N-terminal acetyl group is essential for the inhibitory function of carboxypeptidase Y inhibitor (I(C))

Carboxypeptidase Y (CPY) inhibitor, I(C), a yeast cytoplasmic inhibitor in which the N-terminal amino acid is acetylated, was expressed in Escherichia coli and produced as an unacetylated form of I(C) (unaI(C)). Circular dichroism and fluorescence measurements showed that unaI(C) and I(C) were structurally identical and produce identical complexes with CPY. However, the K(i) values for unaI(C) for anilidase and peptidase activity of CPY were much larger, by 700- and 60-fold, respectively, than those of I(C). The reactivities of phenylmethylsulfonyl fluoride and p-chloromercuribenzoic acid toward the CPY-unaI(C) complex were considerably higher than those toward the CPY-I(C) complex. Thus, the N-terminal acetyl group of I(C) is essential for achieving a tight interaction with CPY and for its complete inactivation.     

Biochemical analysis of the yeast proteinase inhibitor (IC) homolog ICh and its comparison with IC

Carboxypeptidase Y (CPY) inhibitor (I(C)) and its homologous protein (I(C)h) are thought to be members of the phosphatidylethanolamine-binding protein (PEBP) family of Saccharomyces cerevisiae. The biochemical characterization of I(C) and its inhibition mode toward CPY were recently reported, but I(C)h has not been characterized. The molecular mass of I(C)h was determined to be 22,033.7. The N-terminal Met1 was cleaved and the amino group of Ser2 was acetylated. I(C)h is folded as a monomeric beta-protein and is devoid of disulfide bonds. It has no inhibitory activity toward CPY, and it does not form a complex with CPY. I(C)h was exclusively expressed in the early log phase, whereas I(C) was expressed in the logarithmic and stationary phase. The intracellular localization of I(C)h was different from that of I(C). These findings provide insights into the physiological functions of I(C)h.     

The multiple site binding of carboxypeptidase Y inhibitor (IC) to the cognate proteinase. Implications for the biological roles of the phosphatidylethanolamine-binding protein

The serine carboxypeptidase inhibitor in the cytoplasm of Saccharomyces cerevisiae, IC, specifically inhibits vacuolar carboxypeptidase Y (CPY) and belongs to a functionally unknown family of phosphatidylethanolamine-binding proteins (PEBPs). In the presence of 1 M guanidine hydrochloride, a CPY-IC complex is formed and is almost fully activated. The reactivities of phenylmethylsulfonyl fluoride, p-chloromercuribenzoic acid, and diisopropyl fluorophosphate toward the complex are considerably increased in 1 M guanidine hydrochloride, indicating that IC contains a binding site other than its inhibitory reactive site. IC is able to form the complex with diisopropyl fluorophosphate-modified CPY. Tryptic digestion of the complex indicates that two fragments from IC are involved in complex formation with CPY. These findings demonstrate the multiple site binding of IC with CPY. Considering the fact that mouse PEBP has recently been identified as a novel thrombin inhibitor, the binding that characterizes the CPY-IC complex could be a common feature of PEBPs.     

Identification of interaction site of propeptide toward mature carboxypeptidase Y (mCPY) based on the similarity between propeptide and CPY inhibitor (IC)

Both the propeptide in the precursor carboxypeptidase Y (proCPY) and the mature CPY (mCPY)-specific endogenous inhibitor (I(C)) inhibit CPY activity. The N-terminal inhibitory reactive site of I(C) (the N-terminal seven amino acids of I(C)) binds to the substrate-binding site of mCPY and is essential for mCPY inhibition, but the mechanism of mCPY inhibition by the propeptide is poorly understood. In this study, sequence alignment between I(C) and proCPY indicated that a sequence similar to the N-terminal region of I(C) was present in proCPY. In particular, a region including the C-terminus of the propeptide was similar to the N-terminal seven amino acids of I(C). In the presence of peptides identical to the N-terminus of I(C) and the C-terminus of the propeptide, CPY activity was competitively inhibited. The C-terminal region of the propeptide might bind to the substrate-binding site of mCPY.     

Refolding and purification of yeast carboxypeptidase Y expressed as inclusion bodies in Escherichia coli

The genes encoding carboxypeptidase Y (CPY) and CPY propeptide (CPYPR) from Saccharomyces cerevisiae were cloned and expressed in Escherichia coli. Six consecutive histidine residues were fused to the C-terminus of the CPYPR for facilitated purification. High-level expression of CPY and CPYPR-His(6) was achieved but most of the expressed proteins were present in the form of inclusion bodies in the bacterial cytoplasm. The CPY and CPYPR-His(6) produced as inclusion bodies were separated from the cells and solubilized in 6 and 3 M guanidinium chloride, respectively. The denatured CPYPR-His(6) was refolded by dilution 1:30 into the renaturation buffer (50 mM Tris-HCl containing 0.5 M NaCl and 3 mM EDTA, pH 8.0), and the refolded CPYPR-His(6) was further purified to 90% purity by single-step immobilized metal ion affinity chromatography. The denatured CPY was refolded by dilution 1:60 into the renaturation buffer containing CPYPR-His(6) at various concentrations. Increasing the molar ratio of CPYPR-His(6) to CPY resulted in an increase in the CPY refolding yield, indicating that the CPYPR-His(6) plays a chaperone-like role in in vitro folding of CPY. The refolded CPY was purified to 92% purity by single-step p-aminobenzylsuccinic acid affinity chromatography. When refolding was carried out in the presence of 10 molar eq CPYPR-His(6), the specific activity, N-(2-furanacryloyl)-l-phenylalanyl-l-phenylalanine hydrolysis activity per milligram of protein, of purified recombinant CPY was found to be about 63% of that of native S. cerevisiae CPY.     

Structure of the carboxypeptidase Y inhibitor IC in complex with the cognate proteinase reveals a novel mode of the proteinase-protein inhibitor interaction

Carboxypeptidase Y (CPY) inhibitor, IC, shows no homology to any other known proteinase inhibitors and rather belongs to the phosphatidylethanolamine-binding protein (PEBP) family. We report here on the crystal structure of the IC-CPY complex at 2.7 A resolution. The structure of IC in the complex with CPY consists of one major beta-type domain and a N-terminal helical segment. The structure of the complex contains two binding sites of IC toward CPY, the N-terminal inhibitory reactive site (the primary CPY-binding site) and the secondary CPY-binding site, which interact with the S1 substrate-binding site of CPY and the hydrophobic surface flanked by the active site of the enzyme, respectively. It was also revealed that IC had the ligand-binding site, which is conserved among PEBPs and the putative binding site of the polar head group of phospholipid. The complex structure and analyses of IC mutants for inhibitory activity and the binding to CPY demonstrate that the N-terminal inhibitory reactive site is essential both for inhibitory function and the complex formation with CPY and that the binding of IC to CPY constitutes a novel mode of the proteinase-protein inhibitor interaction. The unique binding mode of IC toward the cognate proteinase provides insights into the inhibitory mechanism of PEBPs toward serine proteinases and into the specific biological functions of IC belonging to the PEBP family as well.     

Effects of signal sequences and folding accessory proteins on extracellular expression of carboxypeptidase Y in recombinant Saccharomyces cerevisiae

Carboxypeptidase Y (CPY) is a yeast vacuolar protease with useful applications including C-terminal sequencing of peptides and terminal modification of target proteins. To overexpress CPY with the pro-sequence (proCPY) encoded by the Saccharomyces cerevisiae PRC1 gene in recombinant S. cerevisiae, the proCPY gene was combined with the gene coding for a signal sequence of S. cerevisiae mating factor α (MFα), invertase (SUC2), or Kluyveromyces marxianus inulinase (INU1). Among the three constructs, the MFα signal sequence gave the best specific activity of extracellular CPY. To enhance the CPY expression level, folding accessory proteins of Kar2p, Pdi1p and Ero1p located in the S. cerevisiae endoplasmic reticulum were expressed individually and combinatorially. A single expression of Kar2p led to a 28 % enhancement in extracellular CPY activity, relative to the control strain of S. cerevisiae CEN.PK2-1D/p426Gal1-MFαCPY. Coexpression of Kar2p, Pdi1p and Ero1p gave a synergistic effect on CPY expression, of which activity was 1.7 times higher than that of the control strain. This work showed that engineering of signal sequences and protein-folding proteins would be helpful to overexpress yeast proteins of interest.     

Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle).

Restriction enzymes are well known as reagents widely used by molecular biologists for genetic manipulation and analysis, but these reagents represent only one class (type II) of a wider range of enzymes that recognize specific nucleotide sequences in DNA molecules and detect the provenance of the DNA on the basis of specific modifications to their target sequence. Type I restriction and modification (R-M) systems are complex; a single multifunctional enzyme can respond to the modification state of its target sequence with the alternative activities of modification or restriction. In the absence of DNA modification, a type I R-M enzyme behaves like a molecular motor, translocating vast stretches of DNA towards itself before eventually breaking the DNA molecule. These sophisticated enzymes are the focus of this review, which will emphasize those aspects that give insights into more general problems of molecular and microbial biology. Current molecular experiments explore target recognition, intramolecular communication, and enzyme activities, including DNA translocation. Type I R-M systems are notable for their ability to evolve new specificities, even in laboratory cultures. This observation raises the important question of how bacteria protect their chromosomes from destruction by newly acquired restriction specifities. Recent experiments demonstrate proteolytic mechanisms by which cells avoid DNA breakage by a type I R-M system whenever their chromosomal DNA acquires unmodified target sequences. Finally, the review will reflect the present impact of genomic sequences on a field that has previously derived information almost exclusively from the analysis of bacteria commonly studied in the laboratory.     

Comparison of chloramphenicol acetyltransferase variants in staphylococci. Purification, inhibitor studies and N-terminal sequences.

Four electrophoretic variants of chloramphenicol acetyltransferase (types A, B, C and D) found in chloramphenicol-resistant staphylococci were purified by affinity chromatography. Michaelis constants and the kinetics of inactivation with a variety of reagents for the four variants are virtually identical. Their similar amino acid compositions and near identical N-terminal sequences suggest a high degree of overall sequence homology. The thiol-specific reagents 5,5'-dithiobis-(2-nitrobenzoic acid), 2-nitro-5-thiocyanobenzoic acid and 2,2'-dithiopyridine are without significant effect on enzyme activity, whereas 1-fluoro-2,4-dinitrobenzene, N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and, particularly, bromoacetyl-CoA and diethyl pyrocarbonate are potent inhibitors. Iodoacetate is not an inhibitor. The results of chemical modification studies on the four enzyme variants and the identification of 3-carboxymethylhistidine in acid hydrolysates of one variant (type C) after inactivation with iodoacetamide suggest that a unique histidine residue may be involved in the mechanism of catalysis.     

In vitro reconstitution of intercompartmental protein transport to the yeast vacuole

Toward a detailed understanding of protein sorting in the late secretory pathway, we have reconstituted intercompartmental transfer and proteolytic maturation of a yeast vacuolar protease, carboxypeptidase Y (CPY). This in vitro reconstitution uses permeabilized yeast spheroplasts that are first radiolabeled in vivo under conditions that kinetically trap ER and Golgi apparatus-modified precursor forms of CPY (p1 and p2, respectively). After incubation at 25 degrees C, up to 45% of the p2CPY that is retained in the perforated cells can be proteolytically converted to mature CPY (mCPY). This maturation is specific for p2CPY, requires exogenously added ATP, an ATP regeneration system, and is stimulated by cytosolic protein extracts. The p2CPY processing shows a 5-min lag period and is then linear for 15-60 min, with a sharp temperature optimum of 25-30 degrees C. After hypotonic extraction, the compartments that contain p2 and mCPY show different osmotic stability characteristics as p2 and mCPY can be separated with centrifugation into a pellet and supernatant, respectively. Like CPY maturation in vivo, the observed in vitro reaction is dependent on the PEP4 gene product, proteinase A, which is the principle processing enzyme. After incubation with ATP and cytosol, mCPY was recovered in a vacuole-enriched fraction from perforated spheroplasts using Ficoll step-gradient centrifugation. The p2CPY precursor was not recovered in this fraction indicating that intercompartmental transport to the vacuole takes place. In addition, intracompartmental processing of p2CPY with autoactivated, prevacuolar zymogen pools of proteinase A cannot account for this reconstitution. 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. Finally, reconstitution of p2CPY maturation was not possible with vps mutant perforated cells suggesting that VPS gene product function is necessary for intercompartmental transport to the vacuole in vitro.     

