Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus

Glycolipid glycosyltransferases catalyze the stepwise transfer of monosacch arides from sugar nucleotides to proper glycolipid accepters. They are Golg i resident proteins that colocalize functionally in the organelle, but thei r intimate relationships are not known. Here, we show that the sequentially acting UDP-GalNAc: lactosylceramide/GM3/GD3 beta -1,4-N-acetyl-galactosami nyltransferase and the UDP-Gal:GA2/GM2/GD2 beta -1,3-galactosyltransferase associate physically in the distal Golgi. Immunoprecipitation of the respec tive epitope-tagged versions expressed in transfected CHO-K1 cells resulted in their mutual coimmunoprecipitation. The immunocomplexes efficiently cat alyze the two transfer steps leading to the synthesis of GM1 from exogenous GM3 in the presence of UDP-GalNAc and UDP-Gal, The N-terminal domains (cyt osolic tail, transmembrane domain, and few amino acids of the stem region) of both enzymes are involved in the interaction because (i) they reproduce the coimmunoprecipitation behavior of the full-length enzymes, (ii) they co mpete with the full-length counterpart in both coimmunoprecipitation and GM 1 synthesis experiments, and (iii) fused to the cyan and yellow fluorescent proteins, they localize these proteins to the Golgi membranes in an associ ation close enough as to allow fluorescence resonance energy transfer betwe en them. We suggest that these associations may improve the efficiency of g lycolipid synthesis by channeling the intermediates from the position of pr oduct to the position of acceptor along the transfer steps.