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Protocol for measuring sphingolipid metabolism in budding yeast.

Atsuko Ikeda1,2, Kazuki Hanaoka3, Kouichi Funato1,2,3

  • 1Graduate School of Biosphere Science, Hiroshima University, Kagamiyama 1-4-4, Higashi-Hiroshima 739-8528, Japan.

STAR Protocols
|April 29, 2021
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Summary

This study details a method for analyzing sphingolipid metabolism in yeast. It uses radioactive labeling to track ceramide transport from the endoplasmic reticulum to the Golgi apparatus.

Keywords:
Cell BiologyMetabolismModel OrganismsMolecular Biology

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Area of Science:

  • Cell Biology
  • Biochemistry

Background:

  • Sphingolipid biosynthesis is a complex process occurring across cellular organelles.
  • Ceramide, a key sphingolipid, is synthesized in the endoplasmic reticulum (ER) and subsequently transported to the Golgi apparatus for further modification.

Purpose of the Study:

  • To provide a detailed protocol for analyzing sphingolipid metabolism in budding yeast.
  • To enable the investigation of ceramide transport dynamics within a living yeast cell.

Main Methods:

  • Metabolic labeling of yeast sphingolipids, specifically ceramide and inositolphosphorylceramide (IPC), using radioactive precursors.
  • Development of a step-by-step protocol for executing these analyses in *Saccharomyces cerevisiae*.

Main Results:

  • A reproducible protocol for the metabolic labeling and analysis of yeast sphingolipids was established.
  • The protocol facilitates the study of ceramide transport in an *in vivo* context.

Conclusions:

  • The presented protocol offers a valuable tool for researchers studying sphingolipid metabolism and transport in yeast.
  • This method aids in understanding the intricate pathways of ceramide trafficking and its role in cellular function.