Enhanced peptide secretion by gene disruption of CYM1, a novel protease in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is a widely used host in the production of therapeutic peptides and proteins. Here we report the identification of a novel endoprotease in S. cerevisiae. It is encoded by the CYM1 gene and is specific for the C-terminus of basic residues of heterologously expressed peptides. Gene disruption of CYM1 not only reduced the intracellular proteolysis, but also enhanced the secretion of heterologously expressed peptides such as growth hormone, pro-B-type natriuretic peptide and pro-cholecystokinin. Cym1p resembles metalloendoproteases of the pitrilysin family with the HXXEH(X)E(71-77) catalytic domain as seen in insulysin, nardilysin and human metalloprotease 1. It is a nuclear encoded protease that localizes to mitochondria without a hydrophobic N-terminal signal sequence or a C-terminal tail-anchor. The protease does not require posttranslational processing prior to activation and it contains cytosolic activity that processes peptides designated for the secretory pathway prior to translocation into the endoplasmic reticulum.Keywords: cholecystokinin; growth hormone; metalloprotease; proBNP; yeast.Saccharomyces cerevisiae is often used for industrial production of recombinant peptides [1]. Secretion of heterologously expressed proteins is obtained by expression of fusion proteins in which the protein of interest is fused to the S. cerevisiae a-factor prepropeptide to direct secretion through the secretory pathway. To enrich the secretion of full length peptides, the host strains used are often made protease deficient by inactivation of the vacuolar protease Pep4p and the glycosyl-phophatidylinositol (GPI)-anchored aspartyl protease, Yps1p [2]. Yps1p has been shown to be responsible for both Arg and Lys specific cleavage in the maturation of heterologous peptides such as prosomatostatin, human b-amyloid precursor protein and the elastasespecific inhibitor, pre-elafin [3][4][5]. Protease activity within the secretory pathway is dominated by the serine proteases, Kex2 and Kex1 and the aspartic protease, Yps1. However, outside this pathway another group of proteases, the metallopeptidase family, seems to dominate. Among these are Ste24p and Axl1p involved in the maturation of the a-mating factor [6,7] and the mitochondrial signal peptidases, Mas1 and Mas2 [8].We used human cholecystokinin (CCK) as a substrate to identify additional proteases of budding yeast with Lysspecific activity using a processing-independent assay [9]. Peptide hormones and neuropeptides are synthesized as larger precursors that are proteolytically cleaved in the trans-Golgi network or in the immature secretory vesicles. The excision of mature peptides from prohormones/proproteins is a general event in eukaryotic organisms in which members of the subtilisin family of proteolytic enzymes are responsible. Members of this family are Ca 2+ dependent serine proteases. Kex2p from yeast was the first member of this family to be discovered and is essential in the activation of the a-factor pheromone. In mammals, members of the prohor...

Assessment of Inactivating Stop Codon Mutations in Forty Saccharomyces cerevisiae Strains: Implications for [PSI+] Prion- Mediated Phenotypes

“…A previously identified [ PSI + ] prion-dependent phenotype has been shown to be at least partly attributable to readthrough of the ISCM in the uncharacterized YBR074W ORF (Figure 2). YBR074W is an uncharacterized yeast ORF that is predicted to be a metalloprotease [29]. Although we show a link between this gene and protection against chemicals that induce cell wall stress, we cannot provide any insight to the possible mechanistic role this protein may play in such protection.…”

Targeting Capacity and Conservation of PreP Homologues Localization in Mitochondria of Different Species

“…3,19 showing cleavage of seven N-terminal amino acids from the Cym1 precursor protein. 3,20,21 The reason for the lack of in vitro processing of the Cym1(1-40) GFP construct by MPP is not known; it might be due to a conformation of the precursor with a buried cleavage site. In addition, using a shorter version of Cym1(1-497) to detect processing on an 8% SDS-PAGE, we have observed the processing of the construct that was impaired in a temperaturesensitive mas1 mutant lacking MPP activity (Fig.…”

“…It has previously been reported by Jonson et al that the 30 most N-terminal amino acids of the Cym1 precursor are not necessary to import Cym1 fused to GFP into mitochondria in vivo, suggesting the presence of an internal targeting information 20. However, in vivo experiments do not exclude the possibility that the precursor protein might be associated with the mitochondrial surface.…”

“…16,18,19 The yeast homologue of PreP was first identified as Cym1 protein with a capacity to degrade heterologously expressed peptides in yeast, and gene disruption of CYM1 resulted in decreased intracellular proteolysis and enhanced peptide secretion. 20 Moreover, Cym1 (also termed Mop112) was identified as a PreP homologue, with 29% and 35% sequence identities to AtPreP and hPreP, respectively. Cym1 was shown to be localized in the IMS and to degrade short peptides (generated by proteolysis in the IMS), as well as peptides and presequences, which are transported from the matrix to the IMS by the ABC transporter Mdl1.…”

Identification of genes that enhance cellulase protein production in yeast

“…Deletion of the MON2 gene, the product of which plays a role in endocytosis and vacuole maintenance, enhanced Cypridina noctiluca luciferase (Cluc) activity (Kanjou et al, 2007). Deletion of the CYM1 gene, which encodes a lysine-specific metalloprotease, enhanced human holecystokinin protein secretion (Jonson et al, 2004). On the other hand, overexpression of the SOD1 gene, which encodes a superoxide dismutase, the PDI1 gene, which encodes a protein disulfide isomerase, or the RPP0 gene, which encodes a ribosomal protein, also enhanced heterologous protein production (Raimondi et al, 2008;Robinson et al, 1994;Wentz and Shusta, 2008).…”