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

Area of Science:

• Biochemistry
• Molecular Biology
• Evolutionary Biology

Background:

• The Pro/N-degron pathway utilizes the Gid4 protein to recognize and degrade proteins with N-terminal proline (Nt-Pro) residues.
• In Saccharomyces cerevisiae, key gluconeogenic enzymes like Fbp1 possess Nt-Pro and are degraded via this pathway.
• Mammals and non-Saccharomyces yeasts, such as Kluyveromyces lactis, often lack Nt-Pro in these enzymes, suggesting evolutionary divergence in protein regulation.

Purpose of the Study:

• To investigate the evolutionary changes in the Pro/N-degron pathway's specificity, particularly focusing on the Gid4 N-recognin.
• To determine if the absence of Nt-Pro in Kluyveromyces lactis gluconeogenic enzymes correlates with altered recognition by Gid4.
• To explore the molecular basis for the stability of these enzymes in K. lactis compared to S. cerevisiae.

Main Methods:

• Yeast-based two-hybrid binding assays to assess protein-protein interactions.
• Protein-degradation assays, including a novel assay using the antibiotic blasticidin, to measure protein stability.
• Site-directed mutagenesis to create hybrid Fbp1 proteins with altered N-terminal sequences.

Main Results:

• Kluyveromyces lactis Fbp1, lacking Nt-Pro, is stable in K. lactis due to its N-terminal alanine (Nt-Ala).
• Replacing the N-terminus of K. lactis Fbp1 with the S. cerevisiae Fbp1 sequence (Nt-PTLV) rendered the hybrid protein a short-lived substrate of Gid4 in K. lactis.
• This study represents the first investigation of the ubiquitin system in K. lactis and introduces a new protein-degradation assay for this organism.

Conclusions:

• The N-terminal residue and surrounding sequence of Fbp1 are critical determinants of its stability within the Pro/N-degron pathway.
• Evolutionary changes in N-terminal residues can alter substrate recognition by the Gid4 ubiquitin ligase, impacting protein stability.
• A combination of genetic drift and natural selection likely shaped the evolution of the Pro/N-degron pathway and its substrates across yeast species.