The rat α1,3-fucosyltransferase (rFucT-IV) gene encodes both long and short forms of the enzyme which share the same intracellular location

Abstract

Fucosyltransferase (FucT) activity has been detected on the surface of mouse germ cells and rat Sertoli cells, and has been postulated to play 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 transcription may occur both upstream of the first ATG, as well as between the first two closely spaced, in-frame ATGs. Thus, in certain tissues, notably spleen, significant amounts of both a long and a short form of rFucT-IV would be predicted. This study also determines some basic properties of both the long and short forms of rFucT-IV, and investigates whether the use of alternative ATGs would allow FucT activity to be expressed both on the cell surface and in the Golgi. Plasmids that encode FLAG-epitope-labeled rFucT-IVs that initiate from either of the two ATGs were constructed, and rFucT-IV was expressed either in vitro using cell-free rabbit reticulocyte lysate, or after transfection in tissue culture. The results from these studies demonstrate that rFucT-IV is 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 vitro, to produce enzymatically active glycoproteins. However, no difference in the intracellular localization between the transferase containing a 48 amino acid or a 15 amino acid cytoplasmic tail was detected by immunocytochemistry, as both show the same pattern of Golgi-like staining in several different cell types, with no indication of surface expression. Thus, the additional amino-terminal 33 amino acids of the long form of rFucT-IV do not appear to influence its intracellular location in the cell types investigated.