A point mutation causes mistargeting of Golgi GlcNAc-TV in the Lec4A chinese hamster ovary glycosylation mutant

Jasminder Weinstein, Subha Sundaram, Xuhong Wang, Dora Delgado, Rita Basil, Pamela Stanley

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

The Lec4A and Lec4 Chinese hamster ovary glycosylation mutants lack N- linked glycans with GlcNAcβ(1,6)Manα(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.

Original languageEnglish (US)
Pages (from-to)27462-27469
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number44
DOIs
StatePublished - 1996

Fingerprint

Glycosylation
Cricetulus
Point Mutation
Ovary
Complementary DNA
Endoplasmic Reticulum
Transferases
Membranes
Phenotype
Amino Acids
Mutation
Enzyme activity
Cell membranes
Cell Extracts
Detergents
Open Reading Frames
Polysaccharides
Catalytic Domain
Genes
Cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

A point mutation causes mistargeting of Golgi GlcNAc-TV in the Lec4A chinese hamster ovary glycosylation mutant. / Weinstein, Jasminder; Sundaram, Subha; Wang, Xuhong; Delgado, Dora; Basil, Rita; Stanley, Pamela.

In: Journal of Biological Chemistry, Vol. 271, No. 44, 1996, p. 27462-27469.

Research output: Contribution to journalArticle

Weinstein, Jasminder ; Sundaram, Subha ; Wang, Xuhong ; Delgado, Dora ; Basil, Rita ; Stanley, Pamela. / A point mutation causes mistargeting of Golgi GlcNAc-TV in the Lec4A chinese hamster ovary glycosylation mutant. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 44. pp. 27462-27469.
@article{5a46df0174154275a0af75afcf2857a8,
title = "A point mutation causes mistargeting of Golgi GlcNAc-TV in the Lec4A chinese hamster ovary glycosylation mutant",
abstract = "The Lec4A and Lec4 Chinese hamster ovary glycosylation mutants lack N- linked glycans with GlcNAcβ(1,6)Manα(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.",
author = "Jasminder Weinstein and Subha Sundaram and Xuhong Wang and Dora Delgado and Rita Basil and Pamela Stanley",
year = "1996",
doi = "10.1074/jbc.271.44.27462",
language = "English (US)",
volume = "271",
pages = "27462--27469",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "44",

}

TY - JOUR

T1 - A point mutation causes mistargeting of Golgi GlcNAc-TV in the Lec4A chinese hamster ovary glycosylation mutant

AU - Weinstein, Jasminder

AU - Sundaram, Subha

AU - Wang, Xuhong

AU - Delgado, Dora

AU - Basil, Rita

AU - Stanley, Pamela

PY - 1996

Y1 - 1996

N2 - The Lec4A and Lec4 Chinese hamster ovary glycosylation mutants lack N- linked glycans with GlcNAcβ(1,6)Manα(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.

AB - The Lec4A and Lec4 Chinese hamster ovary glycosylation mutants lack N- linked glycans with GlcNAcβ(1,6)Manα(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.

UR - http://www.scopus.com/inward/record.url?scp=0029861621&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029861621&partnerID=8YFLogxK

U2 - 10.1074/jbc.271.44.27462

DO - 10.1074/jbc.271.44.27462

M3 - Article

VL - 271

SP - 27462

EP - 27469

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 44

ER -