Topology of GPI biosynthesis in the endoplasmic reticulum. Academic Article uri icon

Overview

MeSH

  • Animals
  • Concanavalin A
  • Cytosol
  • Glycolipids
  • Microsomes
  • Phosphatidylinositol Diacylglycerol-Lyase
  • Phosphoinositide Phospholipase C
  • Phosphoric Diester Hydrolases
  • Type C Phospholipases

MeSH Major

  • Endoplasmic Reticulum
  • Glycosylphosphatidylinositols
  • Protozoan Proteins
  • Trypanosoma brucei brucei

abstract

  • Glycosylphosphatidylinositol (GPI) anchors are constructed in the endoplasmic reticulum (ER) through the action of at least seven unique enzymes. Using cell-free systems, mainly derived from African trypanosomes, it has been experimentally possible to re-create many aspects of the GPI biosynthetic pathway in vitro and to obtain a series of glycosylated phosphatidylinositol structures that correspond to biosynthetic intermediates. This approach led to the identification of the biosynthetic donors of individual components of the GPI glycan, and the discovery of unusual fatty acid re-modelling reactions in the GPI pathway in trypanosomes. Despite this progress, questions remain concerning the enzymology of the pathway, particularly the topological distribution of the different assembly steps in the ER membrane. In the work described here we have attempted to define the transbilayer orientation of different GPI biosynthetic intermediates in the ER membrane bilayer. The experiments were performed with a microsomal fraction derived from bloodstream-form Trypanosoma brucei, and standard radiolabeling procedures. The orientation of GPIs was probed with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and the jackbean lectin Concanavalin A. Contrary to expectations based on other ER glycosylation reactions, most notably the reactions involved in the dolichol pathway of N-glycosylation, our results suggest that non-inositol-acylated (PI-PLC-sensitive) GPIs are synthesized in the cytoplasmic leaflet of the ER membrane bilayer and that the final reaction product, a phosphoethanolamine-containing GPI, flips into the luminal leaflet for transfer to protein.

publication date

  • February 1994

has subject area

  • Animals
  • Concanavalin A
  • Cytosol
  • Endoplasmic Reticulum
  • Glycolipids
  • Glycosylphosphatidylinositols
  • Microsomes
  • Phosphatidylinositol Diacylglycerol-Lyase
  • Phosphoinositide Phospholipase C
  • Phosphoric Diester Hydrolases
  • Protozoan Proteins
  • Trypanosoma brucei brucei
  • Type C Phospholipases

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed ID

  • 8081226

Additional Document Info

start page

  • 167

end page

  • 175

volume

  • 27

number

  • 2