The Endoplasmic Reticulum of Mung Bean Cotyledons: ROLE IN THE ACCUMULATION OF HYDROLASES IN PROTEIN BODIES DURING SEEDLING GROWTH

The subcellular localization of two hydrolases (ribonuclease and vicilin peptidohydrolase) which are synthesized de novo in the cotyledons of mung bean seedlings was studied. Earlier experiments had shown that both enzymes accumulate in the protein bodies in the course of seedling growth. Two methods to fractionate subcellular organelles were used to demonstrate that a significant proportion of the enzymes is organelle-associated. This proportion is highest (up to 50% for vicilin peptidohydrolase and 15% for ribonuclease) when synthesis of the enzymes has just started. Evidence obtained with isopycnic sucrose gradients indicates that both hydrolases are associated with membranes rich in NADH-cytochrome c reductase, a marker enzyme for the endoplasmic reticulum (ER). The hydrolases band with the NADH-cytochrome c reductase under conditions where the ribosomes remain attached or are detached from the ER-derived vesicles. Treatment of the ER-derived vesicles with Triton X-100 shows that vicilin peptidohydrolase and vesicle membranes can be physically separated without dissolving the membranes, indicating that the proteinase is soluble within the vesicles. These data support the conclusion that the ER is involved in the transport of ribonuclease and proteinase to the protein bodies.     

Trypsin inhibitor in mung bean cotyledons: purification, characteristics, subcellular localization, and metabolism

Trypsin inhibitor was purified to homogeneity from seeds of the mung bean (Vigna radiata [L.] Wilczek). The protease inhibitor has the following properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel filtration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptidase in the cotyledons of mung bean seedlings.

The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of β-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts.

The intracellular localization of trypsin inhibitor was determined by fractionation of crude extracts on isopycnic sucrose gradients and by cytochemistry with fluorescent antibodies. Both methods indicate that trypsin inhibitor is associated with the cytoplasm and not with the protein bodies where reserve protein hydrolysis occurs. No convincing evidence was obtained which indicates that the catabolism of trypsin inhibitor during germination and seedling growth is causally related to the onset of reserve protein breakdown.

     

Immunocytochemical localization of the soluble NAD-dependent hydrogenase in cells of Alcaligenes eutrophus

Abstract The localization of the soluble NAD-dependent hydrogenase in cells of Alcaligenes eutrophus PHB⁻4 was investigated using the protein A-gold technique as a post-embedding immunoelectron microscopic procedure. The enzyme was found throughout the cytoplasm of the cells. Autotrophic cells harvested in the logarithmic phase of growth exhibited a higher degree of labeling as compared to autotrophic cells from the stationary growth phase. Heterotrophic cells showed an almost identical labeling intensity in all growth phases. In a substrate-shift experiment (from fructose to glycerol, performed in the stationary growth phase), high amounts of newly synthesized enzyme could be observed two hours after the shift. This enzyme was located, as inclusion bodies, in the DNA region of the cells.     

The Protein Body in Dedifferentiating Cels of Tobacco Leaf Explants

Light and electron microscopic observations show that a kind of spherical electron-dense body appears in the dediffercntiating mesophyll cells and their subdivided cells in tobacco leaf explants cultured for more than two days. The larger electron-dense bodies (1.0–1.5μm in diameter) present in vacuoles while the smaller ones (0.1–0.8 m in diameter) in cytoplasm. This implies that the bodies first can be formed in cytoplasm and then secreted into vacuoles. Since the bodies can be fixed with glutaradehyde and 3H- leucine can incorporated into them, they may be recognized as protein bodies. The protein bodies usually closly combined with newly formed cytoplasmic masses so we suggest that they probably play some role in cytoplasmic growth of dedifferentiafing ceils.     

Characterization and subcellular localization of vicilin and phytohemagglutinin,the two major reserve proteins of Phaseolus vulgaris L.

Extracts of bean (Phaseolus vulgaris L. cv. Greensleeves) cotyledons contained two abundant proteins: vicilin and phytohemagglutinin. Vicilin, a 6.9 S protein fraction at neutral pH, associated to an 18.0 S form at pH 4.5 and had 3 non-identical subunits with molecular weights (MW) of 52,000, 49,000 and 46,000. Phytohemagglutinin, a 6.4 S protein fraction, had 2 non-identical subunits with MW of 34,000 and 36,000. Phytohemagglutinin could be separated by isoelectrofocusing into a mitogenic and non-erythroagglutinating protein with a single subunit of MW=34,000, and a mitogenic and erythroagglutinating protein fraction which contained both subunits. Vicilin is apparently identical with the so called glycoprotein II (A. Pusztai and W.B. Watt, Biochim. Biophys. Acta 365, 57–71, 1970) and with globulin G1 (R.C. McLeester, T.C. Hall, S.M. Sun, F.A. Bliss, Phytochem. 2, 85; 1973), while phytohemagglutinin is identical with globulin G2 (McLeester et al., 1973). Since vicilin and phytohemagglutinin are internationally used names there is no need to introduce new names to describe P. vulgaris reserve proteins. Both proteins are catabolized in the course of seedling growth and are located in the protein bodies, indicating that they are reserve proteins. Vicilin isolated in its 18.0 S form from the cotyledons of young seedlings contains substantial quantities of smaller polypeptides, in addition the 3 original ones. We suggest that the presence of these small polypeptides represents partial breakdown of the vicilin prior to its complete catabolism.     

The effects of silicate depletion and subsequent replenishment on the cytoplasmic fine structure of the silica-secreting testate amoeba Netzelia tuberculata in laboratory culture

In the absence of silicate in the growth medium, Netzelia tuberculata cells withdraw their feeding lobopodia, become quiescent, and cease to divide. Upon replenishment of silicate, growth resumes within 18–24 hours. Cytoplasmic changes produced by a low silicate medium result in a zonal arrangement, with siliceous particles at the outer periphery of the cytoplasm in a region rich in Golgi bodies (Region A), a more centrally located layer containing endoplasmic reticulum, lipid reserves, and finely granular cytoplasm (Region B), and a region of partially digested food and waste material fringed by fine rhizopodia extending into the central space of the test (Region C). The reserve siliceous particles in the outer peripheral cytoplasm are foreign particles that contain a fragile deposit of silica and appear to be incomplet. This may be a mechanism for conserving silica in the low-silicate medium by coating particles instead of making particles of solid silica de novo. Upon addition of silicate to the growth medium, new siliceous particles are synthesized within vacuoles in the region of the Golgi apparatus within 2–18 hours. Vacuoles containing fine silica deposits, characteristic of new particle production, are surrounded by Golgi-derived vesicles previously shown to be a source of membrane for the silica-secreting vacuoles. The newly synthesized particles are solid silica as is characteristic of de novo secreted test particles, in contrast to the numerous silica-coated foreign bodies found in quiescent cells produced in low-silicate medium.     

Regulation of a Sulfur-Controlled Protease in Neurospora crassa

Wild-type Neurospora crassa produces and secretes extracellular protease(s) when grown on a medium containing a protein as its principle sulfur source. Readily available sulfur sources, such as sulfate or methionine, repress the synthesis of the proteolytic activity. Preliminary characterization of the proteolytic enzyme shows it to have a molecular weight of about 31,000, a pH optimum of 6 to 9 with casein as substrate, and esterolytic activity against acetyl-tyrosine ethyl ester with a pH optimum of 8.5. The enzyme activity is completely inhibited by diisopropylfluorophosphate, partially inhibited by ethylenediaminetetraacetate, but unaffected by iodoacetate. The proteolytic activity is temperature labile and is reduced by 75% within 15 min at 60 C. Synthesis of the protease activity is induced by proteins, and to a lesser extent by large-molecular-weight polyamino acids, but not at all by small peptides or amino acid mixtures. During conidial out-growth, the protease(s) first appears at about 8 h and continues to increase while the cells are in an active growth phase. When a low concentration of sulfate is present, the protease(s) is not produced until about 18 h, suggesting that the sulfate must first be used by the cells before the protease is either synthesized or released.     

Ultrastructural and biochemical observations on the early changes in apoptotic epithelial cells of the rat prostate induced by castration

The present study describes the sequential ultrastructural changes in the apoptotic cells of the rat ventral and dorsal prostates during the early period of 1-3 days postcastration. The major morphological changes include: (1) condensation of heterochromatin along the nuclear envelope and fragmentation into crescent-shaped micronuclei; (2) formation of membrane-bound cytoplasmic spherical bodies, which contain various organelles and micronuclei, within the apoptotic cells; (3) formation of non-membrane-bound autolytic vacuoles by autolysis of cytoplasm; (4) focal rupture of outer mitochondrial membrane; and (5) phagocytosis of the fragmented cytoplasmic spherical bodies and apoptotic cells by macrophages. The occurrence of both cytoplasmic apoptotic bodies and autolytic vacuoles in apoptotic cells suggests that the cytoplasm of the apoptotic cells could be destroyed by different means. The responsiveness of different prostatic lobes to androgen withdrawal and the time course of the transitory apoptotic activity in different lobes were analyzed by counting the indices of the TUNEL-labeled apoptotic cells against the postcastration periods. The results showed that the ventral lobe responded more rapidly to castration than the lateral and dorsal lobes. The dorsal lobe was the slowest in response to castration among the three lobes. Analysis of protease activities by zymography has identified two Ca(2+)-independent proteases of apparent MW 20 and 24 kDa (expressed in both ventral and dorsolateral lobes), and one Ca(2+)-dependent protease of MW 66.5 kDa (expressed only in the dorsolateral lobe) which became activated at day 3 postcastration. Their expression patterns were different from that of CPP-3 in the castrated prostates, suggesting that the activated proteases were enzymes other than CPP-3. The association of their highest activities with the maximum apoptotic activity at day 3 postcastration and also their loss of activity at day 15 suggest that these protease activities might be related to apoptosis or glandular involution.     

Regulation of the formation of protease inBacillus megaterium

During the growth of the asporogenous variant ofBacillus megaterium KM in medium containing NO₃ ⁻ as nitrogen source, the relative rate of extracellular protease synthesis is higher than in the presence of NH₄ ⁺. It approaches the relative rate of enzyme synthesis at the incubation of cells in nitrogen-free medium with glucose. This supports the suggestion that even amino acids which are synthesized endogenously slow down the protease production. In the postlogarithmic or stationary phase the protease production stops. The interruption of enzyme production does not appear as a result of insufficient aeration in a dense suspension, or of accumulation of amino acids or their metabolites in cells. The non-growing cells retain their ability to renew the enzyme synthesis when transferred into a fresh medium, even into a medium without nitrogen source. In the same way it is possible to “induce” the protease production, if Ca²⁺ is added to cells in the stationary phase when the population was grown in the Ca²⁺ free medium. The amount of enzyme produced at the expense of protein turnover by the non-growing populations is sufficient for the fast hydrolysis of exogenous protein in the medium and for assuring the influx of a sufficient amount of peptides into the cells. In such a case the growth of the culture is therefore very quickly renewed.     

The biosynthesis of ribonuclease and its accumulation in protein bodies in the cotyledons of mung bean seedlings

Germination and seedling growth of mung bean are accompanied by a 7- to 10-fold increase in the ribonuclease content of the cotyledons. The increase occurs during the first 4 days of seedling growth and precedes the senescence of the cotyledons. Separation of the RNases in the cotyledons by polyacrylamide gel electrophoresis indicates the presence of several minor bands in seeds imbibed for 24 hr. On the second day of seedling growth a new major band with an R_f of 0.76 is present. In 4- to 5-day old seedlings this major band accounts for nearly all the RNase activity in the tissue. The characteristics of this RNase show that it is a plant ribonuclease I (pH optimum of 5.0; MW 16,000; activity preferentially inhibited by purine nucleotides; no activity toward DNA; no phosphodiesterase activity). When the seedlings are grown in 66% D₂O the RNase activity undergoes a density shift of 0.61% indicating that the increase in enzyme activity is due to the de novo synthesis of the enzyme molecules. A method is described for the isolation of protein bodies from protoplasts of storage parenchyma cells. Fractionation of protoplast lysates on Ficoll gradients results in the recovery of a high proportion (75%) of intact protein bodies. On these gradients RNase activity comigrates with α-mannosidase, a protein body marker enzyme indicating that the newly synthesized RNase accumulates in the protein bodies. We suggest that the synthesis of RNase in the cotyledons and its accumulation in the protein bodies indicates that protein bodies may function in the degradation of cellular macromolecules other than the reserves stored within them.     

