title: The steady-state distribution of glycosyltransferases between the Golgi apparatus and the endoplasmic reticulum is approximately 90:10.
abstract: Several lines of evidence support a novel model for Golgi protein residency in which these proteins cycle between the Golgi apparatus and the endoplasmic reticulum (ER). However, to preserve the functional distinction between the two organelles, this pool of ER-resident Golgi enzymes must be small. We quantified the distribution for two Golgi glycosyltransferases in HeLa cells to test this prediction. We reasoned that best-practice, quantitative solutions would come from treating images as data arrays rather than pictures. Using deconvolution and computer calculated organellar boundaries, the Golgi fraction for both endogenous beta1,4-galactosyltransferase and UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase 2 fused with green fluorescent protein (GFP) was 91% by fluorescence microscopy. Immunogold labeling followed by electron microscopy and model analysis yielded a similar value. Values reflect steady-state conditions, as inclusion of a protein synthesis inhibitor had no effect. These data strongly suggest that the fluorescence of a GFP chimera with an organellar protein can be a valid indicator of protein distribution and more generally that fluorescent microscopy can provide a valid, rapid approach for protein quantification. In conclusion, we find the ER pool of cycling Golgi glycosyltransferases is small and approximately 1/100 the concentration found in the Golgi apparatus.
authors.affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA.
authors.forename: Sung Wu
authors.initials: SW
authors.lastname: Rhee
authors.forename: Tregei
authors.initials: T
authors.lastname: Starr
authors.forename: Kimberly
authors.initials: K
authors.lastname: Forsten-Williams
authors.forename: Brian
authors.initials: B
authors.lastname: Storrie
mesh_terms: D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure; D016695:Glycosyltransferases / Q000378:metabolism*; D006046:Gold; D006056:Golgi Apparatus / Q000201:enzymology* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron
chemical_list: D006046:Gold; D016695:Glycosyltransferases