Split invertase polypeptides form functional complexes in the yeast periplasm in vivo.
The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.
Schonberger
O
O
Department of Molecular Biology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Knox
C
C
Bibi
E
E
Pines
O
O
D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D010446:Peptide Fragments; D010735:Phosphoglycerate Kinase / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D043324:beta-Fructofuranosidase
D010446:Peptide Fragments; D010735:Phosphoglycerate Kinase; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; D043324:beta-Fructofuranosidase