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

Abstract

Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-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/membrane-anchoring 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 designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) 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, delta tail and delta 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-Leu33 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 transmembrane 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.

Location and Mechanism of α2,6-Sialyltransferase Dimer Formation

“…However, these GalT I dimers, which appear to constitute less than one half of the total enzyme, are quite resistant to standard reducing conditions, suggesting that dimerization may not necessarily involve a disulfide bond (28). Intriguingly, previous work by Aoki et al (30) showed that the replacement of this Cys and His residue in the GalT I transmembrane domain increased cell surface expression of the GalT I. Analysis of the C24A mutant that eliminates the dimerization of the STtyr isoform suggests that dimerization of this form of the enzyme does not significantly alter its exit from the Golgi, as measured by its cleavage and secretion (Fig.…”

The TGN38 glycoprotein contains two non-overlapping signals that mediate localization to the trans-Golgi network.

“…These act to keep proteins in the Golgi and, so far, all Golgi proteins (Machamer and Swift, 1991) and enzymes (Nilsson et al, 1991;Munro, 1991;Aoki et al, 1992;Burke et al, 1992;Colley et al, 1992;Tang et ai., 1992;Teasdale et al, 1992;Wong et al, 1992) have been found to be retained by the membrane-spanning domain. The mechanism of retention is still unclear though two possibilities have been proposed.…”

“…Golgi retention signals were first identified in a viral protein (Swift and Machamer, 1991) and in Golgi glycosylation enzymes (Nilsson et al, 1991;Munro, 1991;Aoki et al, 1992;Burke et al, 1992;Colley et al, 1992;Tang et al, 1992;Teasdale et al, 1992;Wong et al, 1992). In all cases, the retention signal lies in the membrane-spanning domain.…”

Short length transmembrane domains having voluminous exoplasmic halves determine retention of Type II membrane proteins in the Golgi complex

“…Other reports assign importance to the TMD for localization of glycosyltransferases (GTs) in the Golgi (Opat et al, 2001;Tang et al, 1992;Teasdale et al, 1992). This capacity was attributed to particular amino acid residues in the TMD (Aoki et al, 1992;Sousa et al, 2003) or the length of the TMD (Bretscher and Munro, 1993;Rayner and Pelham, 1997;Ronchi et al, 2008). Recent analyses of a large protein dataset showed that TMDs from single-spanning membrane proteins of the Golgi and plasma membrane have distinctive geometric features: Golgi protein TMDs are on average four amino acid residues shorter than those of plasma membrane proteins in vertebrates, and seven residues shorter in fungi.…”