Mutations at the hydrophobic core affect Hal3 trimer stability, reducing its Ppz1 inhibitory capacity but not its...

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Saccharomyces cerevisiae Hal3 (ScHal3) is a moonlighting protein.
• It regulates the Ser/Thr protein phosphatase Ppz1 in its monomeric state.
• ScHal3 also forms a heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme with ScCab3 for CoA biosynthesis.

Purpose of the Study:

• To investigate the structural determinants governing ScHal3's oligomeric state.
• To understand the relationship between Hal3's oligomeric state and its inhibition of Ppz1.
• To characterize the function of mutated Hal3 proteins in PPCDC activity and Ppz1 inhibition.

Main Methods:

• Site-directed mutagenesis of hydrophobic core residues in ScHal3 and Arabidopsis thaliana Hal3 (AtHal3).
• Characterization of mutated proteins' ability to form trimers and function as PPCDC.
• Assessment of mutated ScHal3's interaction with and inhibition of Ppz1.

Main Results:

• Mutations in AtHal3 did not affect trimerization or PPCDC function.
• Mutation of ScHal3 L403 had no effect on its functions.
• ScHal3 L405E mutation disrupted homotrimer formation but retained Cab3 binding, rescuing a synthetic lethal mutation. This mutation also reduced Ppz1 inhibition.

Conclusions:

• The oligomeric state of ScHal3 is critical for its moonlighting functions.
• The monomer-oligomer equilibrium of Hal3 influences its ability to regulate Ppz1.
• Specific residues in the hydrophobic core dictate Hal3's oligomerization and its dual roles in CoA biosynthesis and phosphatase regulation.