ALBERT

Description: Tetraspanins are membrane proteins involved in intra- and/or intercellular signaling, and membrane protein complex formation. In some organisms, their role is associated with virulence and pathogenesis. Here, we investigate known and potential tetraspanins in the human intestinal protozoan parasite Entamoeba histolytica. We conducted sequence similarity searches against the proteome data of E. histolytica and newly identified nine uncharacterized proteins as potential tetraspanins in E. histolytica. We found three subgroups within known and potential tetraspanins, as well as subgroup-associated features in both their amino acid and nucleotide sequences. We also examined the subcellular localization of a few representative tetraspanins that might be potentially related to pathogenicity. The results in this study could be useful resources for further understanding and downstream analyses of tetraspanins in Entamoeba.

Description: 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.