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A novel Mitosomal β-barrel Outer Membrane Protein in Entamoeba.

Santos HJ, Imai K, Makiuchi T, Tomii K, Horton P, Nozawa A, Ibrahim M, Tozawa Y, Nozaki T - Sci Rep (2015)

Bottom Line: Initially identified through in silico analysis, we experimentally confirmed that MBOMP30 is indeed a β-barrel protein.Interestingly, the deletion of the putative β-signal, a sequence believed to guide β-barrel outer membrane protein (BOMP) assembly, did not affect membrane integration, but abolished the formation of a ~240 kDa complex.MBOMP30 represents only the seventh subclass of eukaryotic BOMPs discovered to date and lacks detectable homologs outside Entamoeba, suggesting that it may be unique to Entamoeba mitosomes.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan [2] Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan [3] Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City, 1101 Philippines.

ABSTRACT
Entamoeba possesses a highly divergent mitochondrion-related organelle known as the mitosome. Here, we report the discovery of a novel protein in Entamoeba, which we name Mitosomal β-barrel Outer Membrane Protein of 30 kDa (MBOMP30). Initially identified through in silico analysis, we experimentally confirmed that MBOMP30 is indeed a β-barrel protein. Circular dichroism analysis showed MBOMP30 has a predominant β-sheet structure. Localization to Entamoeba histolytica mitosomes was observed through Percoll-gradient fractionation and immunofluorescence assay. Mitosomal membrane integration was demonstrated by carbonate fractionation, proteinase K digestion, and immunoelectron microscopy. Interestingly, the deletion of the putative β-signal, a sequence believed to guide β-barrel outer membrane protein (BOMP) assembly, did not affect membrane integration, but abolished the formation of a ~240 kDa complex. MBOMP30 represents only the seventh subclass of eukaryotic BOMPs discovered to date and lacks detectable homologs outside Entamoeba, suggesting that it may be unique to Entamoeba mitosomes.

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Prediction of transmembrane β-strands of EhMBOMP30 and multiple alignment with its homologs.Multiple alignment of EhMBOMP30, EnMBOMP30, EdMBOMP30, and EiMBOMP30, built with Clustal omega59 is shown in CLUSTAL format (Eh-Entamoeba histolytica, En-Entamoeba nuttalli, Ed-Entamoeba dispar, Ei-Entamoeba invadens). Predicted transmembrane β-strands of EhMBOMP30 are indicated with yellow arrows. The arrows cover all positions predicted as part of a β-strand by either of two BOMP topology predictors: BOCTOPUS and TMBETAPRED-RBF3233 [See Fig. S1]. Conserved hydrophobic residues are highlighted in green. A red dashed box indicates the region corresponding to the β-signal motif in the last predicted transmembrane β-strand.
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f2: Prediction of transmembrane β-strands of EhMBOMP30 and multiple alignment with its homologs.Multiple alignment of EhMBOMP30, EnMBOMP30, EdMBOMP30, and EiMBOMP30, built with Clustal omega59 is shown in CLUSTAL format (Eh-Entamoeba histolytica, En-Entamoeba nuttalli, Ed-Entamoeba dispar, Ei-Entamoeba invadens). Predicted transmembrane β-strands of EhMBOMP30 are indicated with yellow arrows. The arrows cover all positions predicted as part of a β-strand by either of two BOMP topology predictors: BOCTOPUS and TMBETAPRED-RBF3233 [See Fig. S1]. Conserved hydrophobic residues are highlighted in green. A red dashed box indicates the region corresponding to the β-signal motif in the last predicted transmembrane β-strand.

Mentions: Our MBOMP predictor gives a high probability score for all four Entamoeba MBOMP30 homologs. To assure that this result was not a quirk of our predictor, we analyzed the E. histolytica MBOMP30 sequences using two tools designed for topology prediction of bacterial BOMPs, which may be expected to share some structural properties with MBOMPs. The topology prediction tools BOCTOPUS and TMBETAPRED-RBF3233 predicted the Entamoeba MBOMP30 to contain multiple transmembrane β-strand regions (Fig. 2, Supplementary Fig. S1a), consistent with the premise that MBOMP30 is a mitosomal BOMP. The outside surface of MBOMPs faces a lipid environment and is expected to display a relatively high hydrophobicity. The MBOMP30 sequences indeed share this property, and their predicted β-strand regions and hydrophobic stretches align well (Supplementary Fig. S1b).


