Identification new potential multidrug resistance proteins of Saccharomyces cerevisiae

https://doi.org/10.1016/j.mimet.2020.106029Get rights and content

Highlights

  • STE6 protein is turned out to be the most related to human P-gp protein

  • Yeast BPT1 and YCF1 proteins are shown to be the most phylogenetically close to human MRP1.

  • In the ABCG subfamily of yeast, PDR10, PDR12, PDR15 and PDR18 are turned out to be potential MDR proteins.

Abstract

ABC (ATP-binding cassette) proteins can transport metabolic molecules and removes metabolic products and xenobiotics from the cell. The important problem is to study activity and search inhibitors of ABC proteins. There is a problem that ABC-proteins can transport hydrophobic drugs across the cell membrane due to their high substrate specificity. According to published data, Saccharomyces cerevisiae is an ideal model organism for analysis a lot of functional processes and gene activities of human cells. The aim of the present work is to reveal new potential yeast MDR proteins in S. cerevisiae with novel approach based on the cluster analysis.

According to the cluster analysis of yeast ABCB subfamily, STE6 protein is turned out to be the most related to human P-gp protein. The largest number of homologues with human MDR proteins was found in the yeast ABCC subfamily. Yeast BPT1 and YCF1 proteins are shown to be the most phylogenetically close to human MRP1. In the ABCG subfamily of yeast, PDR10, PDR12, PDR15 and PDR18 are turned out to be potential proteins of multidrug resistance. The future experimental study of these subfamilies should be conducted in order to confirm the role of STE6, YCF1, BPT1, PDR10, PDR12, PDR15 and PDR18 in MDR phenotype of yeast and to study their activity modulators.

Introduction

Members of the ATP binding cassette (ABC) protein superfamily, including human and yeast ABC transporters, transfer a variety of substances including ions, anticancer drugs, antibiotics, peptides, and phospholipids across biological membranes (Linton, 2007). ABC proteins fulfill a stunning variety of functions, ranging from ATP-driven transmembrane transport of great many different molecules, to the regulation of important cellular processes. In particular, ABC proteins can function as ion channels, channel regulators, receptors, proteases, as well as environmental sensors (Higgins, 1995; Dean and Allikmets, 1995; Kuchler and Thorner, 1992).

Most members of the ABC protein family share a similar molecular architecture and domain organization. But the typical ABC transporter contains two transmembrane domains (TMD1, TMD2) and two nucleotide-binding domains (NBD1, NBD2) (Bauer et al., 1999), (Dassa, 2003). The domain architecture of MDR proteins of both species includes full-size transporters (TMS6 − NBD)2 or (NBD − TMS6)2, half-size transporters (TMS6 − NBD) and members lacking obvious TMDs or NBDs (Gaur et al., 2005), (Tusnády et al., 2006). All ABC-transporters contain conserved regions (Hollenstein et al., 2007). NBD is approximately 200 residues in length and possesses six highly conserved motifs: the Walker A, Q-loop, ABC-signature, Walker B, D-loop and H-loop (Saurin et al., 1999). The main function of the NBDs is to bind and hydrolyze ATP or other NTPs, thereby fueling transport processes (Hollenstein et al., 2007). The most conserved features found in any given NBD are the Walker A and B motifs (Higgins, 1992). The Walker A motif has the sequence “GxxGxGKS/T,” where x is any amino acid. Walker B reported the consensus sequence of this motif to be “xxxxD, where D denotes aspartic acid residues respectively, and x represents any of the 20 standard amino acids (Higgins, 1995), (Dean et al., 2001). Walker A and Walker B motifs are involved in the ATP binding (Kerr, 2002). ABC-signature is found only in ABC transporters (Kim and Chen, 2018). In general, particular positions of these motifs are invariantly conserved and occupied by different amino acids in each subfamily (Mishra et al., 2014). Another important domains in ABC-transporters are TMDs. These domains are the primary determinants of substrate specificity through specific substrate-binding sites (Fig. 1) (Hollenstein et al., 2007), (Lewis et al., 2012). Each TMD usually contains six predicted α-helical transmembrane-spanning segments (TMSs), although in some cases four to eight predicted TMSs per TMD are also known (Biemans-Oldehinkel et al., 2006).

Despite their main functions in cell metabolism, ABC transporters can be harmful to humans. For instance, human diseases such as cystic fibrosis, adrenoleukodystrophy, Dubin-Johnson syndrome, Tangier disease are associated with mutations in human genes encoding ABC transporters. But one of the biggest problems with ABC proteins is their overexpression confers the resistance to hundreds of chemically unrelated drugs, including anticancer drugs and many others. Mainly these ABC-transporters localize in the plasma membrane and are called the multidrug resistant (MDR) proteins (Glavinas et al., 2005). For successful chemotherapy, it is necessary to search new inhibitors of MDR proteins in human cells. But the most important requirements are the low toxicity of inhibitors to the human cells. One of the main limitations of using human cells is different cell types need different expensive media to grow and survive, and most of the primary cells in culture have limited number of passages.

