An Entamoeba-Specific Mitosomal Membrane Protein with Potential Association to the Golgi Apparatus

Abstract

The aerobic mitochondrion had undergone evolutionary diversification, most notable among lineages of anaerobic protists. Entamoeba is one of the genera of parasitic protozoans that lack canonical mitochondria, and instead possess mitochondrion-related organelles (MROs), specifically mitosomes. Entamoeba mitosomes exhibit functional reduction and divergence, most exemplified by the organelle’s inability to produce ATP and synthesize iron-sulfur cluster. Instead, this organelle is capable of sulfate activation, which has been linked to amoebic stage conversion. In order to understand other unique features and components of this MRO, we utilized an in silico prediction tool to screen transmembrane domain containing proteins in the mitosome proteome. Here, we characterize a novel lineage-specific mitosomal membrane protein, named Entamoeba transmembrane mitosomal protein of 30 kDa (ETMP30; EHI_172170), predicted to contain five transmembrane domains. Immunofluorescence analysis demonstrated colocalization of hemagglutinin (HA)-tagged ETMP30 with the mitosomal marker, adenosine-5’-phosphosulfate kinase. Mitosomal membrane localization was indicated by immunoelectron microscopy analysis, which was supported by carbonate fractionation assay. Transcriptional gene silencing successfully repressed RNA expression by 60%, and led to a defect in growth and partial elongation of mitosomes. Immunoprecipitation of ETMP30 from ETMP30-HA-expressing transformant using anti-HA antibody pulled down one interacting protein of 126 kDa. Protein sequencing by mass spectrometry revealed this protein as a cation-transporting P-type ATPase, previously reported to localize to vacuolar compartments/Golgi-like structures, hinting at a possible mitosome-vacuole/Golgi contact site.

Import of Entamoeba histolytica Mitosomal ATP Sulfurylase Relies on Internal Targeting Sequences

“…Similar to Giardia, it would not be surprising if future studies reveal a minimalist mechanism of protein targeting and import in this relic mitochondria of Entamoeba that is accompanied by components unique to its genus. This is supported by discoveries of several Entamoeba-specific mitosomal membrane proteins [55] such as the β-barrel outer membrane protein MBOMP30 [25], and the transmembrane protein ETMP30 [29].…”

“…As described previously [29], IFA of amoebic trophozoites was performed by double staining with anti-HA monoclonal antibody (clone 11MO, Covance, Princeton, NJ, USA) to detect hemagluttinin (HA)-tagged proteins and anti-adenosine-5 -phosphosulfate kinase (APSK) (EHI_179080, a mitosomal matrix protein) polyclonal rabbit antiserum [21] to label mitosomes. Cells that expressed HA-tagged proteins were analyzed according to the following three localization categories: mitosome, mitosome and cytosol, and cytosol.…”

Genome-Wide Analysis of Known and Potential Tetraspanins in Entamoeba histolytica

“…To verify the localization of these proteins, we performed immunofluorescence assay. Micrographs of cells stained with anti-HA antibody showed mostly vesicular signals of TSPAN1 and TSPAN2 (Figure 6b), rather than the punctate signal pattern associated with mitosome localization [32,42,43,44,45,46,47]. The anti-HA signals did not colocalize with the mitosome marker (anti-APSK: mitosomal matrix enzyme, [35]) in double-stained cells (Figure 6b, top panel), suggesting TSPAN1 and TSPAN2 are not localized to mitosomes.…”

Golgi apparatus components in Entamoeba histolytica and Entamoeba dispar after monensin treatment

“…Nonetheless, vestigial organelle functionally related to mitochondria, the mitosome, has been reported (Tovar, Fischer, & Clark, 1999). Recently, an in silico and immune‐electron microscopy study reported a new lineage protein specific of mitosomes, previously localized in Golgi‐like vacuoles, suggesting a relation between both structures (Santos, Hanadate, Imai, & Nozaki, 2019).…”