Primers used in this study.

<div><p><i>Hansenula mrakii</i> killer toxin resistant gene 1 (<i>HKR1</i>) is an intronless, single-exon gene that encodes Hkr1, the signaling mucin of the budding yeast <i>Saccharomyces cerevisiae</i>. <i>HKR1</i> overexpression confers <i>S</i>. <i>cerevisiae</i> cells with resistance to the HM-1 killer toxin produced by the killer yeast <i>Hansenula mrakii</i> (currently known as <i>Cyberlindnera mrakii</i>). Hkr1 comprises multiple functional domains and participates in several signal transduction pathways, including the high-osmolarity glycerol (HOG) pathway, the cell wall integrity (CWI) mitogen-activated protein (MAP) kinase pathway, and the filamentation MAP kinase pathway; Hkr1 also controls bud-site selection. In this study, we identified a cryptic promoter in the <i>HKR1</i> exon that regulates the transcription of a shorter transcript encoding a truncated form of Hkr1. This shorter protein still conferred resistance to the HM-1 killer toxin, suggesting that this cryptic promoter helps carry out Hkr1-mediated signal transduction efficiently by producing a specific Hkr1 domain with functions as a signaling messenger. Notably, reporter assays using the fluorescent protein gene <i>mUkG1</i> and the β-galactosidase gene <i>lacZ</i> revealed that the transcriptional activity of this cryptic promoter was modulated by its upstream sequence within the single exon. Hkr1 thus differs from other signaling mucins, whose active C-terminal fragments are generated by post-translational processing, whereas the active C-terminal fragment of Hkr1 is generated by transcription from the exonic promoter. These findings describe a previously unknown example of functional diversification from a single gene, especially for a gene encoding a multidomain, multifunctional protein such as Hkr1.</p></div>