According to literature, Saccharomyces cerevisiae is an ideal model organism for the functional dissection of disease-related genes such as those of the ABC superfamily, because they grow easily, rapidly, cheaply, and have well understood genome (Karathia et al., 2011), (Wolters et al., 2015). Also, for example, up to 30% of genes implicated in human disease may have orthologs in the yeast proteome.

Genome of S. cerevisiae is shown to contain 30 ABC proteins which consists of six subfamilies: MDR (ABCB), MRP/CFTR (ABCC), ALDP (ABCD), RLI (ABCE), YEF3 (ABCF) and PDR5 (ABCG) (Bauer et al., 1999), (Piecuch and Obłak, 2014). Among these subfamilies, there is a network of genes involved in the multiple drug resistance phenotype, called the pleiotropic drug resistance (PDR) proteins in yeast (Piecuch and Obłak, 2014). Currently, yeast PDR network is shown to consist of PDR5 (ABCG), SNQ2 (ABCG), and YOR1 (ABCC) genes (Yibmantasiri et al., 2014). Likely, other ABC subfamily proteins of yeast cells can also perform the function of MDR under certain conditions (Bauer et al., 1999). The function of many ABC-transporters of S. cerevisiae has not been established yet. In the literature is shown that attempts to identify multidrug resistance functions of yeast ABC-proteins are being made by using quantitative reverse transcription PCR (RT-qPCR) analysis, GFP accumulation assay and flow cytometry (Galkina et al., 2018).

Besides the fact that multidrug resistance proteins of ABC superfamily are actively studied in clinical area, some of them are widespread in nature and have agricultural or biotechnological implications including agricultural fungicides, azoles, mycotoxins, herbicides and many others. For example, PDR18 of S. cerevisiae features the resistance to chemical stress agents, including herbicides, agricultural fungicides, and some metals (Teixeira et al., 2012). Also based on a genome-wide screening, PDR18 expression was also found to confer the resistance to the anticancer drugs cisplatin and carboplatin and the antifungal drug nocodazol (Teixeira et al., 2012). Pdr18 was found to play a role in plasma membrane sterol incorporation, and this physiological trait is proposed to contribute to its action as a multidrug resistance determinant (Cabrito et al., 2011). In the same time, PDR18 gene overexpression increases yeast ethanol tolerance and fermentation performance at more highly inhibitory concentrations of ethanol (Cabrito et al., 2011). PDR18 overexpressing in industrial yeast strains appears to be a promising approach to increase the bio-ethanol production (Godinho et al., 2018).

Despite the biological importance of the MDR phenotype, the identification of MDR functions of ABC proteins is carried out either by using time-consuming and expensive methods, or by establishing homology between proteins of different organisms using BLAST algorithms. Conventionally, only one specific part of the polypeptide chain is used for BLAST analysis (for example, the full protein sequence). By this way, the high level of homology of ABC-transporters of S. cerevisiae with Candida albicans, Schizosaccharomyces pombe, Drosophila melanogaster, Mycobacterium tuberculosis, Staphylococcus aureus has shown in previous researches (Shukla et al., 2003), (Christensen et al., 1997), (Braibant et al., 2000), (Burnie et al., 2000). The disadvantage of this method is that the established homology does not allow to assume the functional similarity of the studied proteins, since it relies on one parameter.

In our article, we proposed novel approach for identification potential MDR proteins using cluster analysis. For our analysis we use a number of parameters, namely cell localization and several homology parameters in the functionally important regions of ABC proteins, which are localized in the substrate-binding, ATP-binding and conserved sites of the protein molecules. In addition, to reveal functional similarity among ABC-proteins we use not only yeast ABC proteins that having proven PDR phenotype, but also human ABC proteins with a well-established MDR function, which increases the probability of establishing the MDR activity and functional analogs of yeast ABC proteins.

As a result of applying this approach, we have revealed new functional analogues of human MDR proteins in S. cerevisiae cells, that can be used to model and study the activity of MDR human proteins using cheaper yeast cells. Also, the data obtained might be used to develop new directions of modulating the activity of these proteins in yeast, that is aimed at ethanol bio-production increase.

Base on the literature review this analysis has not been done before and represents a new method identification of intraspecific and interspecies functional analogs of proteins. In addition, this method is easy to use and no time-consuming, and allows to minimize the range of proteins for experimental validation of their predicted functions in future.

Section snippets

Materials and methods

The protein sequence of ABC-transporters of S. cerevisiae and MDR proteins of human identified from UniProt. Information about the amino acid sequence position of NBDs and TMDs of human MDR proteins (p-glycoprotein, MRP1, BCRP) and yeast ABC proteins are presented in the Table A1 (Appendices).

