Indian Journal of Medical Microbiology Home 

BRIEF COMMUNICATION
[Download PDF]
Year : 2020  |  Volume : 38  |  Issue : 2  |  Page : 213--215

Functional studies of Plasmodium falciparum's prohibitin1 and prohibitin 2 in yeast

Savitha Chellappan1, Subarna Roy1, Jyoti M Nagmoti2, Wahida Tabassum3, SL Hoti1, Mrinal Kanti Bhattacharyya3, Praveen Balabaskaran Nina4,  
1 Indian Council of Medical Research - National Institute of Traditional Medicine, Belagavi, Karnataka, India
2 Department of Microbiology, K.L.E. University, Belagavi, Karnataka, India
3 Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India
4 Indian Council of Medical Research - National Institute of Traditional Medicine, Belagavi, Karnataka; Department of Epidemiology and Public Health, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India

Correspondence Address:
Dr. Praveen Balabaskaran Nina
Department of Epidemiology and Public Health, Central University of Tamil Nadu, Thiruvarur - 610 005, Tamil Nadu
India

Abstract

Prohibitins (PHBs) are evolutionarily conserved mitochondrial integral membrane proteins, shown to regulate mitochondrial structure and function, and can be classified into PHB1 and PHB2. PHB1 and PHB2 have been shown to interact with each other, and form heterodimers in mitochondrial inner membrane. Plasmodium falciparum has orthologues of PHB1 and PHB2 in its genome, and their role is unclear. Here, by homology modelling and yeast two-hybrid analysis, we show that putative Plasmodium PHBs (Pf PHB1 and Pf PHB2) interact with each other, which suggests that they could form supercomplexes of heterodimers in Plasmodium, the functional form required for optimum mitochondrial function.

How to cite this article:
Chellappan S, Roy S, Nagmoti JM, Tabassum W, Hoti S L, Bhattacharyya MK, Nina PB. Functional studies of Plasmodium falciparum's prohibitin1 and prohibitin 2 in yeast.Indian J Med Microbiol 2020;38:213-215

How to cite this URL:
Chellappan S, Roy S, Nagmoti JM, Tabassum W, Hoti S L, Bhattacharyya MK, Nina PB. Functional studies of Plasmodium falciparum's prohibitin1 and prohibitin 2 in yeast. Indian J Med Microbiol [serial online] 2020 [cited 2020 Nov 27 ];38:213-215
Available from: https://www.ijmm.org/text.asp?2020/38/2/213/293907

Full Text



 Introduction



Prohibitins (PHBs) cluster into PHB1 and PHB2, and these proteins share >50% similarity, and are evolutionarily conserved across all phyla.[1] PHBs, along with stomatin, flotillin and HflK/C, belong to the SPFH family.[2] Alternating blocks of PHB1 and PHB2 form high-molecular-weight complexes (~1.2 MDa) in the mitochondrial inner membrane.[3] PHBs are made up of an N-terminal transmembrane domain, a conserved PHB domain and a C-terminal coiled-coil domain through which the two PHBs interact with each other.[4] Even though PHBs are considered to be pleiotropic proteins with diverse cellular functions, their role in mitochondrial function has been of considerable interest.[1],[5] In mitochondria, PHBs maintain the copy number and organisation of mitochondrial DNA, support mitochondrial protein synthesis, act as membrane-bound chaperones in assisting respiratory complex assembly, maintain structural integrity, regulate respiratory complex assembly and respiration and are thought to serve as protein and lipid scaffolds in mitochondria.[1],[5]

Mitochondrial electron transport chain (mtETC) in Plasmodium falciparum (Pf) is an established drug target.[6] Atovaquone, an inhibitor of the bc1 complex of the mtETC, is already in clinical use, and drug discovery efforts have led to the advancement of ELQ-300, a mtETC inhibitor as a pre-clinical drug candidate.[7] Given the importance of mtETC as an attractive drug target, it is a high priority that we understand the structure and functional importance of the mitochondrial membrane proteins in Plasmodium. Pf contains two PHBs: Pf PHB1 and Pf PHB2. In addition, stomatin-like protein and unusual PHB-like protein are also present in the Plasmodium genome. PHB1 and PHB2 of Plasmodium berghei (Pb) localise to the mitochondria, and are suggested to be essential.[8] Here, based on homology modelling, and yeast two-hybrid studies, we show that Pf PHB1 and Pf PHB2 are structurally similar to other PHBs, and physically interact with each other, presumably to form heterodimers, required for regulating the structure and function of mitochondria.

