Expression and characterization of rat UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful host for the production of h eterologous proteins through the secretory pathway. However, because of the potential antigenicity of mannan-type sugar chains in humans, yeast cannot be used as a host for the production of glycoprotein therapeutics. To over come this problem, we are trying to breed a yeast which can produce hybrid- or complex-type carbohydrates, UDP-N-acetylglucosamine: alpha-3-D-mannosid e beta-1,2-N-acetylglucosaminyltransferase I (GnT-I) is essential for the c onversion of high mannose-type N-glycans to hybrid- and complex-type ones. As yeast lacks this enzyme, we have introduced the rat GnT-I cDNA into yeas t cells. The transformed yeast cells expressed GnT-I activity in vitro, The expressed GnT-I was localized in all organella, including the endoplasmic reticulum (ER), Golgi apparatus, and vacuole, suggesting that the mammalian Golgi retention signal of GnT-I did not function in yeast cells. Analysis of the GnT-I gene product with a c-Myc epitope tag at the C-terminus elucid ates that the N-terminal region of GnT-I, including the mammalian Golgi ret ention signal, should be removed in the yeast ER.