Abstract
Divergence of paralogous pairs, resulting from gene duplication, plays an important role in the evolution of specialized or novel gene functions. Analysis of selected duplicated pairs has elucidated some of the mechanisms underlying the functional diversification of Saccharomyces cerevisiae (S. cerevisiae) paralogous genes. Similar studies of the orthologous pairs extant in pre-whole genome duplication yeast species, such as Kluyveromyces lactis (K. lactis) remain to be addressed. The genome of K. lactis, an aerobic yeast, includes gene pairs generated by sporadic duplications. The genome of this organism comprises the KlLEU4 and KlLEU4BIS paralogous pair, annotated as putative α-isopropylmalate synthases (α-IPMSs), considered to be the orthologs of the S. cerevisiae ScLEU4/ScLEU9 paralogous genes. The enzymes encoded by the latter two genes are mitochondrially located, differing in their sensitivity to leucine allosteric inhibition resulting in ScLeu4-ScLeu4 and ScLeu4-ScLeu9 sensitive dimers and ScLeu9-ScLeu9 relatively resistant homodimers. Previous work has shown that, in a Scleu4Δ mutant, ScLEU9 expression is increased and assembly of ScLeu9-ScLeu9 leucine resistant homodimers results in loss of feedback regulation of leucine biosynthesis, leading to leucine accumulation and decreased growth rate. Here we report that: (i) K. lactis harbors a sporadic gene duplication, comprising the KlLEU4, syntenic with S. cerevisiae ScLEU4 and ScLEU9, and the non-syntenic KlLEU4BIS, arising from a pre-WGD event. (ii) That both, KlLEU4 and KlLEU4BIS encode leucine sensitive α-IPMSs isozymes, located in the mitochondria (KlLeu4) and the cytosol (KlLeu4BIS), respectively. (iii) That both, KlLEU4 or KlLEU4BIS complement the Scleu4Δ Scleu9Δ leucine auxotrophic phenotype and revert the enhanced ScLEU9 transcription observed in a Scleu4Δ ScLEU9 mutant. The Scleu4Δ ScLEU9 growth mutant phenotype is only fully complemented when transformed with the syntenic KlLEU4 mitochondrial isoform. KlLEU4 and KlLEU4BIS underwent a different diversification pathways than that leading to ScLEU4/ScLEU9. KlLEU4 could be considered as the functional ortholog of ScLEU4, since its encoded isozyme can complement both the Scleu4Δ Scleu9Δ leucine auxotrophy and the Scleu4Δ ScLEU9 complex phenotype.
Highlights
Gene duplication is a source of new or specialized biological functions (Ohno, 1970; Lynch et al, 1991; Force et al, 1999; Kellis et al, 2004)
Paralogous genes can originate from Whole Genome Duplication (WGD) events or from sporadic duplications known as Small Scale Duplications (SSD)
In a Scleu4 ScLEU9 strain, the exclusive presence of the ScLeu9-ScLeu9 leucine-resistant homodimer, results in a growth impaired phenotype, most probably due to the metabolic imbalance produced by the draining of acetyl-CoA to α-IPM and leucine biosynthesis with the consequent depletion of other tricarboxilic acid cycle intermediates (López et al, 2015). This particular phenotype is enhanced by ScLEU9 overexpression in a Scleu4 background (López et al, 2015). These results indicate that in S. cerevisiae, retention and further diversification of the two α-isopropylmalate synthases (α-IPMSs) has resulted in a specific regulatory system that controls the leucine-α-IPM biosynthetic pathway through the different feedback sensitivity of homomeric and heterodimeric isoforms
Summary
Gene duplication is a source of new or specialized biological functions (Ohno, 1970; Lynch et al, 1991; Force et al, 1999; Kellis et al, 2004). Retained duplicate genes (paralogs) can provide increased dosage of the same product, or may go through a process of sub- or neo-functionalization In the former, both copies of the gene lose and/or specialize a subset of their ancestral functions, in the latter, at least one of the copies acquires a new function (Ohno, 1970; Lynch et al, 1991; Force et al, 1999). The lineage that gave rise to Kluyveromyces lactis (K. lactis) diverged from the Saccharomyces cerevisiae (S. cerevisiae) lineage before the WGD event (Kellis et al, 2004) This yeast exhibits much less overall genetic redundancy (Conde e Silva et al, 2009), suggesting that the paralogs genes present in K. lactis originated from independent SSD events (Dujon et al, 2004; Huerta-Cepas et al, 2014). Subfunctionalization of paralogs pairs can be achieved through various non-exclusive molecular mechanisms such as modifications of the coding sequence leading to: (i) changes in the kinetic parameters (DeLuna et al, 2001; Rojas-Ortega et al, 2018), (ii) differential subcellular localization (Marques et al, 2008), (iii) formation of hetero-oligomeric isozymes with emerging biochemical properties (DeLuna et al, 2001; López et al, 2015), or (iv) modifications of the regulatory region determining differential expression of each copy (Avendaño et al, 2005; Quezada et al, 2008; González et al, 2017)
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