Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica.

Abstract

Processing and secretion of the alkaline extracellular protease (AEP) from the yeast Yarrowia lipolytica was studied by pulse-chase and immunoprecipitation experiments. Over half of newly synthesized AEP was secreted by 6 min. Over 99% of AEP activity which was external to the cytoplasmic membrane was located in the supernatant medium. Polypeptides of 55, 52, 44, 36, and kifodaltons derived from the proregion of AEP indicated that one major processing pathway was 55K-*52K-*32K. The gene coding for AEP (XPR2) was cloned and sequenced. The sequence and the immunoprecipitation results suggest that AEP is originally synthesized with an additional preprol-proII-proIlI amino-terminal region. Processing definitely involves cleavage(s) after pairs of basic amino acids and the addition of one N-linked oligosaccharide. Signal peptidase cleavage, dipeptidyl aminopeptidase cleavages, and at least one additional proteolytic cleavage may also be involved.

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“…- [4, (Yaver et al, 1992). AEP antiserum was prepared as described previously (Matoba et al, 1988).…”

Dipeptidyl aminopeptidase processing and biosynthesis of alkaline extracellular protease from Yarrowia lipolytica

“…1). The prepro-region contains a consensus sequence for N-linked glycosylation and ends with a LysArg pair of basic amino acids (Davidow et al, 1987;Matoba et al, 1988 ;Nicaud et al, 1989). Removal of the signal peptide and addition of 2 kDa of N-linked carbohydrate in the pro-region are thought to occur as prepro-AEP is translocated across the ER membrane yielding a 55 kDa AEP precursor (Matoba et al, 1988;Fabre et al, 1991).…”

“…The dimorphic yeast Yarrowia lipolytica secretes high levels of a 32 kDa subtilisin-like alkaline extracellular protease (AEP) (Tobe et al, 1976;Ogrydziak & Scharf, 1982;Matoba et al, 1988). AEP has been used as a reporter molecule for in uiuo studies of functions of the signal recognition particle of Y .…”

“…Removal of the signal peptide and addition of 2 kDa of N-linked carbohydrate in the pro-region are thought to occur as prepro-AEP is translocated across the ER membrane yielding a 55 kDa AEP precursor (Matoba et al, 1988;Fabre et al, 1991). Removal of the X-Ala, X-Pro dipeptides by dipeptidyl aminopeptidase (DPAPase) activity, which yields a 52 kDa precursor, and cleavage after Lys-Arg at the end of the pro-region by the KEX2-like XPR6 processing endoprotease, which yields 32 kDa mature AEP, presumably occur in the late Golgi (Matoba et al, 1988;Matoba & Ogrydziak, 1989;Fabre et al, 1991;Enderlin & Ogrydziak, 1993;Lopez et al, 1994). The role of the nine X-Ala, X-Pro dipeptides (dipeptide stretch) in AEP processing and secretion is not clear.…”

“…PMSF, casein (Hammersten), polypropylene glycol, DMSO, Triton X-100, EDTA, Protein ASepharose 4 Fast Flow, endoglycosidase H and 5-fluoroorotic acid (FOA) were obtained from the same sources as described previously (Yaver et al, 1992). AEP antiserum was prepared as described previously (Matoba et al, 1988 Enderlin et al (1993) Mutagenesis Kit manual). Y .…”