 Methods



Homology modelling

Phyre2 (Protein Homology/Analog Y Recognition Engine)[9] server was used to predict the protein structure of PfPHB1 and Pf PHB2 (http://www.sbg.bio.ic.ac.uk/phyre2). The output of Phyre2 was analysed using RasMol as shown in [Figure 1]. After Phyre2 prediction, Pf PHB1 and Pf PHB2 were modelled based on the crystal structure of a core stomatin domain (chain c) of c3bk6c from Pyrococcus horikoshii. The confidence rate of prediction is >99.9%, with 57% and 51% sequence coverage for Pf PHB1 and Pf PHB2, respectively.{Figure 1}

Interacting sites in PfPHB1 and PfPHB2 modelled protein structures were determined in the Phyre2 web portal for protein modelling, prediction and analysis,[9] and the interacting residues are shown in red for Pf PHB1 and Pf PHB2 [Figure 1]c and [Figure 1]d. Two continuous interface regions are seen in Pf PHB1 and Pf PHB2, and the sequences are FQTPYIY-IK/HLSYGK-A and FERSIIY-VR/HLSFSN-E, respectively. These amino acids could facilitate interaction with one another and with other proteins.

Yeast two-hybrid analysis

Yeast two-hybrid experiments were carried out to identify the interaction between Pf PHB1 and Pf PHB2. Yeast two-hybrid assay was done as described by James et al.[10] using the pGADC1 and pGBDUC1 plasmids encoding GAL4 activation domain and DNA-binding domain, respectively.

The constructs, pGADC1:Pf PHB1 and pGBDUC1:Pf PHB2, were transformed into PJ69-4A yeast cells using the lithium acetate method.[11] The cells transformed with empty pGADC1 and pGBDUC1 were used as negative control. Two-hybrid interactions were tested with the yeast strain PJ69-4A which has the ADE2 reporter gene. The transformed strains were grown in SC-Ura-Leu medium till log phase at 30°C. Tenfold serial dilutions were prepared starting with an equal optical density of 0.5 OD ml −1 and spotted on the control plate (Sc-Ura-Leu) and experimental plate (Sc-Ura-Leu-Ade) to test the strength of the protein–protein interaction. The plates were incubated at 30°C for 72 h. The colonies on the control plate were observed from the 2nd day of incubation. The colonies in the experimental plate (Sc-Ura-Leu-Ade) were observed after 3 days, and their growth was slow compared to that of the control plate (Sc-Ura-Leu). Growth was measured in liquid media, and the number of cells in the media was equalised before spotting. Transformation efficiency and expression of URA3 and LEU2 genes in the plasmids were assessed by the growth of colonies in the SC-Ura-Leu plates. Two random clones were taken to test the strength of protein–protein interaction, and both the clones responded equally. Both the clones grew till the third dilution (10−3) on SC-Ura-Leu-Ade plates, which suggests a strong interaction between Pf PHB1 and Pf PHB2, as shown in [Figure 1]e.

 Results and Discussion



PHB1 and PHB2 have been found to be integral membrane proteins of the mitochondrial inner membrane.[3] PHB1 and PHB2 physically interact, and the PHB complex is considered to be the physiologically active form as loss of one subunit leads to the degradation of the other.[12] Yeast has been shown to be an excellent heterologous model system to study the function of Plasmodium proteins.[13] Homology modelling predicts the presence of putative interacting sites in Pf PHBs. Yeast two-hybrid experiments show that Pf PHB1 and Pf PHB2 strongly interact with each other. These experiments strongly suggest that Pf PHBs could make similar physiological interactions in the Plasmodium mitochondrion. Native page experiments on isolated Plasmodium mitochondria will help us identify the size of the PHB complex.

