NARF is 31% identical and 41% similar to yeast Nar1. Due to this structural homology, it has been suggested that NARF performs a function similar to that of Nar1. A complementation assay performed in the study of Balk et al. (2004) was unable to prove this hypothesis (Balk et al., 2004). The authors tested both NARFL and NARF, but found that neither of them appears to have an evolutionarily conserved function. Since the authors were unable to present the expression and functionality of the NARF/NARFL proteins used in their complementation assay, I repeated this experiment. In my study, I was able to overexpress the human homologues in Nar1-depleted yeast, but they failed to rescue yeast growth. Systematic studies on yeast-human orthologous gene pairs have revealed that sequence similarity can be helpful in predicting functional rescue: sequence similarity is higher for complementing pairs (45%) than non-complementing pairs (29%) (Sun et al., 2016). At the same time, it has also been shown that the human non-orthologous protein may act as a functional substitute for the yeast protein as well (Hamza et al., 2015), suggesting that complementation cannot be completely
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predicted by sequence similarity (Hamza et al., 2015; Kachroo et al., 2015). Failure in the complementation assay could also be attributable to incorrect conformation of tagged human proteins, which in turn could cause the expression of non-functional proteins in yeast cells. As was demonstrated in this study, both the NARF and the NARFL proteins tagged with BFP2 exhibited untypical aggregation in the cytoplasm. Tagging of proteins with fluorescent epitopes can be helpful in determining the expression and proper localisation of the introduced protein; however, it can also lead to a decreased expression level and/or toxicity of the heterologous protein (Tugendreich et al., 2001). It is also known that complementation efficiency strongly depends on the cellular function of proteins. Systematic analyses indicate that a function-specific group of proteins can be almost completely replaceable (e.g.
metabolic enzymes, which have over 90% replaceability), poorly replaceable (e.g. DNA replication and repair genes, which have 35% replaceability), or not replaceable at all (e.g.
cell growth and dead genes, which have only 3% replaceability) (Kachroo et al., 2015), suggesting species-specific pathways that cannot be reproduced in different organisms.
Considering that NARF contributes also to the maintenance of genomic stability and DNA repair, it falls into a group of proteins that is difficult to replace. Another species-specific feature that can prevent complementation is the protein-protein interactions network. This was illustrated in the example of human and yeast proteins involved in meiotic recombination, mitotic DNA repair, and telomere maintenance. The human MRE11B gene, which is a homologue of the yeast MRE11 gene, was not able to rescue mre11 mutants due to the lack of necessary interactions with yeast proteins (Chamankhah et al., 1998). Homologous proteins can contain conserved domains ensuring their conserved function among different organisms, but they can differ in terms of the remaining structure of the protein, and this part of the protein is species-specific. Such a situation has been described in proteins engaged in splicing and cell cycle control: Prp16 and Prp17, containing conserved WD repeats motifs in the C-terminal of proteins. The human PRP16 and PRP17 genes have both failed to exhibit the ability to rescue yeast prp16 and prp17 mutants, respectively. The generation of chimeric proteins composed of yeast N-terminal and human conserved C-terminal parts, however, has been found to result in functional replacement in both cases, indicating a species-specific role of the N-terminal part of yeast proteins (Ben Yehuda et al., 1998; Zhou and Reed, 1998). In the case of the examined Nar1, NARF, and NARFL homologues, all three share conserved C-terminal cysteine residues coordinating the H-cluster characteristic for iron-only hydrogenases (Balk et al., 2004). The generation of chimeric proteins containing the C-terminal H-cluster of NARF and NARFL fused with the yeast-specific N-terminus could potentially replace Nar1,
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culminating in improved functional complementation in yeast. Moreover, it has been proposed that selecting an adequate promoter for protein expression can be important in complementation assays. The commonly used constitutive or inducible promoters lead to artificial overexpression of the tested proteins; this, in turn, can disrupt the balance required for the proper protein function, thus leading to a lack of complementation (Lo Presti et al., 2009). In my study, all proteins used in the complementation assay were expressed from the inducible Met25 promoter. To rule out the possibility that suboptimal concentrations of NARF and NARFL block complementation of the Nar1 function in yeast, it may be advisable to try using native promoters. In general, there is no available set of common conditions that can ensure successful complementation, because each human-yeast gene pair is unique (Hamza et al., 2015). NARFL and Nar1 seem to be more closely related, as both are important players in the cytosolic iron-sulphur cluster assembly (CIA) machinery in mammals and yeast, respectively, but NARFL is still unable to substitute for the Nar1 function. The lower organisms, such as yeast or nematodes, possess only one homologue of this hydrogenase-like protein (Nar1 or oxy-4, respectively), whereas two (NARF and NARFL) are presented in higher eukaryotes, such as mammals. Many examples demonstrate that more complex organisms possess more than one homologue of essential genes. An excellent example is a group of genes involved in the NER mechanism, including the yeast RAD6 gene and its human homologues, HHR6A and HHR6B, and the yeast RAD23 gene and its human homologues, HHR23A and HHR23B (Koken et al., 1991; van der Spek et al., 1994). One can imagine that the NAR1 gene might have split its function into two homologues during evolution. In this way, emerging proteins with structural homology can fulfil different functions. This would explain why neither NARF nor NARFL can replace the Nar1 function separately, raising the question of whether the two together would be able to replace the Nar1 function. Another interesting experiment would be to perform the complementation assay the other way around, using human and/or mouse cell lines lacking expression of the NARF/Narf and NARFL/Narfl genes to test the potential of the yeast NAR1 or the nematode OXY-4 gene to rescue the phenotype. Such an assay would allow for testing the ability of invertebrate proteins to complement mammalian proteins’ functions. On the example of centromere proteins, it has been demonstrated that functional complementation can be examined in both ways because the yeast Cse4p homologue can substitute for a lack of the human CENP-A protein induced by RNAi in cells (Wieland et al., 2004).
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