Autofluorescent proteins for monitoring the intracellular distribution of glycosyltransferases

Methods Enzymol. 2006;416:102-20. doi: 10.1016/S0076-6879(06)16007-3.

Abstract

To analyze the Golgi compartmentalization of glycosyltransferases (GTs), we generated versions of several enzymes fused to either the enhanced green fluorescent protein (EGFP) or the red fluorescent protein from Discosoma sp. reef coral (DsRed2) and examined their intracellular distribution by confocal fluorescence microscopy in living cells. In a previous work, we have shown that the N-terminal peptides of GTs, encompassing the cytosolic and the transmembrane domains (CTDs), can serve as Golgi-targeting signals to localize the enzymes to their corresponding compartments within the Golgi apparatus (Zerfaoui et al., 2002). Using sialyl-Lewis x synthesis and selectin binding as functional assays, we show here that by swapping CTDs between GTs, it is possible to mislocalize an enzyme from a Golgi compartment to another, thereby altering the overall cellular glycosylation. On the other hand, we demonstrate that the use of an autofluorescent tag such as EGFP offers numerous advantages including the possibility of (1) facilitating sorting by fluorescence-activated cell sorter (FACS) of stably transfected polyclonal cell population, (2) constantly monitoring the expression of the enzymes in live cells, (3) establishing a direct relationship between the fluorescence intensity and the enzyme activities in vivo and in vitro, (4) establishing a visual relationship between function and intracellular distribution of a given GT, as well as co-localization with cognate protein acceptors by confocal microscopy, and (5) detecting proteins on blots with highly sensitive commercially available antibodies.

MeSH terms

  • Glycosyltransferases / chemistry*
  • Glycosyltransferases / metabolism*
  • Golgi Apparatus / chemistry
  • Golgi Apparatus / enzymology*
  • Green Fluorescent Proteins / chemistry*
  • Intracellular Fluid / chemistry
  • Intracellular Fluid / enzymology
  • Luminescent Proteins / chemistry*
  • Microscopy, Confocal

Substances

  • Luminescent Proteins
  • enhanced green fluorescent protein
  • red fluorescent protein
  • Green Fluorescent Proteins
  • Glycosyltransferases