The Lec4A CHO glycosylation mutant arises from miscompartmentalization of a Golgi glycosyltransferase.
Two CHO glycosylation mutants that were previously shown to lack N-linked carbohydrates with GlcNAc beta 1,6Man alpha 1,6 branches, and to belong to the same genetic complementation group, are shown here to differ in the activity of N-acetylglucosaminyltransferase V (GlcNAc-TV) (UDP-GlcNA: alpha 1,6mannose beta-N-acetylglucosaminyltransferase V). One mutant, Lec4, has no detectable GlcNAc-TV activity whereas the other, now termed Lec4A, has activity equivalent to that of parental CHO in detergent cell extracts. However, Lec4A GlcNAc-TV can be distinguished from CHO GlcNAc-TV on the basis of its increased sensitivity to heat inactivation and its altered subcellular compartmentalization. Sucrose density gradient fractionation shows that the major portion of GlcNAc-TV from Lec4A cells cofractionates with membranes of the ER instead of Golgi membranes where GlcNAc-TV is localized in parental CHO cells. Other experiments show that Lec4A GlcNAc-TV is not concentrated in lysosomes, or in a post-Golgi compartment, or at the cell surface. The altered localization in Lec4A cells is specific for GlcNAc-TV because two other Lec4A Golgi transferases cofractionate at the density of Golgi membranes. The combined data suggest that both lec4 and lec4A mutations affect the structural gene for GlcNAc-TV, causing either the loss of GlcNAc-TV activity (lec4) or its miscompartmentalization (lec4A). The identification of the Lec4A defect indicates that appropriate screening of different glycosylation-defective mutants should enable the isolation of other mammalian cell trafficking mutants.
Chaney
W
W
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
Sundaram
S
S
Friedman
N
N
Stanley
P
P
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005816:Genetic Complementation Test; D005838:Genotype; D005964:Glucosyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases; D010053:Ovary; D011499:Protein Processing, Post-Translational
D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase