Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain

Daisuke Aoki, Ni Lee, Naoto Yamaguchi, Craig Dubois, Michiko N. Fukuda

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111 Citations (Scopus)

Abstract

Galactosyltransferase (GT; UDPgalactose:β-D-N-acetylglucosaminide β-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membraneanchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was desiged to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin α subunit (hCGα) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, Δ tail and Δ stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu 33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29 → Ser29 and His32 → Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31 → Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32 → Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrance domain of GT contributes to the Golgi retention signal and that particularly Cys29 and His32 in this region are critical for GT to be retained in the Golgi. (.

Original languageEnglish
Pages (from-to)4319-4323
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume89
Issue number10
Publication statusPublished - 1992

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Galactosyltransferases
Chorionic Gonadotropin
Histidine
Transferrin Receptors
Cysteine
Membrane Proteins
Membranes
Cell Membrane
Lactose Synthase
Amino Acids
COS Cells
Fluorescence Microscopy
Blood Proteins
Catalytic Domain
Peptides
Mutation
Antibodies

Keywords

  • Glycosylation
  • Glycosyltransferase
  • Intracellular targeting transport
  • Mutagenesis
  • Oligomerization

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

@article{78945279745842b6850c94861284f82d,
title = "Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain",
abstract = "Galactosyltransferase (GT; UDPgalactose:β-D-N-acetylglucosaminide β-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membraneanchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was desiged to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin α subunit (hCGα) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, Δ tail and Δ stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu 33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29 → Ser29 and His32 → Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31 → Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32 → Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrance domain of GT contributes to the Golgi retention signal and that particularly Cys29 and His32 in this region are critical for GT to be retained in the Golgi. (.",
keywords = "Glycosylation, Glycosyltransferase, Intracellular targeting transport, Mutagenesis, Oligomerization",
author = "Daisuke Aoki and Ni Lee and Naoto Yamaguchi and Craig Dubois and Fukuda, {Michiko N.}",
year = "1992",
language = "English",
volume = "89",
pages = "4319--4323",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
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TY - JOUR

T1 - Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain

AU - Aoki, Daisuke

AU - Lee, Ni

AU - Yamaguchi, Naoto

AU - Dubois, Craig

AU - Fukuda, Michiko N.

PY - 1992

Y1 - 1992

N2 - Galactosyltransferase (GT; UDPgalactose:β-D-N-acetylglucosaminide β-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membraneanchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was desiged to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin α subunit (hCGα) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, Δ tail and Δ stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu 33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29 → Ser29 and His32 → Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31 → Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32 → Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrance domain of GT contributes to the Golgi retention signal and that particularly Cys29 and His32 in this region are critical for GT to be retained in the Golgi. (.

AB - Galactosyltransferase (GT; UDPgalactose:β-D-N-acetylglucosaminide β-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membraneanchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was desiged to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin α subunit (hCGα) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, Δ tail and Δ stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu 33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29 → Ser29 and His32 → Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31 → Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32 → Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrance domain of GT contributes to the Golgi retention signal and that particularly Cys29 and His32 in this region are critical for GT to be retained in the Golgi. (.

KW - Glycosylation

KW - Glycosyltransferase

KW - Intracellular targeting transport

KW - Mutagenesis

KW - Oligomerization

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JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

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