The Cytoplasmic Tail of α1,3-Galactosyltransferase Inhibits Golgi Localization of the Full-length Enzyme

Abstract

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 ...

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