Knockdown of PHBs in other organisms has been shown to disrupt the mitochondrial morphology.[14],[15] Processes regulating mitochondrial morphology and mitochondrial DNA organisation and maintaining copy number in Plasmodium are poorly understood, and PfPHBs may play an important role. Attempts to knockout PHB1 and PHB2 in Pb have been unsuccessful.[8] Whole-genome saturation screen in Pf suggests that PHB2 is non-mutable in the coding sequence.[16] Overall, we provide an important mechanical insight into the functioning of Pf PHB1 and Pf PHB2 by showing that they physically interact with each other. Future studies should focus on understanding the role of PHB1 and PHB2 in Pf biology.

Acknowledgement for financial support

We thank Department of Science and Technology (DST) for financial support.

Financial support and sponsorship

This study was financially supported by DST through DSTWOS A funds to Ms. Savitha Chellappan and Inspire faculty research funds to Dr. Praveen Balabaskaran Nina.

Conflicts of interest

There are no conflicts of interest.

References

1Osman C, Merkwirth C, Langer T. Prohibitins and the functional compartmentalization of mitochondrial membranes. J Cell Sci 2009;122:3823-30.
2Browman DT, Hoegg MB, Robbins SM. The SPFH domain-containing proteins: More than lipid raft markers. Trends Cell Biol 2007;17:394-402.
3Tatsuta T, Model K, Langer T. Formation of membrane-bound ring complexes by prohibitins in mitochondria. Mol Biol Cell 2005;16:248-59.
4Winter A, Kämäräinen O, Hofmann A. Molecular modeling of prohibitin domains. Proteins 2007;68:353-62.
5Thuaud F, Ribeiro N, Nebigil CG, Désaubry L. Prohibitin ligands in cell death and survival: Mode of action and therapeutic potential. Chem Biol 2013;20:316-31.
6Sheiner L, Vaidya AB, McFadden GI. The metabolic roles of the endosymbiotic organelles of Toxoplasma and Plasmodium spp. Curr Opin Microbiol 2013;16:452-8.
7Frueh L, Li Y, Mather MW, Li Q, Pou S, Nilsen A, et al. Alkoxycarbonate ester prodrugs of preclinical drug candidate ELQ-300 for prophylaxis and treatment of malaria. ACS Infect Dis 2017;3:728-35.
8Matz JM, Goosmann C, Matuschewski K, Kooij TW. An unusual prohibitin regulates malaria parasite mitochondrial membrane potential. Cell Rep 2018;23:756-67.
9Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 2015;10:845-58.
10James P, Halladay J, Craig EA. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 1996;144:1425-36.
11Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol 1983;153:163-8.
12Coates PJ, Nenutil R, McGregor A, Picksley SM, Crouch DH, Hall PA, et al. Mammalian prohibitin proteins respond to mitochondrial stress and decrease during cellular senescence. Exp Cell Res 2001;265:262-73.
13Bergman LW, Kaiser K, Fujioka H, Coppens I, Daly TM, Fox S, et al. Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites. J Cell Sci 2003;116:39-49.
14Artal-Sanz M, Tsang WY, Willems EM, Grivell LA, Lemire BD, van der Spek H, et al. The mitochondrial prohibitin complex is essential for embryonic viability and germline function in Caenorhabditis elegans. J Biol Chem 2003;278:32091-9.
15Osman C, Haag M, Potting C, Rodenfels J, Dip PV, Wieland FT, et al. The genetic interactome of prohibitins: Coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria. J Cell Biol 2009;184:583-96.
16Zhang M, Wang C, Otto TD, Oberstaller J, Liao X, Adapa SR, et al. Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis. Science 2018;360:eaap7847.