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Tuning Degradation to Achieve Specific and Efficient Protein Depletion
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Rapid COG Depletion in Mammalian Cell by Auxin-Inducible Degradation System

Farhana Taher Sumya1, Irina D Pokrovskaya1, Vladimir V Lupashin2

  • 1Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

Methods in Molecular Biology (Clifton, N.J.)
|December 13, 2022
PubMed

Insights

The Conserved Oligomeric Golgi (COG) complex is vital for cell function. A new auxin-inducible degradation system rapidly depletes COG subunits, revealing immediate cellular defects in intra-Golgi trafficking.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The Conserved Oligomeric Golgi (COG) complex regulates intra-Golgi retrograde trafficking and macromolecule glycosylation.
  • The precise mechanism of COG function remains incompletely understood.
  • Previous research using prolonged protein depletion methods may introduce indirect cellular effects.

Purpose of the Study:

  • To develop a rapid depletion system for COG subunits in human cells.
  • To investigate the immediate cellular consequences of acute COG complex dysfunction.
  • To establish a versatile protocol for studying vesicle tethering complexes and intracellular trafficking.

Main Methods:

  • Utilized the auxin-inducible degradation system for rapid and efficient depletion of COG subunits.
  • Employed human cell lines engineered for inducible protein degradation.
  • Observed and analyzed the accumulation of COG complex-dependent (CCD) vesicles.

Main Results:

  • Achieved fast and efficient depletion of COG subunits using the auxin-inducible degradation system.
  • Successfully accumulated COG complex-dependent (CCD) vesicles upon acute COG subunit depletion.
  • Identified initial cellular defects associated with the rapid loss of COG complex function.

Conclusions:

  • The auxin-inducible degradation system provides a robust method for studying COG complex function.
  • Acute COG depletion allows for the investigation of early defects in intra-Golgi retrograde trafficking.
  • This protocol is adaptable for studying other vesicle tethering complexes and intracellular transport pathways.
Keywords:
AuxinCOG complexGlycosylationGolgiMembrane traffickingVesicle tethering

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