The Golgi apparatus has a central role in the glycosylation of proteins and lipids. There is a sequential addition of carbohydrates by glycosyltransferases that are distributed within the Golgi in the order in which the glycosylation occurs. The mechanism of glycosyltransferase retention is considered to involve their transmembrane domains and flanking regions, although we have shown that the cytoplasmic tail of ␣1,2-fucosyltransferase is important for its Golgi localization. Here we show that the removal of the ␣1,2-fucosyltransferase cytoplasmic tail altered its function of fucosylation and its localization site. When the tail was removed, the enzyme moved from the Golgi to the trans Golgi network, suggesting that the transmembrane is responsible for retention and that the cytoplasmic tail is responsible for localization. The cytoplasmic tail of ␣1,2-fucosyltransferase contains 8 amino acids (MWVPSRRH), and mutating these to alanine indicated a role for amino acids 3 to 7 in localization with a particular role of Ser 5 . Mutagenesis of Ser 5 to amino acids containing an hydroxyl (Tyr and Thr) demonstrated that the hydroxyl at position 5 is important. Thus, the cytoplasmic tail, and especially a single amino acid, has a predominant role in the localization and thus the function of ␣1,2-fucosyltransferase.Glycosylation of proteins and lipids occurs in the endoplasmic reticulum and the Golgi apparatus and is mediated by glycosyltransferases and glycosidases that are resident there. The enzymes are ordered so that carbohydrates can be sequentially added to and removed from proteins and lipids (1-3). The mechanism for retention of the enzymes in the appropriate subcompartment and the domains of the enzymes that direct this localization are not clear. At present there are two proposed mechanisms for Golgi glycosyltransferase localization, kin recognition (4, 5), where like gycosyltransferases form aggregates within the Golgi, and the lipid bilayer model, where the hydrophobic transmembrane domain retains the transferases in the Golgi (6). Neither the kin-recognition nor the lipid bilayer models fully explain the specific and functional localization of transferases to discrete sites within the Golgi; we now demonstrate that the cytoplasmic tail is a key element in the specific localization of ␣1,2-fucosyltransferase.Glycosyltransferases are type II integral membrane proteins, with the catalytic domain residing in the lumen of the Golgi (7) and the N terminus in the cytoplasm (cytoplasmic tail). Several glycosyltransferases have been localized in compartments of the Golgi by electron microscopy (for review see Ref. 8). Studies of glycosyltransferases have demonstrated the importance of the transmembrane domain and its flanking sequences (9 -14) and the luminal domain (15) for localization.We examined two glycosyltransferases that compete for the same acceptor, N-acetyllactosamine (NAcLac), 1 and showed that when there is expression of ␣1,3-galactosyltransferase (GT) together with ␣1,2-fucosyltransferase (FT) within a ...

It is currently under debate whether the mechanism of Golgi retention of different glycosyltransferases is determined by sequences in the transmembrane, luminal, or cytoplasmic domains or a combination of these domains. We have shown that the cytoplasmic domains of ␣1,3-galactosyltransferase (GT) and ␣1,2-fucosyltransferase (FT) are involved in Golgi localization. Here we show that the cytoplasmic tails of GT and FT are sufficient to confer specific Golgi localization. Further, we show that the expression of only the cytoplasmic tail of GT can lead to displacement or inhibition of binding of the whole transferase and that cells expressing the cytoplasmic tail of GT were not able to express fulllength GT or its product, Gal␣1,3Gal. Thus, the presence of the cytoplasmic tail prevented the localization and function of full-length GT, suggesting a possible specific Golgi binding site for GT. The effect was not altered by the inclusion of the transmembrane domain. Although the transmembrane domain may act as an anchor, these data show that, for GT, only the cytoplasmic tail is involved in specific localization to the Golgi.Proteins and lipids are transported through the endoplasmic reticulum and Golgi where carbohydrates are sequentially added by glycosyltransferase enzymes. The glycosyltransferases are considered to reside in the Golgi in a spatial configuration that allows the sequential specific addition of carbohydrates (1), although the mechanisms governing their ordered localization and retention is not clear. Glycosyltransferases are type II integral membrane proteins with a C-terminal catalytic domain in the lumen of the Golgi and an N-terminal cytoplasmic tail (2). Two mechanisms of glycosyltransferase localization in the Golgi have been suggested: kin recognition (3), where similar proteins form aggregates within the Golgi, and the lipid bilayer model (4), where the length of the hydrophobic transmembrane domain retains the transferases. However, neither mechanism fully explains the localization of transferases to specific sites within the Golgi. Thus far, no amino acid consensus sequence for localization has been described, and there is difficulty in competitively saturating the glycosyltransferase retention mechanisms (5). We and others have proposed a third mechanism of retention involving the cytoplasmic domain of proteins resident in the Golgi. Indeed, there is a growing body of evidence implicating the cytoplasmic domain of Type I (6) and Type II resident Golgi proteins (7-11), coiled-coil proteins (12, 13), and viral proteins (14 -17). One of the most convincing studies showed that Myc and FLAG tags placed at the N terminus of the cytoplasmic tails of ␣2,6-sialyltransferase and N-acetylglucosaminyltransferase I disrupted Golgi localization (18). A 20-amino acid spacer between the tag at the N terminus and the cytoplasmic tail sequence was able to restore Golgi localization, suggesting the importance of the cytoplasmic tail in localization (18,19). However, the currently accepted view in the field is ...

Carbohydrate structures influence many aspects of cell biology. Manipulating the glycosyltransferase enzymes, that sequentially add carbohydrate moieties to proteins and lipids as they pass through the Golgi and secretory pathway, can alter these carbohydrate epitopes. We previously demonstrated that the eight amino acid cytoplasmic tail of alpha1,2fucosyltransferase (FT) contained a sequence for Golgi localisation. In this study, we examined the localisation of the closely related secretor type alpha1,2fucosyltransferase (Sec) which has a smaller, yet apparently unrelated, five amino acid cytoplasmic tail. In contrast to the Golgi localisation of FT, Sec displayed atypical cytoplasmic vesicular-like staining. However, replacing just the five amino acid tail of Sec with FT was sufficient to relocalise the enzyme to a perinuclear region with Golgi-like staining. The biological significance of this relocalisation was this chimaeric enzyme was more effective than FT at competing for N-Acetyl-lactosamine and thus was superior in reducing expression of the Galalpha(1,3)Gal xenoepitope.