Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Kex2 protease processes pro-alpha-factor in a late Golgi compartment in Saccharomyces cerevisiae. The first approximately 30 residues of the 115 amino acid CO2H-terminal cytosolic tail (C-tail) of the Kex2 protein (Kex2p) contain a Golgi retention signal that resembles coated-pit localization signals in mammalian cell surface receptors. Mutation of one (Tyr713) of two tyrosine residues in the C-tail or deletion of sequences adjacent to Tyr713 results in loss of normal Golgi localization. Surprisingly, loss of the Golgi retention signal resulted in transport of C-tail mutant Kex2p to the vacuole (yeast lysosome), as judged by kinetics of degradation and by indirect immunofluorescence. Analysis of the loss of Kex2 function in vivo after shutting off expression of wild-type or mutant forms proved that mutations that cause rapid vacuolar turnover do so by increasing the rate of exit of the enzyme from the pro-alpha-factor processing compartment. The most likely explanation for these results is that mutation of the Golgi retention signal in the C-tail results in transport of Kex2p to the vacuole by default. Wild-type Kex2p also was transported to the vacuole at an increased rate when overproduced, although apparently not due to saturation of a Golgi-retention mechanism. Instead, the wild-type and C-tail mutant forms of Kex2p may follow distinct paths to the vacuole.
Mol Biol Cell 1992 Dec
PMID:Mutation of a tyrosine localization signal in the cytosolic tail of yeast Kex2 protease disrupts Golgi retention and results in default transport to the vacuole. 149 34

The K1 killer toxin of Saccharomyces cerevisiae consists of 103- and 83-residue alpha and beta components whose derivation, from a 316-residue precursor preprotoxin, requires processing at the alpha N-terminus (after ProArg-44), the alpha C-terminus (after ArgArg-149) and at the beta N-terminus (after LysArg-233). These processing events occur after translocation to the Golgi and have been investigated using beta-lactamase fusions. Signal peptidase cleavage of the precursor, predicted to occur after Ala-26, was confirmed by N-terminal sequence analysis of Ala-34 and Ile-52 fusions. Cleavage at all of the other predicted processing sites, including ProArg-44, is dependent on activity of the Kex2 protease. A fourth Kex2-dependent cleavage occurs at LysArg-188. Implications for the specificity of Kex2 cleavage and preprotoxin processing are discussed.
Mol Microbiol 1992 Feb
PMID:Kex2-dependent processing of yeast K1 killer preprotoxin includes cleavage at ProArg-44. 156 Jul 80

Several putative peptide-processing endoproteases have been identified by homology to the yeast Kex2 endoprotease, including furin, PC2, and PC1. However, the question is still open as to which might be involved in peptide posttranslational processing. To enable detailed comparisons of physiological changes in peptide processing with biochemical and molecular biological studies, we cloned rat pituitary cDNAs for PC1 and PC2. The amino acid sequence homologies among rat, human, and mouse PC1, PC2, and furin are consistent with each being a highly conserved but distinct member of a larger family of mammalian subtilisin-like proteases. PC1 and PC2 mRNAs show a restricted distribution among rat tissues and cultured cell lines, consistent with a role in tissue-specific peptide processing; the occurrence of furin mRNA among these tissues and cell lines is much more widespread, being high in many nonneuroendocrine tissues. In the neurointermediate pituitary, PC1 and PC2 mRNAs are strikingly regulated in response to dopaminergic agents, in parallel with mRNAs for POMC, peptidylglycine alpha-amidating monooxygenase, and carboxypeptidase-H. In AtT-20 cells, PC1 mRNA is coregulated with POMC and peptidylglycine alpha-amidating monooxygenase mRNAs in response to CRH and glucocorticoids. When the endogenous PC1 mRNA level in AtT-20 cells is significantly and specifically decreased by stable expression of antisense RNA to PC1, biosynthetic labeling of newly synthesized POMC-derived peptides shows a substantial blockade of normal POMC processing. These data are consistent with a role for PC1 protein in endoproteolysis, either as a processing endoprotease or as the activator of the actual processing endoprotease(s).
Mol Endocrinol 1991 Dec
PMID:Prohormone-converting enzymes: regulation and evaluation of function using antisense RNA. 179 45

Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322. The Ste2 and beta la components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in beta la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of beta la; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in beta la secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of beta la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells.
Mol Cell Biol 1991 May
PMID:In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions. 201 68