Proteases of nuclei of testicular cells assayed with fluorogenic peptides

Nuclei of seminiferous epithelial cells (SEC) of rat testis exhibit protease activity when assayed with fluorogenic peptides or with [3H]histones. At pH 8 the nuclear protease rapidly hydrolyzes BOC-Val-Pro-Arg-7-Amino-4-Methyl Coumarin (BVPAC) and Glu-Gly-Arg-7-Amino-4-Methyl Coumarin (GGAC), but this enzyme does not hydrolyze CBZ-Arg-7-Amino-4-Methyl Coumarin (CAC) or Glu-Phe-7-Amino-4-Methyl Coumarin (GPC). The cytoplasm of these cells hydrolyzes each of these substrates. The protease activity versus BVPAC can be extracted from cytoplasm and nuclei with 0.1 M H2SO4. The extracted activity from cytoplasm is lost during storage for 5 days at either pH 3 or pH 8 at -25 degrees, but the activity extracted from nuclei is maintained under these conditions. The nuclear protease activity is found in SEC of young rats prior to the appearance of acrosin.     

A method of isolation of partially purified alkaline peptide hydrolase from Drosophila melanogaster larvae

A semipreparative method is developed for preparing peptidohydrolase from Drosophila melanogaster larvae which involves the stages of extraction, salting-out, gel-filtration and ion-exchange chromatography. It is established that the maximal (up to 81%) yield of the enzyme is observed with the single extraction in the alkaline medium. The main bulk of the enzyme is salted-out in the low acid 3 M ammonium sulphate solution. Gel-filtration on column with Sephadex-25 provides complete salting-out of the enzyme-containing fraction, and ion exchange chromatography on CM-cellulose--a considerable purification of the enzyme under study. A degree of the obtained purification of the enzyme under study. A degree of the obtained peptidohydrolase preparation purity in acid and alkaline medium is determined by the method of electrophoresis in PAAG. At all stages of the preparation the enzyme possesses the casein-lytic activity and is able of hydrolyzing the ethyl ester and benzoyl arginine p-nitroanilide.     

Coilin Shuttles between the Nucleus and Cytoplasm In Xenopus Oocytes

Coiled bodies are discrete nuclear organelles often identified by the marker protein p80-coilin. Because coilin is not detected in the cytoplasm by immunofluorescence and Western blotting, it has been considered an exclusively nuclear protein. In the Xenopus germinal vesicle (GV), most coilin actually resides in the nucleoplasm, although it is highly concentrated in 50-100 coiled bodies. When affinity-purified anti-coilin antibodies were injected into the cytoplasm of oocytes, they could be detected in coiled bodies within 2-3 h. Coiled bodies were intensely labeled after 18 h, whereas other nuclear organelles remained negative. Because the nuclear envelope does not allow passive diffusion of immunoglobulins, this observation suggests that anti-coilin antibodies are imported into the nucleus as an antigen-antibody complex with coilin. Newly synthesized coilin is not required, because cycloheximide had no effect on nuclear import and subsequent targeting of the antibodies. Additional experiments with myc-tagged coilin and myc-tagged pyruvate kinase confirmed that coilin is a shuttling protein. The shuttling of Nopp140, NO38/B23, and nucleolin was easily demonstrated by the targeting of their respective antibodies to the nucleoli, whereas anti-SC35 did not enter the germinal vesicle. We suggest that coilin, perhaps in association with Nopp140, may function as part of a transport system between the cytoplasm and the coiled bodies.     

