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Sugar nucleotides dissipate ATP-generated transmembrane pH gradient in Golgi vesicles from suspension-cell

M Gogarten-Boekels1, J P Gogarten, F W Bentrup

  • 1Botanisches Institut I der Justus-Liebig-Universität, Senckenbergstraße 17-21, D-6300, Giessen, Federal Republic of Germany.

Planta
|November 14, 2013
PubMed

Insights

Isolated Golgi vesicles (GV) from Chenopodium rubrum exhibit an ATP-driven pH gradient, crucial for UDP-glucose incorporation. This process involves a specific translocator and anion channel, offering insights into plant Golgi function.

Area of Science:

  • Plant Cell Biology
  • Membrane Transport
  • Biochemistry

Background:

  • The Golgi apparatus plays a vital role in post-translational modification and protein sorting in plant cells.
  • Understanding the specific functions of isolated Golgi vesicles (GV) is key to elucidating cellular processes.
  • Photoautotrophic suspension-cell protoplasts of Chenopodium rubrum provide a model system for studying plant Golgi vesicles.

Purpose of the Study:

  • To isolate and characterize Golgi vesicles (GV) from Chenopodium rubrum.
  • To investigate the mechanism of pH gradient formation and its role in vesicle function.
  • To identify key components involved in UDP-glucose transport and incorporation within the Golgi.

Main Methods:

  • Isolation of Golgi vesicles (GV) enriched with latent inosine diphosphatase (IDPase) from Chenopodium rubrum protoplasts.
  • Measurement of pH gradients using acridine orange accumulation in response to ATP.
  • Equilibrium density centrifugation and Percoll gradient analysis for vesicle characterization.
  • Enzyme inhibition studies using various compounds (ADP, Pi, orthovanadate, monensin, nigericin, N-ethylmaleimide, KNO3, DIDS).
  • Analysis of UDP-glucose and UDP-galactose effects on pH gradients and Pi release.
  • Radioisotope labeling ([U-(14)C]glucose) to track UDP-glucose incorporation into macromolecules.

Main Results:

  • Isolated GVs exhibited an ATP-dependent pH gradient, sensitive to MgATP concentration and competitively inhibited by ADP.
  • The pH gradient was dissipated by ionophores (monensin, nigericin) and partially by KNO3 and N-ethylmaleimide.
  • UDP-glucose and UDP-galactose dissipated the pH gradient and promoted Pi release, with UDP-glucose incorporation into macromolecules inhibited by DIDS.
  • A UDP-glucose/uridine-5'-monophosphate translocator and a phosphate-permeable anion channel are proposed to operate in these GVs.

Conclusions:

  • Golgi vesicles from Chenopodium rubrum possess an active proton pump generating a pH gradient essential for their function.
  • The study identifies a potential UDP-glucose transport system and anion channel involved in Golgi-mediated biosynthesis.
  • These findings contribute to a model for Golgi vesicle function in plants, particularly in cell wall polysaccharide synthesis.

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