G C A T T A C G G C A T genes Article An Entamoeba-Specific Mitosomal Membrane Protein with Potential Association to the Golgi Apparatus Herbert J. Santos 1,2,3 , Yuki Hanadate 2,3, Kenichiro Imai 4,5 and Tomoyoshi Nozaki 1,2,3,* 1 Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; [email protected] 2 Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan; [email protected] 3 Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan 4 Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan; [email protected] 5 Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan * Correspondence: [email protected] Received: 9 March 2019; Accepted: 10 May 2019; Published: 13 May 2019 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. Keywords: Entamoeba histolytica; mitosome; Entamoeba-specific transmembrane mitosomal protein of 30 kDa; secretory pathway calcium ATPase 1. Introduction The mitochondrion is a vital organelle in the cell as it is involved in key metabolic processes such as ATP generation via oxidative phosphorylation, iron-sulfur cluster formation, the tricarboxylic acid cycle, β-oxidation, heme biosynthesis, phospholipid, vitamin, steroid and amino acid metabolism, calcium homeostasis, and programmed cell death [1,2]. However, certain eukaryotic organisms that inhabit anoxic or hypoxic environments, lack canonical mitochondria, and instead possess highly Genes 2019, 10, 367; doi:10.3390/genes10050367 www.mdpi.com/journal/genes Genes 2019, 10, 367 2 of 14 divergent and often degenerate mitochondrion-related organelles (MROs). Particularly, a few species of parasitic protists contain MROs, which perform diversified functions and mechanisms that contribute to their parasitic lifestyle [3]. One example is the mitosome of the anaerobic protozoan Entamoeba histolytica, the etiological agent of amoebiasis, which causes an estimated 100,000 deaths annually [4]. Amoebiasis is a disease characterized by diarrhea and dysentery, which could lead to extra-intestinal complications such as abscess formation in the liver, lungs, and brain. Apart from having components that are essential to the cell’s survival [5,6], the E. histolytica mitosome contains enzymes that carry out sulfate activation [7] which leads to the formation of cholesteryl sulfate, a molecule linked to stage conversion from the metabolically active trophozoite form to the infective cyst form [8]. Such features highlight the importance of mitosomes not only on parasite proliferation but also on disease transmission with its involvement in amoebic cell differentiation. Unlike the canonical aerobic mitochondria, the E. histolytica mitosomal membranes have minimal and mostly divergent components, including transport systems for proteins and metabolites [9,10]. Only a few mitosomal outer membrane proteins have been identified, such as the pore forming β-barrel component of the translocase of the outer membrane (TOM) complex, Tom40 [11] involved in mitochondrial protein import [12], the central component of the sorting and assembly machinery (SAM) complex, Sam50 [11], involved in the assembly of β-barrel proteins on the outer membrane [12], and a lineage-specific mitosomal β-barrel outer membrane protein of 30 kDa, MBOMP30 [6]. On the inner mitosomal membrane, only the sodium/sulfate transporter [7] and two proteins that belong to the mitochondrial carrier family (MCF), namely ATP-ADP carrier protein [13], and phosphate carrier [11], are the known channel proteins reported. In our previous works, we developed in silico tools to predict membrane proteins, including a specialized prediction pipeline for proteins possessing α-helical transmembrane domains [14], and successfully confirmed our predictions through supporting cell biological and biochemical evidences [6,15]. Using this predictor, we found additional novel lineage-specific mitosomal membrane protein candidates, namely EHI_170120, which was confirmed to be localized to the mitosomal membranes, and EHI_099350, which demonstrated dual localization to mitosomes and the ER [14]. Here, we report another Entamoeba-specific mitosomal membrane protein predicted to possess five transmembrane domains, which we name Entamoeba transmembrane mitosomal protein of 30 kDa (ETMP30). It appears to interact with a cation-transporting P-type ATPase (EHI_065670) previously reported to be localized to vacuolar compartments that are postulated to be Golgi-like structures [16] in E. histolytica. 2. Materials and Methods 2.1. In Silico Predictions and Analyses For the prediction of Entamoeba-specific membrane proteins, we used a prediction pipeline developed in a previous study, which combined highly-sensitive homolog search methods (JackHMMER [17] and HHblits [18]) and a novel transmembrane domain prediction method [14]. Tofind ETMP30 homologs in other Entamoeba species, we performed sequence similarity searches against the genome and proteome sequences of E. dispar, E. moshkovskii, E. invadens, and E. nuttalli in the Amoebozoa resource database, AmoebaDB [19], using blastp and tblastx [20]. For the analysis of transmembrane regions, we used DG prediction for transmembrane insertion [21]. For structural similarity prediction and sequence repeat analysis, we used HHpred [22] and HHrepID [23], respectively. 2.2. Amoeba Cultivation, Plasmid Construction, and Amoeba Transfection Axenic cultures of Entamoeba histolytica HM-1:IMSS cl6 [24] and G3 [25] were maintained in Diamond BI-S-33 medium [24]. Total RNA extraction from E. histolytica HM1:IMSS clone 6, followed by mRNA purification, and cDNA synthesis, were performed following previously described protocols [6]. The E. histolytica ETMP30 gene (EHI_172170) was PCR-amplified from cDNA using Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA) using the appropriate primer pair listed in Genes 2019, 10, 367 3 of 14 Table S1. After restriction digestion with BglII, amplified fragments were ligated into the expression vector containing the hemagglutinin (HA) tag, pEhEx- HA [26] using Ligation-Convenience Kit (Nippongene, Tokyo, Japan), to produce pEhEx-ETMP30-HA. For gene silencing of ETMP30, a plasmid was constructed by inserting a 420-bp-long 5’ end of the ETMP30 protein-coding region that was amplified by PCR, using cDNA as template and the primer pair indicated in Table S1. The PCR-amplified DNA fragment was digested with StuI and SacI and ligated into StuI and SacI-double digested silencing vector, pSAP2-Gunma [10], to produce pSAP2-ETMP30. Transfection of E. histolytica cl6 strain for overexpression, and G3 strain for gene silencing was performed by lipofection as described previously [7]. Selection was performed by daily replacement of medium supplemented with Geneticin/G418 (Gibco/Life Technologies, Waltham, MA, USA) at increasing concentration, until control cells (transfected without plasmid) failed to survive the antibiotic challenge. 2.3. Immunoflourescence Assay Indirect immunofluorescence assay (IFA) was conducted as previously described [7] with anti-HA monoclonal antibody (clone 11MO, Covance, Princeton, NJ, USA) to detect HA-tagged ETMP30 diluted 1:500 in 0.1% bovine serum albumin in Tris-buffered saline-Tween 20 (BSA-TBST) and anti-adenosine-5’-phosphosulfate kinase (APSK; EHI_179080; a mitosomal matrix enzyme involved in sulfate activation) polyclonal rabbit antiserum [10] diluted 1:300 in 0.1% BSA-TBST to stain mitosomes. Visualization