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

Tian Xie1, Feitong Dong1, Gongshe Han2

  • 1Department of Chemical Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China.

Cell Reports
|January 29, 2024
PubMed

Insights

Yeast serine palmitoyltransferase (SPT) activity is regulated by Orm dephosphorylation and ceramide binding. Ceramide stabilizes the inhibitory conformation of Orm2, while phosphorylation destabilizes it, revealing a unique yeast regulatory mechanism.

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.
Keywords:
CP: MetabolismCP: Molecular biology

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