Loss of a GPI-anchored membrane protein Aah3p causes a defect in vacuolar protein sorting in Schizosaccharomyces pombe

Schizosaccharomyces pombe has four alpha-amylase homologs (Aah1p-Aah4p) with a glycosylphosphatidylinositol (GPI) modification site at the C-terminal end. Disruption mutants of aah genes were tested for mislocalization of vacuolar carboxypeptidase Y (CPY), and aah3Delta was found to secrete CPY. The conversion rate from pro- to mature CPY was greatly impaired in aah3Delta, and fluorescence microscopy inidicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. These results indicate that aah3Delta had a defect in the retrograde transport of Vps10p, and that Aah3p is the first S. pombe specific protein required for vacuolar protein sorting.     

Identification of a functionally important conformation-sensitive region of the secretory Na+-K+-2Cl- cotransporter (NKCC1)

The secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a member of a small gene family of electroneutral salt transporters that play essential roles in salt and water homeostasis in many mammalian tissues. We have identified a highly conserved residue (Ala-483) in the sixth membrane-spanning segment of rat NKCC1 that when mutated to cysteine renders the transporter sensitive to inhibition by the sulfhydryl reagents 2-aminoethyl methanethiosulfonate (MTSEA) and 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The mutation of Ala-483 to cysteine (A483C) results in little or no change in the affinities of NKCC1 for substrate ions but produces a 6-fold increase in sensitivity to the inhibitor bumetanide, suggesting a specific modification of the bumetanide binding site. When residues surrounding Ala-483 were mutated to cysteine, only I484C was sensitive to inhibition by MTSEA and MTSET. Surprisingly I484C showed increased transport activity in the presence of low concentrations of mercury (1-10 microm), whereas A483C showed inhibition. The inhibition of A483C by MTSEA was unaffected by the presence or absence of sodium and potassium but required the presence of extracellular chloride. Taken together, our results indicate that Ala-483 lies at or near an important functional site of NKCC1 and that the exposure of this site to the extracellular medium is dependent on the conformation of the transporter. Specifically, our results indicate that the cysteine introduced at residue 483 is only available for interaction with MTSEA when chloride is bound to NKCC1 at the extracellular surface.     

Biochemical Analysis of the Yeast Proteinase Inhibitor (IC) Homolog ICh and Its Comparison with IC

Carboxypeptidase Y (CPY) inhibitor (I^C) and its homologous protein (I^Ch) are thought to be members of the phosphatidylethanolamine-binding protein (PEBP) family of Saccharomyces cerevisiae. The biochemical characterization of I^C and its inhibition mode toward CPY were recently reported, but I^Ch has not been characterized. The molecular mass of I^Ch was determined to be 22,033.7. The N-terminal Met1 was cleaved and the amino group of Ser2 was acetylated. I^Ch is folded as a monomeric β-protein and is devoid of disulfide bonds. It has no inhibitory activity toward CPY, and it does not form a complex with CPY. I^Ch was exclusively expressed in the early log phase, whereas I^C was expressed in the logarithmic and stationary phase. The intracellular localization of I^Ch was different from that of I^C. These findings provide insights into the physiological functions of I^Ch.     

