Intracellular transit of a yeast protease is rescued by trans-complementation with its prodomain.
The alkaline extracellular protease (AEP) of the yeast Yarrowia lipolytica is synthesized as a preproprotein. The precursor undergoes a complex maturation during its intracellular transit, successively involving signal peptide cleavage, dipeptidyl aminopeptidase processing, and cleavage at a dibasic site which results in the extracellular release of the active enzyme. It was previously shown that various deletions within the proregion affect the intracellular transit of the protease. Prodeleted precursors are translocated and have their signal sequences removed, but they accumulate in the secretion apparatus. We show here that the secretion of partially active proteins is restored when the prodomain is supplied in trans as an independent peptide. The secretion rescue and maturation processing that are reconstituted by the free propeptide do not reach wild type efficiency. The results of pulse-chase experiments indicate that a rate-limiting step occurs during the intracellular transit of the rescued precursors, before Kex2p proteolytic cleavage. This delayed maturation seems to be responsible for an overall slower release of the rescued polypeptides. Propeptide and AEP were secreted in equimolar amounts by both wild type and trans-complemented strains, but none could be detected in the supernatant when expressed alone. These experiments suggest that the prodomain of AEP initially acts as a crucial folding aid for the early secretory transit of the translocated precursor. They further suggest that the prodomain is also required for a second structural change of the AEP precursor during its activation.
Fabre
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E
Laboratoire de Génétique des Microorganismes, Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique, Thiverval Grignon, France.
Tharaud
C
C
Gaillardin
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D016521:Electrophoresis, Gel, Pulsed-Field; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D012697:Serine Endopeptidases; C067468:Yarrowia lipolytica alkaline extracellular protease