Disease relevance of PEP4

• Two were pepstatin-sensitive carboxyl proteinases (porcine pepsin and proteinase A from baker's yeast) and two were pepstatin-insensitive carboxyl proteinases (from Pseudomonas sp. 101 (pseudomonapepsin; PCP) and Xanthomonas sp. T-22 (xanthomonapepsin; XCP)) [1].
• The toxicity of HBsAg in the secretion pathway of pep4 strains can be progressively reduced in modified SD media containing lower concentrations of ammonium sulphate [2].
• Previously we reported a procedure to enrich NI transformants that are not inhibited by cytotoxic expression of hepatitis B virus surface antigen in the secretion pathway of the protease-A-deficient (pep4) strain [3].

High impact information on PEP4

• The vps34 and vps15 mutants displayed additional phenotypes such as defects in transport of proteinase A and proteinase B, implying the existence of another PtdIns 3-kinase complex(es) [4].
• Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for the PEP4 gene (which encodes the principal CPY processing enzyme, proteinase A) and acceptor membranes from a PEP4 yeast strain, indicating intercompartmental transfer [5].
• Chs2p degradation depends on the vacuolar protease encoded by PEP4, indicating that Chs2p is destroyed in the vacuole [6].
• However, yeast mutant strains defective in endocytosis (end3, end4 and chc1-ts) and vacuolar degradation (pep4) exhibit copper-dependent Ctr1p degradation, indicating that internalization and delivery to the vacuole is not the principal mechanism responsible for degradation [7].
• NOP4 is a single copy essential gene present on chromosome XVI, between RAD1 and PEP4 [8].

Biological context of PEP4

• The amino acid sequence deduced from the gene shows extensive homology to a number of aspartyl proteases including the PEP4 and BAR1 gene products from S. cerevisiae [9].
• The trifunctional FAS2 gene encoding subunit alpha of the Saccharomyces cerevisiae fatty acid synthetase complex was mapped on the left arm of chromosome XVI 24 centimorgans proximal to GAL4 and 39 centimorgans distal to PEP4 relative to the centromere [10].
• The open reading frames of the PEP4 gene (1263 bp encoding a protein of 420 amino acids) and the PRBI gene (1683 bp encoding a protein of 560 amino acids) were found [11].
• Both PEP4 and PRBI genes were disrupted in the C. boidinii genome by one-step gene disruption [11].
• Biogenesis of the yeast vacuole (lysosome). Active site mutation in the vacuolar aspartate proteinase yscA blocks maturation of vacuolar proteinases [12].

Anatomical context of PEP4

• These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolytically cleaved in a PEP4-dependent manner [13].
• When the 22 degrees C culture was shifted to 34 degrees C, Ste2-3p was removed from the plasma membrane and degraded by a PEP4-dependent mechanism with a 24-min half-life; the wild-type Ste2p displayed a 72-min half-life [14].
• Stimulation of in vitro processing with energy and cytosol took place efficiently when the expression of PEP4, under control of the GAL1 promoter, was induced then completely repressed before radiolabeling spheroplasts [15].
• We found that the 76-amino-acid preprosegment of PrA contains at least two sorting signals: an amino-terminal signal peptide that is cleaved from the protein at the level of the endoplasmic reticulum followed by the prosegment which functions as a vacuolar protein-sorting signal [16].
• These vat mutant strains accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. The kinetics of secretion suggests that missorting occurs in the Golgi complex or in post-Golgi vesicles [17].

Associations of PEP4 with chemical compounds

• When a multicopy PEP4 transformant of a prb1 mutant was grown in the presence of the aspartyl protease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium [18].
• This degradation is blocked by inhibiting the vacuolar proteolytic pathway with the protease inhibitor phenyl methyl sulphonyl fluoride or by mutation of the PEP4 gene [19].
• One of these activities was a PEP4-dependent carboxypeptidase that was sensitive to phenylmethylsulfonyl fluoride [20].
• Proteinase yscA deficiency leads to rapid cell death when glucose-grown cells starve for nitrogen or other nutrients [21].
• We investigated the relationship between acidification and protein sorting in yeast by treating yeast cells with ammonium chloride and found that this lysosomotropic agent caused the mislocalization of a substantial fraction of the newly synthesized vacuolar (lysosomal) enzyme proteinase A (PrA) to the cell surface [22].

Physical interactions of PEP4

• Compared to pep4 cells, pep4 doa4 cells have huge amounts of membrane-bound ubiquitin conjugates [23].
• Degradation occurs in the vacuole, as Hxt6/7 were stabilized in proteinase A-deficient mutant cells [24].

Regulatory relationships of PEP4

• The proteinase yscA-triggered maturation process of pro-carboxypeptidase yscY leads to an intermediate mol. wt form of the enzyme which is, however, fully active [25].
• Sephadex G-100 gel filtration of a purified mixture of tryptophan-synthase-inactivating enzymes I and II from Saccharomyces cerevisiae, which had previously been identified as yeast proteinases A and B, yields two coincident peaks of tryptophan-synthase-inactivating activity and proteinase A and B activities [26].
• At pH 5, the carboxypeptidase Y-inhibitor complex is activated both by proteinase A (EC 3.4.23.6) and B. Yeast proteinases are activated in a crude extract by incubation at pH 5 [3] [27].

Other interactions of PEP4

• Degradation was not observed in strains defective in the END3/END4 endocytic pathway or in the production of vacuolar proteases (PEP4) [28].
• In cells with a deletion in the vacuolar protease PEP4, high iron medium leads to the accumulation of Fet3p and Ftr1p in the vacuole [29].
• Both PEP4 or PRC1 gene disruptions resulted in a lower frequency of mitochondrial DNA escape [30].
• However, mature proteinase yscB is not stable in the absence of proteinase yscA [31].
• Additional mutations in the carboxy-terminus of Vps10p, including a deletion of a putative retention/recycling signal (FYVF), also result in CPY missorting and PEP4-dependent receptor instability [32].

Analytical, diagnostic and therapeutic context of PEP4

• To elucidate the activation process of proteinase yscA the catalytically active amino acid Asp294 of the enzyme was exchanged with Ala294 using site directed mutagenesis [12].
• Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts [33].
• Proteinase A produced by Saccharomyces cerevisiae carrying the gene PEP4 (which encodes the prepro-proteinase A on multicopy plasmids) was isolated and characterized by means of SDS/PAGE, laser desorption m.s. (l.d.m.s.) and high-performance capillary electrophoresis [34].
• Moreover, the end1 mutant and all of the previously reported pep mutants, with the exception of pep4, were found to exhibit a profound vacuolar protein sorting defect, and complementation tests between the PEP, VPL VPT and END1 groups demonstrated that there are extensive overlaps between these groups [35].
• Likewise, by immunofluorescence, we observe that Ste6p accumulates in the vacuole in the doa4 mutant, as it does in the vacuolar protease-deficient pep4 mutant [36].

References