Rapid purification of dehydrogenases by affinity chromatography with ternary complexes

The rapid purification of dehydrogenases by a modification of affinity chromatography was investigated. A ternary complex enzyme-NAD(H)-inhibitor (E-NADH-I) was formed by the addition of coenzyme and a substrate-competitive inhibitor to the dehydrogenases initially separated from nondehydrogenases by an NAD-affinity column. The enzyme in the ternary complex cannot rebind to the NAD-agarose column in the presence of inhibitor. As all other dehydrogenases do, this yields a highly purified enzyme-inhibitor complex. Aldehyde dehydrogenases in the presence of chloral hydrate and alcohol dehydrogenase with pyrazole were purified as their E-NAD⁺-I ternary complexes, while lactic dehydrogenase in the presence of oxamate was purified as the E-NADH-I complex. This technique allows for the rapid separation of a specific dehydrogenase from other dehydrogenases. The technique should be applicable to the purification of other enzymes exhibiting ordered sequential binding.     

Chemical modification of a beta-glucosidase from Schizophyllum commune: evidence for essential carboxyl groups

The beta-glucosidase from Schizophyllum commune was purified to homogeneity by a modified procedure that employed Con A-Sepharose. The participation of carboxyl groups in the mechanism of action of the enzyme was delineated through kinetic and chemical modification studies. The rates of beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside were determined at 27 degrees C and 70 mM ionic strength over the pH range 3.0-8.0. The pH profile gave apparent pK values of 3.3 and 6.9 for the enzyme-substrate complex and 3.3 and 6.6 for the free enzyme. The enzyme is inactivated by Woodward's K reagent and various water-soluble carbodiimides; chemical reagents selective for carboxyl groups. Of these reagents, 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide in the absence of added nucleophile was the most effective and a kinetic analysis of the modification indicated that one molecule of carbodiimide is required to bind to the beta-glucosidase for inactivation. Employing a tritiated derivative of the carbodiimide, 44 carboxyl groups in the enzyme were found to be labelled while the competitive inhibitor deoxynojirimycin protected three residues from modification. Treatment of the enzyme with tetranitromethane resulted in the modification of five tyrosine residues with approx. 28% diminution of enzymic activity. Titration of denatured enzyme with dithiobis(2-nitro-benzoic acid) indicated the absence of free thiol groups. Reaction of the enzyme with diethyl pyrocarbonate resulted in the modification of four histidine residues with the retention of 78% of the original enzymatic activity. The divalent transition metals Cu2+ and Hg2+ were found to be potent inhibitors of the enzyme, binding in an apparent irreversible manner.     

Ribonuclease inhibitor from pig brain: purification, characterization, and direct spectrophotometric assay

The ribonuclease inhibitor from pig brain has been purified 1,500-fold by a combination of ammonium sulfate fractionation, ion-exchange chromatography, hydroxylapatite chromatography, and gel filtration. The inhibitor has a Mr 50,000. It is a noncompetitive inhibitor for pancreatic ribonuclease A with a Ki of 1 nM, forming a 1:1 complex. Both ribonuclease A and B, but not ribonuclease U1 and T1, are inactivated by the inhibitor. The inhibition capacity was abolished by sulfhydryl reagents such as p-chloromercuribenzoate. Incubation of the enzyme-inhibitor complex with the sulfhydryl reagent caused dissociation into active ribonuclease and inactive inhibitor. Dithiothreitol was required during purification to retain the activity of the inhibitor.     

Rhodopsin-G-protein interactions monitored by resonance energy transfer

Resonance energy transfer measurements were implemented to monitor the specific interactions between G-protein and rhodopsin in phospholipid vesicles reconstituted with the purified proteins. Fluorescently labeled G-protein was extracted from bleached rod outer segments (ROS) reacted with several sulfhydryl reagents: N-(1-pyrenyl)maleimide (P), monobromobimane (B), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (C), and N-(4-anilino-1-naphthyl)maleimide (A). Limited labeling of ROS, resulting in the modification of less than a single -SH residue per G-protein molecule and less than 0.2 residue per rhodopsin, did not impair the specific in situ interactions between rhodopsin and G-protein. This was demonstrated by preservation of their light-activated tight association and Gpp(NH)p binding and their fast dissociation with excess GTP. The distribution of fluorescent label among the three subunits of G-protein revealed a highly reactive -SH group in the gamma subunit accessible to labeling when G-protein was bound specifically to bleached rhodopsin. Recombination of purified fluorescent derivatives of G-protein with purified rhodopsin reconstituted in lipid vesicles restored the light-activated Gpp(NH)p binding to a level comparable to that measured with unlabeled G-protein. Similar observations were obtained with ROS depleted of peripheral proteins. Likewise, modification of up to two -SH groups per rhodopsin molecule with the fluorescent reagents did not affect the functional recombination of G-protein with rhodopsin in reconstituted lipid vesicles or in depleted ROS. Interactions between rhodopsin and G-protein were monitored by resonance energy transfer measurements, with the following fluorescent conjugates as donor/acceptor couples: P-rhodopsin/C-G-protein, P-rhodopsin/B-G-protein, and P-G-protein/C-rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast.

