Disease relevance of KEX2

• Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 prohormone endoprotease or a human structural homologue (fur gene product) contained an elevated level of a membrane-associated endoproteolytic activity that could cleave at pairs of basic amino acids (-LysArg- and -ArgArg-) [1].
• Spodoptera frugiperda Sf9 insect cells infected with a recombinant baculovirus, containing the complete KEX2 gene which encodes the Kex2p protease (814 amino acids), accumulate an 120-kDa functional form of the enzyme [2].
• Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2 [3].
• In addition to known kex2 phenotypes, such as pH and calcium hypersensitivity, the mutants grew in cellular aggregates and were found to be hypersensitive to several antifungal drugs that target the cell membrane, including azoles, amorolfine and amphotericin B [4].
• Human type 2 preprorelaxin was coexpressed in human kidney 293 cells with the candidate prohormone convertase-processing enzymes mPC1 or mPC2, both cloned from the mouse pituitary tumor AtT-20 cell line, or with the yeast kex2 alpha-mating factor-converting enzyme from Saccharomyces cerevisiae [5].

High impact information on KEX2

• The normal KEX2 gene was isolated by complementation of three of the phenotypes conferred by the kex2-1 mutation [6].
• Gene dosage effects suggest that KEX2 is the structural gene for the endopeptidase [6].
• Intracellular activation is regulated by an unusual 10-amino-acid insert sandwiched between the pro- and catalytic-domains of stromelysin-3, which is encrypted with an Arg-X-Arg-X-Lys-Arg recognition motif for the Golgi-associated proteinase, furin, a mammalian homologue of the yeast Kex2 pheromone convertase [7].
• The prohormone-processing endoprotease (KEX2 gene product) of the yeast Saccharomyces cerevisiae is a membrane-bound, 135,000-dalton glycoprotein, which contains both asparagine-linked and serine- and threonine-linked oligosaccharide and resides in a secretory compartment [8].
• This result suggest that clathrin is required for the retention of Kex2p in the Golgi apparatus [9].

Chemical compound and disease context of KEX2

• The two serine proteases Kex2 from Saccharomyces cerevisiae and subtilisin from Bacillus subtilis are used as examples to illustrate how the method finds the catalytic residues for both enzymes [10].
• Taking into account this similarity, a synthetic peptide modelling the cleavage site of HIV-1 envelope glycoprotein precursor was used as substrate for Kex2p [11].

Biological context of KEX2

• Mutations in the yeast KEX2 gene cause a Vma(-)-like phenotype: a possible role for the Kex2 endoprotease in vacuolar acidification [12].
• The cloned KEX1 gene of K. lactis has low but significant sequence homology with the KEX2 gene of Saccharomyces cerevisiae [13].
• In contrast to the BAR1 gene product, the novel aspartyl protease (YAP3 for Yeast Aspartyl Protease 3) contains a C-terminal serine/threonine-rich sequence and potential transmembrane domain similar to those found in the KEX2 gene product [14].
• SOI1 suppressor alleles reduced the efficiency of localization of wild-type Kex2p to the TGN, implying an impaired ability to discriminate between the normal TLS and a mutant TLS. soi1 mutants also exhibited a recessive defect in vacuolar protein sorting [15].
• KEX2 mutations suppress RNA polymerase II mutants and alter the temperature range of yeast cell growth [16].

Anatomical context of KEX2

• The KEX2-encoded endoprotease of Saccharomyces cerevisiae resides in the Golgi complex where it participates in the maturation of alpha-factor mating pheromone precursor [17].
• Furthermore, when the secretion of alpha Gal was directed by the invertase (SUC2) SP, the intracellular enzyme localized to the endoplasmic reticulum (ER), whereas use of the MF alpha 1 SP caused the intracellular enzyme to be outer-chain-glycosylated and processed by the KEX2 endoproteinase, implying that it had passed the ER [18].
• Mutation of TLS1 results in rapid transit of Kex2p to the vacuole [19].
• Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p [20].
• Organelle fractionation by sucrose density gradient centrifugation shows that Kex2p is not retained in the Golgi complex of lam1 cells [17].

Associations of KEX2 with chemical compounds

• In addition, KEX2 was specifically inhibited by the mutant alpha 1-antitrypsin but not by other serine protease inhibitors [21].
• For secretion by yeast cells of HGH with the same NH2 terminus as native HGH, an artificial Lys-Arg linker, which is one of the potential KEX2 recognition sequences, was introduced at the junction between the M. pusillus rennin secretion leader and mature HGH [22].
• This processing did not take place when form A was obtained from protoplasts lysed in the presence of either EDTA or leupeptin, two wel-characterized inhibitors of KEX2 endoprotease from Saccharomyces cerevisiae [23].
• The enzyme activity was inhibited by serine-protease inhibitors, such as DFP and PMSF, indicating that the KEX2 endopeptidase belongs to a serine-protease family [24].
• Loss of QDS1/KEX2 function results in quinidine sensitivity [25].

Physical interactions of KEX2

• In contrast, inactivation of the clathrin heavy-chain gene CHC1 results in transport of Kex2p and other Golgi membrane proteins to the cell surface [26].

Co-localisations of KEX2

• A Green Fluorescent Protein-Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p [27].

Regulatory relationships of KEX2

• After inactivation of clathrin heavy chain, vacuolar protease-dependent degradation of all forms of Kex2p was blocked by a sec1 mutation, which is required for secretory vesicle fusion to the plasma membrane, indicating that transport to the cell surface was required for degradation by vacuolar proteolysis [26].
• The gene isolated in this way was shown also to be essential for the KEX2-independent processing of propheromone overproduced from plasmid-borne MF alpha 1 [14].
• Moreover, Ypt31/32 and Rcy1 regulate the recycling of the furin-homolog Kex2 to the Golgi [28].

Other interactions of KEX2

• In mutant cells carrying a temperature-sensitive allele of CHC1 (chc1-ts), alpha-factor precursor appears in the culture medium within 15 min, and Kex2p and DPAP A reach the cell surface within 30 min, after imposing the nonpermissive temperature [29].
• These results suggested that Soi1p functions at two steps in the cycling of Kex2p and other proteins between the TGN and prevacuolar compartment (PVC) [19].
• In double-immunofluorescence labeling experiments, significant colocalization of Sec7 and Kex2 proteins was found [30].
• A grd19 null mutation causes rapid mislocalization of the late-Golgi membrane proteins A-ALP and Kex2p to the vacuole [31].
• The toxins encoded by the M satellite RNAs are processed by enzymes (KEX1 and KEX2, for killer expression) whose study led to discovery of mammalian hormone-processing enzymes [32].

Analytical, diagnostic and therapeutic context of KEX2

• The transmembrane Kex2 protease, which processes precursors of secreted peptides within the yeast secretory pathway, is also localized by indirect immunofluorescence to multiple structures in the yeast cell (Redding, K., and R. Fuller, manuscript submitted for publication) [30].
• Site-specific mutagenesis of the cDNA gene establishes that one of the two potential KEX2 cleavage sites is critical for toxin action but not for immunity [33].
• Characterization of the POMC-related immunoreactive peptides by gel permeation and reversed-phase high pressure liquid chromatography showed that the KEX2 enzyme was active and capable of carrying out cleavage of POMC to release the authentic maturation product beta-endorphin(1-31) [34].
• Because KEX2 proteinase is located within the Golgi compartment, it may be isolated by differential centrifugation of broken cells at 7000g for 15 min and at 20,000g for 15 min [35].
• Absence of this activity cosegregates with the other phenotypes of a kex2 lesion in genetic crosses [6].

References