrogenosomal enzymes enter the secretory pathway, as a peptide secretion signal is absent in these proteins (Kucknoor et al 2007), and previous cell localization studies of PFO, malic enzyme, and SCS subunits have visualized these proteins exclusively in hydrogenosomes (Brugerolle et al 2000;Dolezal et al 2005;Garg et al 2015;Hrd y et al 2004;Rada et al 2011Rada et al , 2015Smutna et al 2009;Smutn a et al 2014;Sutak et al 2004;Zimorski et al 2013).…”

Targeting of tail‐anchored proteins to Trichomonas vaginalis hydrogenosomes

“…The cells were prepared for immunofluorescence microscopy as described elsewhere (Rada et al ., ). The recombinant proteins were detected in trichomonads using a mouse monoclonal anti‐HA antibody or a rabbit monoclonal anti‐V5 antibody (Abcam).…”

“…The cells were fixed on high‐precision cover glasses (Carl Zeiss, Germany) for super‐resolution microscopy and prepared for immunofluorescence microscopy as described elsewhere (Rada et al ., ). The recombinant proteins were detected in trichomonads using a mouse monoclonal anti‐HA antibody (Exbio, Czech Republic).…”