The glycosylation defect in solute carrier SLC35A2/SLC35A3 double knockout cells is rescued by SLC35A2-SLC35A3 and SLC35A3-SLC35A2 hybrids.

SLC35A2 and SLC35A3 are homologous proteins with postulated nucleotide sugar transporting activities. Unlike SLC35A2, whose specificity for UDP-Gal is well-established, the UDP-GlcNAc transporting activity initially attributed to SLC35A3 has recently been put into question. In this study, we constructed two hybrid proteins (SLC35A2-SLC35A3 and SLC35A3-SLC35A2) and expressed them in a previously generated SLC35A2/SLC35A3 double knockout HEK293T cell line. Our idea was to force equivalent stoichiometry of the two proteins in the cells in order to reproduce the behavior of the SLC35A2/SLC35A3 complexes in the Golgi membrane. The hybrid proteins were able to fully restore glycosylation in the double knockout. In contrast, the expression of SLC35A3 alone in these cells improved galactosylation only to a limited extent. Our study shows that the proper glycosylation requires a balanced cooperation between SLC35A2 and SLC35A3.