SELECTIVE REENTRY OF RECYCLING CELL-SURFACE GLYCOPROTEINS TO THE BIOSYNTHETIC-PATHWAY IN HUMAN HEPATOCARCINOMA HEPG2 CELLS

Return of cell surface glycoproteins to compartments of the secretory pathway has been examined in HepG2 cells comparing return to the trans -Golgi network (TGN), the trans/medial- and cis-Golgi, Transport to th ese sites was studied by example of the transferrin receptor (TfR) and the serine peptidase dipeptidylpeptidase IV (DPPIV) after labeling th ese proteins with the N-hydroxysulfosuccinimide ester of biotin on the cell surface, This experimental design allowed to distinguish between glycoproteins that return to these biosynthetic compartments from the cell surface and newly synthesized glycoproteins that pass these comp artments during biosynthesis en route to the surface. Re-entry to the TGN was measured in that surface glycoproteins were desialylated with neuraminidase and were monitored for resialylation during recycling. R eturn to the trans-Golgi was traced measuring the transfer of [H-3]fuc ose residues to recycling surface proteins by fucosyltransferases. To study return to the cis-Golgi, surface proteins were metabolically lab eled in the presence of the mannosidase I inhibitor deoxymannojirimyci n (dMM), As a result surface proteins retained N-glycans of the oligom annosidic type. Return to the site of mannosidase I in the medial/cis- Golgi was measured monitoring conversion of these glycans to those of the complex type after washout of dMM, Our data demonstrate that DPPIV does return from the cell surface not only to the TGN, but also to th e trans-Golgi thus linking the endocytic to the secretory pathway. In contrast, no reentry to sites of mannosidase I could be detected indic ating that the early secretory pathway is not or is only at insignific ant rates accessible to recycling DPPIV. In contrast to DPPIV, TfR was very efficiently sorted from endosomes to the cell surface and did no t return to the TGN or to other biosynthetic compartments in detectabl e amounts, indicating that individual surface proteins are subject to different sorting mechanisms or sorting efficiencies during recycling.