We have investigated the role of clathrin in vacuolar protein sorting using yeast strains harboring a temperature-sensitive allele of clathrin heavy chain (chc1-ts). After a 5 min incubation at the non-permissive temperature (37 degrees C), the chc1-ts strains displayed a severe defect in the sorting of lumenal vacuolar proteins. Sorting of a vacuolar membrane protein, alkaline phosphatase, and transport to the surface of a cell wall protein, was not affected at 37 degrees C. In chc1-ts cells incubated at 37 degrees C, secretion of the missorted lumenal vacuolar protein carboxypeptidase Y (CPY) was blocked by the sec1 mutation which prevents fusion of secretory vesicles to the plasma membrane. Unexpectedly, chc1-ts cells incubated for extended periods at 37 degrees C regained the ability to sort CPY. Cells carrying deletions of the CHC1 gene (chc1 delta) also sorted CPY to the vacuole even when subjected to temperature shifts. Vacuolar delivery of CPY in chc1 delta cells was not blocked by sec1 suggesting that transport does not occur by secretion and endocytosis. These results provide in vivo evidence that clathrin plays a role in the Golgi complex in sorting of vacuolar proteins from the secretory pathway. With time, however, yeast cells lacking functional clathrin heavy chains are able to adapt in a way that allows restoration of vacuolar protein sorting in the Golgi complex. These conclusions clarify previous studies of chc1 delta cells which raised the possibility that clathrin is not involved in vacuolar protein sorting.     

Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT-1

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1. Aqualysin I was purified, and its apparent relative molecular mass was determined to be 28 500. The enzyme contained four Cys residues (probably as two cystines), and its amino acids composition was similar to those of cysteine-containing serine proteases (proteinase K, etc.) as well as those of subtilisins. The NH2-terminal sequence of aqualysin I showed homology with those of the microbial serine proteases. The optimum pH for the proteolytic activity of aqualysin I was around 10.0. Ca2+ stabilized the enzyme to heat treatment, and the maximum proteolytic activity was observed at 80 degrees C. Aqualysin I was stable to denaturing reagents (7 M urea, 6 M guanidine.HCl and 1% SDS) at 23 degrees C for 24 h. The enzyme hydrolyzed the ester bond of an alanine ester and succinyl-Ala-Ala-Ala p-nitroanilide, a synthetic substrate for mammalian elastase. The cleavage sites for aqualysin I in oxidized insulin B chain were not specific when it was digested completely.     

Interaction of Cibacron Blue 3GA and Remazol Brilliant Blue R with the nucleotide binding site of lactate dehydrogenase and (Na+ + K+)-ATPase

The interaction of Cibacron Blue 3GA (C.I.2) and Remazol Brillant Blue R (C.I.19) with purified preparations of Lactate dehydrogenase (LDH) and (Na+ + K+)-ATPase was studied by means of the enzyme kinetics method. LDH was found to be inhibited by both C.I.2 and C.I.19, with the former being a stronger inhibitor. This may be explained by the fact that in contrast to C.I.19, C.I.2 resembles the whole molecule of the specific cofactor. (Na+ + K+)-ATPase activity was inhibited by both dyes to approximately similar degree. C.I.2 and C.I.19 resemble the ATP molecule to approximately similar extent, particularly as concerns the molecule shape and size. The results obtained confirmed the applicability of C.I.2 and C.I.19 as nucleotide-specific ligands.