Regulated use of alternative Transcription Start Sites controls the production of cytosolic or mitochondrial forms of branched-chain aminotransferase in Kluyveromyces marxianus

Following a whole genome duplication (WGD) event approximately 100 million years ago, the yeast lineage from which the model Saccharomyces cerevisiae derives maintained two copies of genes where it was necessary to synthesise proteoforms with different sub-cellular localisation. In contrast, yeasts that did not undergo the WGD event have a single gene that must encode both proteoforms. We adopted an integrated in silico and experimental approach to study how this is achieved with BAT1, a gene that encodes mitochondrial and cytosolic forms of a branched chain aminotransferase (BCAT) in pre-WGD yeast such as Kluyveromyces marxianus. We determined that condition-specific regulation of alternative transcription sites gives rise to mRNA isoforms that differ at the 5end and that, when decoded, generate a mitochondrial or cytosolic proteoform. Furthermore, targeted mutants lacking specific transcription factors were generated to establish how this differentiation was regulated. As in S. cerevisiae, Gcn4 and Leu3 activated expression of the mRNA encoding the mitochondrial proteoform under conditions when branched chain amino acid synthesis was required. Unlike S. cerevisiae, however, K. marxianus lacked tight regulation of the mRNA encoding the cytosolic proteoform supporting the hypothesis that maintaining paralogous genes in post-WGD yeasts facilitated development of more sophisticated expression control mechanisms.