Overexpression and mislocalization of a tail-anchored GFP redefines the identity of peroxisomal ER

Traffic. 2003 Jul;4(7):491-501. doi: 10.1034/j.1600-0854.2003.00107.x.

Abstract

Peroxisomal ascorbate peroxidase (APX) sorts indirectly via a subdomain of the ER (peroxisomal ER) to the boundary membrane of peroxisomes in tobacco Bright Yellow 2 cells. This novel subdomain characteristically appears as fluorescent reticular/circular compartments distributed variously in the cytoplasm. Further characterizations are presented herein. A peptide possessing the membrane targeting information for peroxisomal APX was fused to GFP (GFP-APX). Transiently expressed GFP-APX sorted to peroxisomes and to reticular/circular compartments; in both cases, the GFP moiety faced the cytosol. Of particular interest, both homotypic and heterotypic aggregates of peroxisomes, mitochondria, and/or plastids were formed. The latter two organelles comprised the circular portion of the reticular/circular compartments, apparently as a consequence of oligomerization (zippering) of the GFP moieties after insertion into the outer membranes of the affected organelles. These results, coupled with the accumulation of endogenous peroxisomal APX in cytoplasmic, noncircular compartment(s) following treatment with brefeldin A, indicate that authentic peroxisomal ER is composed only of a reticular compartment(s). Equally important, the data show that overexpressed, membrane-targeted GFP fusion proteins have a propensity to form organelle aggregates that may lead to misinterpretations of sorting pathways of trafficked proteins.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Ascorbate Peroxidases
  • Brefeldin A / metabolism
  • Cell Line
  • Cell Membrane / metabolism
  • Cytoplasm
  • Endoplasmic Reticulum / metabolism*
  • Green Fluorescent Proteins
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Peroxidases / genetics
  • Peroxidases / metabolism*
  • Peroxisomes / metabolism*
  • Plant Proteins / genetics
  • Plant Proteins / metabolism
  • Protein Synthesis Inhibitors / metabolism
  • Protein Transport / physiology
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Tobacco / cytology
  • Tobacco / metabolism

Substances

  • Luminescent Proteins
  • Plant Proteins
  • Protein Synthesis Inhibitors
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Brefeldin A
  • Peroxidases
  • Ascorbate Peroxidases