THE RAT ALPHA-1,3-FUCOSYL-TRANSFERASE (RFUCT-IV) GENE ENCODES BOTH LONG AND SHORT FORMS OF THE ENZYME WHICH SHARE THE SAME INTRACELLULAR LOCATION

Fucosyltransferase (FucT) activity has been detected on the surface of mouse germ cells and rat Sertoli cells, and has been postulated to pl ay a role in cell-cell interactions. A recently cloned rat FucT (rFucT -IV) is expressed in the testes, and thus is a candidate for encoding the cell-surface FucT activity. This study maps the 5'-ends of several rFuc-T-IV mRNAs, and these results suggest that initiation of transcr iption may occur both upstream of the first ATG, as well as between th e first two closely spaced, in-frame ATGs. Thus, in certain tissues, n otably spleen, significant amounts of both a long and a short form of rFucT-IV would be predicted. This study also determines some basic pro perties of both the long and short forms of rFucT-IV, and investigates whether the use of alternative ATGs would allow FucT activity to be e xpressed both on the cell surface and in the Golgi. Plasmids that enco de FLAG-epitope-labeled rFucT-IVs that initiate from either of the two ATGs were constructed, and rFucT-IV was expressed either in vitro usi ng cell-free rabbit reticulocyte lysate, or after transfection in tiss ue culture. The results from these studies demonstrate that rFucT-IV i s a glycosylated, transmembrane protein with a short cytoplasmic tail, and that either of the two ATGs in the 5' region of the rFucT-IV gene are capable of acting as functional initiators of translation in vitr o, to produce enzymatically active glycoproteins. However, no differen ce in the intracellular localization between the transferase containin g a 48 amino acid or a 15 amino acid cytoplasmic tail was detected by immunocytochemistry, as both show the same pattern of Golgi-like stain ing in several different cell types, with no indication of surface exp ression. Thus, the additional ami no-terminal 33 amino acids of the lo ng form of rFucT-IV do not appear to influence its intracellular locat ion in the cell types investigated.