In mammalian cells, the transmembrane beta-amyloid peptide precursor (beta-APP) undergoes a complex series of alternative proteolytic processing steps that result in the secretion of varying proportions of its extra-cellular domain (protease nexin II) and beta-amyloid peptide. The protein is also reinternalized and degraded in the endosomal-lysosomal system. The relative efficiencies of these competing processes determine the yield of beta-amyloid peptide. Several proteases have been implicated in this complex processing pathway, although none has been identified to date. The yeast secretory system contains proteases homologous to mammalian pro-hormone convertases and is susceptible to genetic manipulation. We therefore investigated the expression and processing of the beta-amyloid peptide precursors (beta-APP-695 and beta-APP-751) in Saccharomyces cerevisiae transformed with human beta-APP cDNA's. beta-APP (695 or 751) cDNA either with its authentic signal sequence or the yeast-derived prepro-alpha-factor leader, was inserted into a glucose-regulated expression vector and transfected into a protease-deficient yeast strain. In all instances, expression of beta-APP was about 1% of total protein. Protease protection studies indicated that either the natural human signal sequence or the alpha-factor leader sequence targetted beta-APP to the endoplasmic reticulum and inserted it with the amino-terminal domain in the lumen. All of the beta-APP fused to the alpha-factor leader proceeded to the trans-Golgi, where Kex2 endopeptidase removed the leader and released the normal amino-terminus of beta-APP. About one-half of the beta-APP was also cleaved at the "alpha-secretase" site in the middle of the beta-peptide sequence, 12 residues before the membrane-spanning sequence. A fraction of the alpha-secretase-cleaved beta-APP appeared in the culture medium; however, most of it associated with the exterior of the cells. The carboxyl-terminal fragments formed by cleavage at the alpha-secretase site accumulated in the membranes. Other proteolytic processes generated membrane-associated carboxyl-terminal fragments that also resembled those found in mammalian cells. These results indicate that the secretory system of S. cerevisiae possesses proteases with specificities similar to the mammalian enzymes that process beta-APP.
Cell Mol Biol Res 1994
PMID:The expression and processing of human beta-amyloid peptide precursors in Saccharomyces cerevisiae: evidence for a novel endopeptidase in the yeast secretory system. 786 29

Kex2 protease (Kex2p) and Ste13 dipeptidyl aminopeptidase (Ste13p) are required in Saccharomyces cerevisiae for maturation of the alpha-mating factor in a late Golgi compartment, most likely the yeast trans-Golgi network (TGN). Previous studies identified a TGN localization signal (TLS) in the C-terminal cytosolic tail of Kex2p consisting of Tyr-713 and contextual sequences. Further analysis of the Kex2p TLS revealed similarity to the Ste13p TLS. Mutation of the Kex2p TLS results in transport of Kex2p to the vacuole by default. When expression of a GAL1 promoter-driven KEX2 gene is shut off in MAT(alpha) cells, the TGN becomes depleted of Kex2p, resulting in a gradual decline in mating competence which is greatly accelerated by TLS mutations. To identify the genes involved in localization of Kex2p, we isolated second-site suppressors of the rapid loss of mating competence observed upon shutting off expression of a TLS mutant form of Kex2p (Y713A). Seven of 58 suppressors were allele specific, suppressing point mutations at Tyr-713 but not deletions of the TLS or entire C-terminal cytosolic tail. By linkage analysis, the allele-specific suppressors defined three genetic loci, SOI1, S0I2, and S0I3. Pulse-chase analysis demonstrated that these suppressors increased net TGN retention of both Y713A Kex2p and a Ste13p-Pho8p fusion protein containing a point mutation in the Ste13p TLS. 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. Suppressor alleles of S0I2 were dominant. These results suggest that the SOI1 and S0I2 genes encode regulators or components of the TLS recognition machinery.
Mol Cell Biol 1996 Nov
PMID:Allele-specific suppression of a defective trans-Golgi network (TGN) localization signal in Kex2p identifies three genes involved in localization of TGN transmembrane proteins. 888 51

Localization of Kex2 protease (Kex2p) to the yeast trans-Golgi network (TGN) requires a TGN localization signal (TLS) in the Kex2p C-terminal cytosolic tail. Mutation of the TLS accelerates transport of Kex2p to the vacuole by an intracellular (SEC1-independent) pathway. In contrast, inactivation of the clathrin heavy-chain gene CHC1 results in transport of Kex2p and other Golgi membrane proteins to the cell surface. Here, the relationship of the two localization defects was assessed by examining the effects of a temperature-sensitive CHC1 allele on trafficking of wild-type (WT) and TLS mutant forms of Kex2p. Inactivation of clathrin by shifting chc1-ts cells to 37 degrees C caused WT and TLS mutant forms of Kex2p to behave identically. All forms of Kex2p appeared at the plasma membrane within 30-60 min of the temperature shift. TLS mutant forms of Kex2p were stabilized, their half-lives increasing to that of wild-type Kex2p. 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. Finally, after clathrin inactivation, all forms of Kex2p were degraded in part by a vacuolar protease-independent pathway. After inactivation of both chc1-ts and sec1-ts, Kex2 was degraded exclusively by this pathway. We conclude that the effects of clathrin inactivation on Kex2p localization are independent of the Kex2p C-terminal cytosolic tail. Although these results neither prove nor rule out a direct interaction between the Kex2 TLS and a clathrin-dependent structure, they do imply that clathrin is required for the intracellular transport of Kex2p TLS mutants to the vacuole.
Mol Biol Cell 1996 Nov
PMID:The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p. 893 Aug 91

Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway. The enzymes themselves are made as inactive precursors, and we investigated the activation process by studying the maturation of Krp1, a kexin from the fission yeast Schizosaccharomyces pombe. Using a cell-free translation-translocation system prepared from Xenopus eggs, we found that Krp1 is made as a preproprotein that loses the presequence during translocation into the endoplasmic reticulum. The prosequence is also rapidly cleaved in a reaction that is autocatalytic and probably intramolecular and is inhibited by disruption of the P domain. Prosequence cleavage normally occurs at Arg-Tyr-Lys-Arg102/ (primary cleavage site) but can occur at Lys-Arg82 (internal cleavage site) and/or Trp-Arg99 when the basic residues are removed from the primary site. Cleavage of the prosequence is necessary but not sufficient for activation, and Krp1 is initially unable to process substrates presented in trans. Full activation is achieved after further incubation in the extract and is coincident with the addition of O-linked sugars. O glycosylation is not, however, essential for activity, and the crucial event appears to be cleavage of the initially cleaved prosequence at the internal site. Our results are consistent with a model in which the cleaved prosequence remains noncovalently associated with the catalytic domain and acts as an autoinhibitor of the enzyme. Inhibition is then relieved by a second (internal) cleavage of the inhibitory prosequence. Further support for this model is provided by our finding that overexpression of a Krp1 prosequence lacking a cleavable internal site dramatically reduced the growth rate of otherwise wild-type S. pombe cells, an effect that was not seen after overexpression of the normal, internally cleavable, prosequence or prosequences that lack the Lys-Arg102 residues.
Mol Cell Biol 1998 Jan
PMID:Activation of the kexin from Schizosaccharomyces pombe requires internal cleavage of its initially cleaved prosequence. 941 87

PC2 and PC3 are neuroendocrine specific members of the eukaryotic subtilisin-like proprotein convertase (PC) family. Both are sorted via the regulated secretory pathway into secretory granules. In order to identify sequences in PC2 which are involved in targeting to the regulated secretory pathway we expressed a series of PC2 cDNAs containing mutations in the C terminal or propeptide domains in the mouse corticotrophic AtT20 cell line. Sorting of endogenous PC3 was used as a control. PC2 and PC3 were secreted with similar kinetics and sorted to secretory granules with similar efficiencies. Deletions of up to 50 amino acids from the C-terminus of proPC2 had no effect on secretion or sorting, but larger deletions completely prevented maturation or secretion. Two large deletions within the propeptide also prevented secretion. Smaller deletions between the primary and secondary cleavage sites, or of the primary cleavage site, reduced the amount of protein secreted but did not affect sorting to secretory granules. Replacement of the propeptide of PC2 with that of the endogenous PC3 also had no effect on secretion or sorting. The results indicate that targeting of proPC2 to the regulated secretory pathway is dependent on more than one region within the proPC2 molecule.
J Mol Endocrinol 1998 Oct
PMID:Sorting of PC2 to the regulated secretory pathway in AtT20 cells. 980 64

A number of precursors for neuropeptides have recently been cloned in amphibians, but little is known concerning the endoproteases responsible for the processing of these precursors. Here we report on the molecular cloning of the cDNA encoding the proprotein convertase PC2 and the distribution of the corresponding mRNA in the European green frog Rana ridibunda. The full cDNA structure (2125 bp) was obtained from the analysis of the PCR products combined with the sequence from a clone isolated from a frog pituitary cDNA library. The deduced amino acid sequence revealed that frog PC2 comprises 636 amino acid residues including a 22-residue signal peptide. RT-PCR analysis showed that PC2 is expressed not only in the brain and pituitary but also in various peripheral organs including the pancreas, stomach, intestine, liver, kidney and testis. In situ hybridization histochemistry revealed that, in the central nervous system, PC2 mRNA is widely distributed, the highest concentrations being found in the pallium, the anterior preoptic area, the hypothalamus and the medial amygdala. High levels of PC2 mRNA were also detected in the intermediate lobe of the pituitary. The overall distribution of PC2 mRNA in the frog brain is consistent with its involvement in the processing of a number of neuropeptide and hormone precursors.
Brain Res Mol Brain Res 1998 Dec 10
PMID:Characterization of the cDNA encoding the prohormone convertase PC2 and localization of the mRNA in the brain of the frog Rana ridibunda. 983 19


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