Genetic dissection of human ABCE1 in yeast reveals separable requirements for ribosome recycling and suppression of aberrant reinitiation.

The ATP-binding cassette E1 (ABCE1) protein is an essential factor for ribosome recycling, splitting post-termination ribosomes into subunits for subsequent rounds of translation. Despite high conservation, human ABCE1 (hABCE1) fails to functionally complement the depletion of its essential yeast homolog, Rli1. In this study, we leveraged this species-specificity to dissect the functional architecture of ABCE1. Through analysis of yeast-human chimeric proteins, we identified the N-terminal nucleotide-binding domain (NBD1) as the primary determinant of this incompatibility. To further investigate, we isolated multiple hABCE1 point mutants (revertants) that successfully restored yeast growth. We then developed a novel dual-luciferase reporter assay to quantify aberrant translation reinitiation in the 3'UTR, an event recognized as a direct consequence of ABCE1 deficiency. Notably, while the revertant mutants rescued yeast viability, they failed to suppress aberrant reinitiation, exhibiting levels equivalent to the nonfunctional hABCE1. This genetic uncoupling of viability from the suppression of reinitiation suggests that the canonical ribosome recycling function required for cell growth and the role in preventing aberrant reinitiation have distinct functional thresholds or are genetically separable aspects of ABCE1 activity.