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Previous studies have shown that when the cytosolic domains of the type I membrane proteins TGN38 and lysosomal glycoprotein 120 (lgp120) are added to a variety of reporter molecules, the resultant chimeric molecules are localized to the trans-Golgi network (TGN) and to lysosomes, respectively. In the present study we expressed chimeric constructs of rat TGN38 and rat lgp120 in HeLa cells. We found that targeting information in the cytosolic domain of TGN38 could be overridden by the presence of the lumenal and transmembrane domains of lgp120. In contrast, the presence of the transmembrane and cytosolic domains of TGN38 was sufficient to deliver the lumenal domain of lgp120 to the trans-Golgi network. On the basis of steady-state localization of the various chimeras and antibody uptake experiments, we propose that there is a hierarchy of targeting information in each molecule contributing to sorting within the endocytic pathway. The lumenal and cytosolic domains of lgp120 contribute to sorting and delivery to lysosomes, whereas the transmembrane and cytosolic domains of TGN38 contribute to sorting and delivery to the trans-Golgi network.

TGN38 is a heavily glycosylated, type I integral membrane protein which is predominantly localized to the trans Golgi network (TGN), but which constitutively traffics between the TGN and the cell surface. The trafficking of TGN38 has been extensively studied in non-polarized cells, and a short, tyrosine-based, peptide motif within the cytosolic domain of the protein has been shown to be necessary and sufficient for its rapid internalization from the cell surface and efficient delivery to the TGN. Such tyrosine-based motifs have also been shown to act as basolateral targeting signals, whilst N-linked glycans (as occur on the extracytosolic domain of TGN38) can act as apical targeting signals. TGN38 has previously been shown to be sorted to the basolateral surface of polarized canine MDCK cells; a polarized cell line in which biosynthetic sorting decisions concerning the eventual destination of apical or basolateral targeted plasma membrane proteins are made at the TGN. We now show that TGN38 is targeted exclusively to the basolateral domain of polarized human Caco-2 cells, a cell line in which newly synthesized membrane proteins destined for either the apical or basolateral plasma membrane may be sorted for delivery to their final destination either at the TGN or at the cell surface. These data also demonstrate that the heavily glycosylated, extracytosolic domain of TGN38 does not contain a dominant apical targeting signal.