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The Steady‐State Distribution of Glycosyltransferases Between the Golgi Apparatus and the Endoplasmic Reticulum is Approximately 90:10
Author(s) -
Rhee Sung Wu,
Starr Tregei,
ForstenWilliams Kimberly,
Storrie Brian
Publication year - 2005
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/j.1600-0854.2005.00333.x
Subject(s) - golgi apparatus , endoplasmic reticulum , biology , organelle , secretory pathway , glycosyltransferase , microbiology and biotechnology , green fluorescent protein , immunogold labelling , fluorescence microscope , glycosylation , cellular compartment , cytoplasm , biochemistry , fluorescence , enzyme , cell , ultrastructure , gene , physics , anatomy , quantum mechanics
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 β1,4‐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.