Evolution of Substrates and Components of the Pro/N-Degron Pathway.

Gid4, a subunit of the ubiquitin ligase GID, is the recognition component of the Pro/N-degron pathway. Gid4 targets proteins in particular through their N-terminal (Nt) proline (Pro) residue. In Saccharomyces cerevisiae and other Saccharomyces yeasts, the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 bear Nt-Pro and are conditionally destroyed by the Pro/N-degron pathway. However, in mammals and in many non-Saccharomyces yeasts, for example, in Kluyveromyces lactis, these enzymes lack Nt-Pro. We used K. lactis to explore evolution of the Pro/N-degron pathway. One question to be addressed was whether the presence of non-Pro Nt residues in K. lactis Fbp1, Icl1, and Mdh2 was accompanied, on evolutionary time scales (S. cerevisiae and K. lactis diverged ∼150 million years ago), by a changed specificity of the Gid4 N-recognin. We used yeast-based two-hybrid binding assays and protein-degradation assays to show that the non-Pro (Ala) Nt residue of K. lactis Fbp1 makes this enzyme long-lived in K. lactis. We also f...