Collaborative regulation of yeast SPT-Orm2 complex by phosphorylation and ceramide.

Area of Science:

• Biochemistry
• Molecular Biology
• Yeast Genetics

Background:

• Serine palmitoyltransferase (SPT) activity is crucial for sphingolipid biosynthesis.
• Regulation of SPT in yeast involves Orm protein N-terminal phosphorylation, absent in higher eukaryotes.
• Ceramide feedback inhibition of the SPT-ORM/ORMDL complex is conserved in higher eukaryotes, but its yeast mechanism is unclear.

Purpose of the Study:

• To elucidate the structural basis of SPT regulation by Orm2 in yeast.
• To investigate the interplay between phosphorylation and ceramide in SPT regulation.
• To identify conserved regulatory elements in the yeast SPT-Orm2 complex.

Main Methods:

• X-ray crystallography to determine the structure of the yeast SPT-Orm2 complex.
• Biochemical assays to assess SPT activity in response to ceramide and phosphorylation states.
• Site-directed mutagenesis to create dephosphomimetic and phosphomimetic Orm2 variants.

Main Results:

• The structure of the yeast SPT-Orm2 complex in a dephosphomimetic state was determined.
• An evolutionarily conserved ceramide-sensing site was identified.
• Ceramide binding stabilizes an inhibitory conformation of dephosphomimetic Orm2 via an intramolecular β-sheet.
• A phosphomimetic mutation destabilizes this inhibitory conformation.

Conclusions:

• Both Orm dephosphorylation and ceramide binding are essential for suppressing SPT activity in yeast.
• Yeast employs a distinctive regulatory mechanism involving collaborative phosphorylation and ceramide actions on SPT.
• Findings provide insights into the evolution of SPT regulation across eukaryotes.