SPLIT INVERTASE POLYPEPTIDES FORM FUNCTIONAL COMPLEXES IN THE YEAST PERIPLASM IN-VIVO

The assembly of functional proteins from fragments in vivo has been re cently described for several proteins, including the secreted maltose binding protein in Escherichia coil. Here we demonstrate for the first time that split gene products can function within the eukaryotic secr etory system. Saccharomyces cerevisiae strains able to use sucrose pro duce the enzyme invertase, which is targeted by a signal peptide to th e central secretory pathway and the periplasmic space. Using this enzy me as a model we find the following: (i) Polypeptide fragments of inve rtase, each containing a signal peptide, are independently translocate d into the endoplasmic reticulum (ER) are modified by glycosylation, a nd travel the entire secretory pathway reaching the yeast periplasm. ( ii) Simultaneous expression of independently translated and translocat ed overlapping fragments of invertase leads to the formation of an enz ymatically active complex, whereas individually expressed fragments ex hibit no activity. (iii) An active invertase complex is assembled in t he ER, is targeted to the yeast periplasm, and is biologically functio nal, as judged by its ability to facilitate growth on sucrose as a sin gle carbon source. These observations are discussed in relation to pro tein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.