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 language | English |
|---|---|
| Pages (from-to) | 4319-4323 |
| Number of pages | 5 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 89 |
| Issue number | 10 |
| Publication status | Published - 1992 |
Fingerprint
Keywords
- Glycosylation
- Glycosyltransferase
- Intracellular targeting transport
- Mutagenesis
- Oligomerization
ASJC Scopus subject areas
- Genetics
- General
Cite this
Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain. / Aoki, Daisuke; Lee, Ni; Yamaguchi, Naoto; Dubois, Craig; Fukuda, Michiko N.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 89, No. 10, 1992, p. 4319-4323.Research output: Contribution to journal › Article
}
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
UR - http://www.scopus.com/inward/record.url?scp=0026607683&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0026607683&partnerID=8YFLogxK
M3 - Article
C2 - 1584766
AN - SCOPUS:0026607683
VL - 89
SP - 4319
EP - 4323
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
SN - 0027-8424
IS - 10
ER -