Folding of active beta-lactamase in the yeast cytoplasm before translocation into the endoplasmic reticulum.
Polypeptides targeted to the yeast endoplasmic reticulum (ER) posttranslationally are thought to be kept in the cytoplasm in an unfolded state by Hsp70 chaperones before translocation. We show here that Escherichia coli beta-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in living Saccharomyces cerevisiae cells. beta-Lactamase is a globular trypsin-resistant molecule in authentic form. For these studies, it was linked to the C terminus of a yeast polypeptide Hsp150delta, which conferred posttranslational translocation and provided sites for O-glycosylation. We devised conditions to retard translocation of Hsp150delta-beta-lactamase. This enabled us to show by protease protection assays that an unglycosylated precursor was associated with the cytoplasmic surface of isolated microsomes, whereas a glycosylated form resided inside the vesicles. Both proteins were trypsin resistant and had similar beta-lactamase activity and Km values for nitrocefin. The enzymatically active cytoplasmic intermediate could be chased into the ER, followed by secretion of the activity to the medium. Productive folding in the cytoplasm occurred in the absence of disulfide formation, whereas in the ER lumen, proper folding required oxidation of the sulfhydryls. This suggests that the polypeptide was refolded in the ER and consequently, at least partially unfolded for translocation.
Paunola
E
E
Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.
Suntio
T
T
Jämsä
E
E
Makarow
M
M
D001692:Biological Transport; D003593:Cytoplasm / Q000201:enzymology*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism; D006023:Glycoproteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin / Q000378:metabolism; D015003:Yeasts / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004792:Enzyme Precursors; D006023:Glycoproteins; C074229:HSP150 protein, S cerevisiae; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin; D001618:beta-Lactamases