A novel Mitosomal β-barrel Outer Membrane Protein in Entamoeba.

Santos HJ, Imai K, Makiuchi T, Tomii K, Horton P, Nozawa A, Ibrahim M, Tozawa Y, Nozaki T - Sci Rep (2015)

Prediction of transmembrane β-strands of EhMBOMP30 and multiple alignment with its homologs.Multiple alignment of EhMBOMP30, EnMBOMP30, EdMBOMP30, and EiMBOMP30, built with Clustal omega59 is shown in CLUSTAL format (Eh-Entamoeba histolytica, En-Entamoeba nuttalli, Ed-Entamoeba dispar, Ei-Entamoeba invadens). Predicted transmembrane β-strands of EhMBOMP30 are indicated with yellow arrows. The arrows cover all positions predicted as part of a β-strand by either of two BOMP topology predictors: BOCTOPUS and TMBETAPRED-RBF3233 [See Fig. S1]. Conserved hydrophobic residues are highlighted in green. A red dashed box indicates the region corresponding to the β-signal motif in the last predicted transmembrane β-strand.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4339806&req=5

f2: Prediction of transmembrane β-strands of EhMBOMP30 and multiple alignment with its homologs.Multiple alignment of EhMBOMP30, EnMBOMP30, EdMBOMP30, and EiMBOMP30, built with Clustal omega59 is shown in CLUSTAL format (Eh-Entamoeba histolytica, En-Entamoeba nuttalli, Ed-Entamoeba dispar, Ei-Entamoeba invadens). Predicted transmembrane β-strands of EhMBOMP30 are indicated with yellow arrows. The arrows cover all positions predicted as part of a β-strand by either of two BOMP topology predictors: BOCTOPUS and TMBETAPRED-RBF3233 [See Fig. S1]. Conserved hydrophobic residues are highlighted in green. A red dashed box indicates the region corresponding to the β-signal motif in the last predicted transmembrane β-strand.
Mentions: Our MBOMP predictor gives a high probability score for all four Entamoeba MBOMP30 homologs. To assure that this result was not a quirk of our predictor, we analyzed the E. histolytica MBOMP30 sequences using two tools designed for topology prediction of bacterial BOMPs, which may be expected to share some structural properties with MBOMPs. The topology prediction tools BOCTOPUS and TMBETAPRED-RBF3233 predicted the Entamoeba MBOMP30 to contain multiple transmembrane β-strand regions (Fig. 2, Supplementary Fig. S1a), consistent with the premise that MBOMP30 is a mitosomal BOMP. The outside surface of MBOMPs faces a lipid environment and is expected to display a relatively high hydrophobicity. The MBOMP30 sequences indeed share this property, and their predicted β-strand regions and hydrophobic stretches align well (Supplementary Fig. S1b).

Bottom Line: Initially identified through in silico analysis, we experimentally confirmed that MBOMP30 is indeed a β-barrel protein.Interestingly, the deletion of the putative β-signal, a sequence believed to guide β-barrel outer membrane protein (BOMP) assembly, did not affect membrane integration, but abolished the formation of a ~240 kDa complex.MBOMP30 represents only the seventh subclass of eukaryotic BOMPs discovered to date and lacks detectable homologs outside Entamoeba, suggesting that it may be unique to Entamoeba mitosomes.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan [2] Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan [3] Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City, 1101 Philippines.

ABSTRACT
Entamoeba possesses a highly divergent mitochondrion-related organelle known as the mitosome. Here, we report the discovery of a novel protein in Entamoeba, which we name Mitosomal β-barrel Outer Membrane Protein of 30 kDa (MBOMP30). Initially identified through in silico analysis, we experimentally confirmed that MBOMP30 is indeed a β-barrel protein. Circular dichroism analysis showed MBOMP30 has a predominant β-sheet structure. Localization to Entamoeba histolytica mitosomes was observed through Percoll-gradient fractionation and immunofluorescence assay. Mitosomal membrane integration was demonstrated by carbonate fractionation, proteinase K digestion, and immunoelectron microscopy. Interestingly, the deletion of the putative β-signal, a sequence believed to guide β-barrel outer membrane protein (BOMP) assembly, did not affect membrane integration, but abolished the formation of a ~240 kDa complex. MBOMP30 represents only the seventh subclass of eukaryotic BOMPs discovered to date and lacks detectable homologs outside Entamoeba, suggesting that it may be unique to Entamoeba mitosomes.

Show MeSH
Related in: MedlinePlus