The full sequences, NBDs, TMDs, Walker A and Walker B motifs, Q-loop were used for local alignment withBLASTp.InformationabouttheaminoacidsequencepositionofABCsignature, Walker A and Walker B motifs,

Identification of potential MDR proteins of yeast ABCB subfamily

Four ABC proteins of S. cerevisiae MDL1, MDL2, ATM1 and STE6 belong to ABCB subfamily. MDL1exports peptides formed upon proteolysis of mitochondrial proteins, whereas the function of MDL2 remains unknown (Paumi et al., 2009). ATM1 performs biogenesis of iron‑sulfur (Fe/S) clusters, STE6takes part in mating of α factor secretion protein (Srinivasan et al., 2014), (Kölling and Hollenberg, 1994). Recently, functioning of members of ABCB subfamily as MDR proteins has not been covered in the

Discussion

The most convenient approach for the identification of potential MDR proteins has been found because there is the growth of data on the functions of ABC-transporters in human cells, namely, their participation in drug transfer. The disadvantages of using animal and human cell lines are the necessity of standardization of the environment, difficulties of medium preparation and a limited number of passages. An attempt of conduct a cluster analysis of three subfamilies of ABC-transporters of S.

Funding

Our publication was supported for this work by the Belarusian Republican Foundation for Fundamental Research (grant agreement № М19МС-033 (02.05.2019)). Registration number 20200121.

Author statement

All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript.

Declaration of Competing Interest

Authors declare that they have no conflict of interests.

References (64)

  • I.D. Kerr

    Structure and association of ATP-binding cassette transporter nucleotide-binding domains

    Biochim. Biophys. Acta Biomembr.

    (2002)
  • A. Kolaczkowska et al.

    Compensatory activation of the multidrug transporters Pdr5p, Snq2p, and Yor1p by Pdr1p in Saccharomyces cerevisiae

    FEBS Lett.

    (2008)
  • I. Leier et al.

    The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates

    J. Biol. Chem.

    (1994)
  • Z.S. Li et al.

    The yeast cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump

    J. Biol. Chem.

    (1996)
  • M. Liesa et al.

    Mitochondrial ABC transporters function: the role of ABCB10 (ABC-me) as a novel player in cellular handling of reactive oxygen species

    Biochim. Biophys. Acta, Mol. Cell Res.

    (2012)
  • M. Marek et al.

    The yeast plasma membrane ATP binding cassette (ABC) transporter Aus1: purification, characterization, and the effect of lipids on its activity

    J. Biol. Chem.

    (2011)
  • K. Park et al.

    Mode of membrane insertion of individual transmembrane segments in Mdl1 and Mdl2, multi-spanning mitochondrial ABC transporters

    FEBS Lett.

    (2014)
  • X. Ren et al.

    Functional Comparison between YCF1 and MRP1 Expressed in Sf21 Insect Cells

    (2000)
  • G.E. Tusnády et al.

    Membrane topology of human ABC proteins

    FEBS Lett.

    (2006)
  • D.S. Wilks

    Cluster analysis

    Int. Geophys.

    (2011)
  • E. Balzi et al.

    Yeast multidrug resistance: the PDR network

    J. Bioenerg. Biomembr.

    (1995)
  • P. Borst et al.

    A family of drug transporters: the multidrug resistance-associated proteins

    J. Natl. Cancer Inst.

    (2000)
  • J.P. Burnie et al.

    Identification of an immunodominant ABC transporter in methicillin- resistant Staphylococcus aureus infections

    Infect. Immun.

    (2000)
  • T.R. Cabrito et al.

    The yeast ABC transporter Pdr18 (ORF YNR070w) controls plasma membrane sterol composition, playing a role in multidrug resistance

    Biochem. J.

    (2011)
  • P.U. Christensen et al.

    The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor

    Mol. Gen. Genet.

    (1997)
  • E. Dassa

    Phylogenetic and functional classification of ABC (ATP-binding cassette) systems

  • R. Egner et al.

    Endocytosis and vacuolar degradation of the plasma membrane-localized Pdr5 ATP-binding cassette multidrug transporter in Saccharomyces cerevisiae

    Mol. Cell. Biol.

    (1995)
  • K.V. Galkina et al.

    Penetrating cations induce pleiotropic drug resistance in yeast

    Sci. Rep.

    (2018)
  • M. Gaur et al.

    Complete inventory of ABC proteins in human pathogenic yeast, Candida albicans

    J. Mol. Microbiol. Biotechnol.

    (2005)
  • H. Glavinas et al.

    The Role of ABC Transporters in Drug Resistance

    Metab.Toxicity. Curr Drug Deliv.

    (2005)
  • C.P. Godinho et al.

    Pdr18 is involved in yeast response to acetic acid stress counteracting the decrease of plasma membrane ergosterol content and order

    Sci. Rep.

    (2018)
  • K. Gulshan et al.

    Vacuolar import of phosphatidylcholine requires the ATP-binding cassette transporter ybt1

    Traffic.

    (2011)
  • Cited by (4)

    • Molecular study on the role of vacuolar transporters in glycyrrhetinic acid production in engineered Saccharomyces cerevisiae

      2022, Biochimica et Biophysica Acta - Biomembranes
      Citation Excerpt :

      This is a part of the nucleotide binding domain (NBD) which is responsible for ATP hydrolysis [50]. In addition, there were four motifs that belong to the six-transmembrane helical domain (TMD) (Fig. S3) that involves the identification of substrate by means of unique substrate binding sites [49]. The docking results analysis showed that GA was located within the cavity of the transporter formed by the α-helices of the transmembrane domains in all six proteins (Fig. 2e–j).

    View full text