A point mutation causes mistargeting of Golgi GlcNAc-TV in the Lec4A Chinese hamster ovary glycosylation mutant.
The Lec4A and Lec4 Chinese hamster ovary glycosylation mutants lack N-linked glycans with GlcNAcbeta(1,6)Manalpha(1,6) branches that are initiated by the transferase termed GlcNAc-TV. Detergent extracts of Lec4 cells have no detectable GlcNAc-TV activity, but Lec4A extracts have activity equivalent to that of parental Chinese hamster ovary cells. This discrepancy occurs because Lec4A GlcNAc-TV activity co-localizes with membranes of the endoplasmic reticulum (ER) instead of with Golgi membranes (Chaney, W., Sundaram, S., Friedman, N., and Stanley, P. (1989) J. Cell. Biol. 109, 2089-2096). cDNAs from the coding region of the GlcNAc-TV gene have now been isolated from each mutant line. Lec4 GlcNAc-TV cDNA was found to possess two insertions, the first of which shifts the open reading frame and codes for a truncated transferase missing 585 amino acids from the catalytic domain. By contrast, Lec4A GlcNAc-TV cDNA possesses a single point mutation from T to G, which results in a change from Leu to Arg at position 188. When transfected into Lec4 cells, both cDNAs gave the appropriate phenotype; Lec4 cDNA was unable to restore GlcNAc-TV activity, whereas Lec4A cDNA converted Lec4 cells to the Lec4A phenotype, with an active GlcNAc-TV mislocalized to ER membranes. Moreover, Lec4A cDNA cured of its mutation restored a functional, Golgi-localized GlcNAc-TV to Lec4 cells. The results demonstrate that a single change in the 740 amino acids of GlcNAc-TV serves to functionally inactivate the transferase in an intact cell by causing it to localize to the ER instead of the Golgi compartment. The mislocalized transferase retains full enzyme activity, showing that it is well folded and stable and suggesting that the L188R mutation either prevents association with exit complexes from the ER or causes retrograde transport from a Golgi compartment.
Amgen Inc., Thousand Oaks, California 91320, USA.
J
J
Weinstein
S
S
Sundaram
X
X
Wang
D
D
Delgado
R
R
Basu
P
P
Stanley
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D017931:DNA Primers; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis* / Q000737:chemistry*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D051381:Rats; D015183:Restriction Mapping
D017931:DNA Primers; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase