Role of the proregion in the production and secretion of the Yarrowia lipolytica alkaline extracellular protease.
The yeast Yarrowia lipolytica secretes an alkaline extracellular protease (AEP). It is first synthesized as a precursor comprising a putative signal peptide, a stretch of 10 X-Ala or X-Pro sequences that are substrates for a dipeptidyl aminopeptidase, a large pro-region that contains a glycosylation site and two Lys-Arg sites that can be cleaved by a KEX2-like endoprotease and finally the mature protease itself. A defect in the XPR6 (KEX2-like) gene results in the secretion of an inactive proenzyme (Matoba, S., and Ogrydziak, D. M. (1989) J. Biol. Chem. 264, 6037-6043), showing that the proregion inhibits protease activity. To determine whether the proregion plays an additional role in protease secretion, we have generated deletions and point mutations in the corresponding region of the structural gene. In this paper we examine the effects of these mutations on AEP secretion and maturation and show that the proregion is essential for its secretion. All deletions affecting the proregion resulted in the intracellular accumulation of unprocessed precursors. Deletion of the glycosylation site in the proregion resulted in the production of an unglycosylated precursor that was secreted and matured correctly at 18 degrees C but accumulated in the cells at 28 degrees C. From these results, we propose that the AEP prosequence plays an additional essential role in guiding the proper folding of the protein into a conformation compatible with secretion.
Fabre
E
E
Institut National Agronomique, Centre de Biotechnologie Agro-Industrielle, Thiverval, Grignon, France.
Nicaud
J M
JM
Lopez
M C
MC
Gaillardin
C
C
D000595:Amino Acid Sequence; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D013696:Temperature; D015003:Yeasts / Q000201:enzymology*
D015345:Oligonucleotide Probes; D012697:Serine Endopeptidases; C067468:Yarrowia lipolytica alkaline extracellular protease