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

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. (.