Processing of pulmonary surfactant protein B by napsin and cathepsin H

Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. SP-B is synthesized in alveolar type II cells as a preproprotein and processed to the mature peptide by the cleavage of NH2- and COOH-terminal peptides. An aspartyl protease has been suggested to cleave the NH2-terminal propeptide resulting in a 25-kDa intermediate. Napsin, an aspartyl protease expressed in alveolar type II cells, was detected in fetal lung homogenates as early as day 16 of gestation, 1 day before the onset of SP-B expression and processing. Napsin was localized to multivesicular bodies, the site of SP-B proprotein processing in type II cells. Incubation of SP-B proprotein from type II cells with a crude membrane extract from napsin-transfected cells resulted in enhanced levels of a 25-kDa intermediate. Purified napsin cleaved a recombinant SP-B/EGFP fusion protein within the NH2-terminal propeptide between Leu178 and Pro179, 22 amino acids upstream of the NH2 terminus of mature SP-B. Cathepsin H, a cysteine protease also implicated in pro-SP-B processing, cleaved SP-B/EGFP fusion protein 13 amino acids upstream of the NH2 terminus of mature SP-B. Napsin did not cleave the COOH-terminal peptide, whereas cathepsin H cleaved the boundary between mature SP-B and the COOH-terminal peptide and at several other sites within the COOH-terminal peptide. Knockdown of napsin by small interfering RNA resulted in decreased levels of mature SP-B and mature SP-C in type II cells. These results suggest that napsin, cathepsin H, and at least one other enzyme are involved in maturation of the biologically active SP-B peptide.     

Growth pattern of Rickettsia tsutsugamushi in irradiated L cells.

Irradiated L cells infected with Rickettsia tsutsugamushi were studied under the electron microscope to define the morphological growth pattern of the organism. For 2 days after inoculation, no rickettsiae were found either extra- or intracellularly; after 2 days multiple rickettsiae appeared within the host cells without morphological evidence of their entry. These observations showed that the rickettsiae within the cell were assembled in situ by segregation of portions of the granular cytoplasm and subsequent internal differentiation and surface membrane assembly of the segregated bodies. The protoplasmic (P) bodies, which seemed to be formed by shedding infected-cell granular cytoplasm, consistently appeared on the surface and within the phagosomes of the host cells. Rickettsiae were occasionally seen entering host cells in the later phase of infection; these were apparently the ones assembled within the P bodies. This suggested that the P bodies, and not the rickettsiae, were the major infectious particles that transmitted the rickettsial genetic substance among the host cells. On the basis of the present morphological observations, viral-type multiplication for R. tsutsugamushi is proposed.     

Identification of a membrane-associated cysteine protease with possible dual roles in the endoplasmic reticulum and protein storage vacuole

SH-EP is a vacuolar cysteine proteinase from germinated seeds of Vigna mungo. The enzyme has a C-terminal propeptide of 1 kDa that contains an endoplasmic reticulum (ER) retention signal, KDEL. The KDEL-tail has been suggested to function to store SH-EP as a transient zymogen in the lumen of the ER, and the C-terminal propeptide was thought to be removed within the ER or immediately after exit from the ER. In the present study, a protease that may be involved in the post-translational processing of the C-terminal propeptide of SH-EP was isolated from the microsomes of cotyledons of V. muno seedlings. cDNA sequence for the protease indicated that the enzyme is a member of the papain superfamily. Immunocytochemistry and subcellular fractionation of cotyledon cells suggested that the protease was localized in both the ER and protein storage vacuoles as enzymatically active mature form. In addition, protein fractionations of the cotyledonary microsome and Sf9 cells expressing the recombinant protease indicated that the enzyme associates with the microsomal membrane on the luminal side. The protease was named membrane-associated cysteine protease, MCP. The possibility that a papain-type enzyme, MCP, exists as mature enzyme in both ER and protein storage vacuoles will be discussed.     

