EXPRESSION AND SECRETION OF BETA-GALACTOSIDASE IN SACCHAROMYCES-CEREVISIAE USING THE SIGNAL SEQUENCES OF GGP-I, THE MAJOR YEAST GLYCOSYLPHOSPHATIDYLINOSITOL-CONTAINING PROTEIN

New secretory signals and strategies can be attempted to improve the s ecretion of heterologous proteins of biotechnological interest which e ncounter difficulties being exported in yeast. The GGPI gene of Saccha romyces cerevisiae codes for a 125 kDa glycoprotein transported throug h the secretory pathway and anchored to the plasma membrane by means o f a glycosylphosphatidylinositol, The regions coding for the secretory signal or also for the first 46 amino acids were tested for efficienc y in secretion by fusion to the lacZ gene of Escherichia coli resultin g in the synthesis of the endoplasmic reticulum-targeted I-22- and I-6 8-GgpIp/beta-gal hybrids, A cytoplasmic form was also examined. The I- 22 beta gal is partially transported to the cell surface and in the me dium in an unglycosylated form. The I-68 beta gal is completely retain ed in the intracellular membranes and is N-glycosylated in the GgpIp m oiety. The amount of hybrid protein produced is similar and independen t from its targeted site, suggesting that translocation through endopl asmic reticulum is not a limiting step, whereas the amount of active e nzyme is from 50 to 80% lower for the endoplasmic reticulum forms comp ared with the cytoplasmic form. BiP/KarZp putative precursor is accumu lated in cells expressing the endoplasmic reticulum-targeted forms but not in those producing the cytosolic beta-galactosidase or overexpres sing an endogenous secretory protein, Thus, glycosylation and abnormal folding rather than overexpression are among the factors responsible for the decreased activity and exit of beta-galactosidase from the end oplasmic reticulum and for induction of pip The results obtained indic ate that the sole secretory signal of GgpIp is suitable to drive secre tion of foreign products with complex folding and point to the importa nce of the endoplasmic reticulum quality control in the secretion of h eterologous proteins in yeast.