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

Kevin A. Morano^2,

Institute of Marine Resources, University of California, Davis, CA 95616, USA
Section of Microbiology, University of California, Davis, CA 95616, USA

¹ Author for correspondence: David M. Ogrydziak. Tel: +1 916 752 2506. Fax: + 1 916 752 4759. e-mail: dmogrydziak@ucdavis.edu

ABSTRACT

Alkaline extracellular protease (AEP) from Yarrowia lipolytica is synthesized as a precursor with a 157 aa prepro-region. Signal peptide cleavage was shown to occur after Ala₁₅ by N-terminal amino acid radiosequencing of the largest intracellular AEP precursor. AEP proteolytic activity was not required for AEP processing. After a change of the putative active site Ser to Ala, inactive AEP with the same mobility on SDS-PAGE as wild-type mature AEP was secreted. The role of dipeptidyl aminopeptidase (DPAPase) activity in AEP processing was also investigated. Mutations early in the -X-Ala- and -X-Pro- dipeptide stretch (Pro₁₇ to Met which should prevent DPAPase processing and Ala₁₉ to Val which should allow removal of only the first dipeptide) did not prevent synthesis of active mature AEP nor did use of the DPAPase inhibitor Pro-boroPro. Deletion of the entire dipeptide stretch (Ala₁₆ to Pro₃₃) resulted in intracellular accumulation of an AEP precursor, which surprisingly was not glycosylated, and little or no secretion of AEP-related polypeptides. Expression of AEP in wild-type and dpp1 dap2 Saccharomyces cerevisiae strains (lacking both the Golgi and vacuolar DPAPases) resulted in secretion of only mature AEP and no AEP precursors. Transit times and levels of AEP secretion were similar for both strains. These results indicate that the KEX2-like cleavage after Lys₁₅₆-Arg₁₅₇, which yields mature active AEP can occur in the absence of DPAPase processing and that DPAPase processing is not necessary for secretion of mature active AEP.

Present address: Department of Biological Chemistry, University of Michigan Medical School Ann Arbor MI 48109-0606, USA.

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