Secretion-dependent proteolysis of heterologous protein by recombinant Escherichia coli is connected to an increased activity of the energy-generating dissimilatory pathway

The synthesis of a proteolytically unstable protein, originally designed for periplasmic export in recombinant Escherichia coli BL21(DE3), a strain naturally deficient for the ATP-dependent protease Lon (or La) and the outer membrane protease OmpT, is associated with a severe growth inhibition. This inhibition is not observed in BL21(DE3) synthesizing a closely related but proteolytically stable protein that is sequestered into inclusion bodies. It is shown that the growth inhibition is mainly caused by a slower cell division rate and a reduced growth yield and not by a general loss of cell division competence. Cells proceed with their normal growth characteristics when exposed again to conditions that do not sustain the expression of the heterologous gene. The performance of cells synthesizing either the stable or the degraded protein was also studied in high cell density cultures by employing a new method to calculate the actual specific growth rate, the biomass yield coefficient, and the dissimilated fraction of the carbon substrate in real-time. It is shown that the growth inhibition of cells synthesizing the proteolytically degraded protein is connected to an increased dissimilation of the carbon substrate resulting in a concomitant reduction of the growth rate and the biomass yield coefficient with respect to the carbon source. It is postulated that the increased dissimilation of the carbon substrate by lon-deficient Bl21(DE3) cells synthesizing the proteolytically unstable protein may result from a higher energy demand required for the in vivo degradation of this protein by ATP-dependent proteases different from the protease Lon.     

Assembly and intracellular localization of the bluetongue virus core protein VP3

The bluetongue virus (BTV) core protein VP3 plays a crucial role in the virion assembly and replication process. Although the structure of the protein is well characterized, much less is known about the intracellular processing and localization of the protein in the infected host cell. In BTV-infected cells, newly synthesized viral core particles accumulate in specific locations within the host cell in structures known as virus inclusion bodies (VIBs), which are composed predominantly of the nonstructural protein NS2. However, core protein location in the absence of VIBs remains unclear. In this study, we examined VP3 location and degradation both in the absence of any other viral protein and in the presence of NS2 or the VP3 natural associate protein, VP7. To enable real-time tracking and processing of VP3 within the host cell, a fully functional enhanced green fluorescent protein (EGFP)-VP3 chimera was synthesized, and distribution of the fusion protein was monitored in different cell types using specific markers and inhibitors. In the absence of other BTV proteins, EGFP-VP3 exhibited distinct cytoplasmic focus formation. Further evidence suggested that EGFP-VP3 was targeted to the proteasome of the host cells but was dispersed throughout the cytoplasm when MG132, a specific proteasome inhibitor, was added. However, the distribution of the chimeric EGFP-VP3 protein was altered dramatically when the protein was expressed in the presence of the BTV core protein VP7, a normal partner of VP3 during BTV assembly. Interaction of EGFP-VP3 and VP7 and subsequent assembly of core-like particles was further examined by visualizing fluorescent particles and was confirmed by biochemical analysis and by electron microscopy. These data indicated the correct assembly of EGFP-VP3 subcores, suggesting that core formation could be monitored in real time. When EGFP-VP3 was expressed in BTV-infected BSR cells, the protein was not associated with proteasomes but instead was distributed within the BTV inclusion bodies, where it colocalized with NS2. These findings expand our knowledge about VP3 localization and its fate within the host cell and illustrate the assembly capability of a VP3 molecule with a large amino-terminal extension. This also opens up the possibility of application as a delivery system.     

Localization of Chitin synthase in Mucor rouxii by an autoradiographic method

The localization of chitin synthase in the cells of Mucor rouxii was studied by a method which combined permeabilization of the cells with toluene/ethanol and incubation with the radioactive substrate UDP-[3H]GlcNAc followed by high resolution autoradiography. By this technique it was demonstrated that most of the chitin synthesized by these cells was located within the cytoplasm, and only a small amount of the enzyme product appeared at the cell surface. It was concluded that most of the chitin synthase of M. rouxii is located in the cytoplasm of the cells.     

Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity

Noninvasive real-time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease's natural milieu. We developed a protease activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, on protease-mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV as well as in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high-throughput screening of 39,000-compound small molecule libraries, leading to identification of furin inhibitors. Furthermore, this strategy was used to identify inhibitors of another Golgi protease, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE). BACE cleavage of the APP leads to formation of the Abeta peptide, a key event that leads to Alzheimer's disease. In conclusion, we describe a customizable noninvasive technology for real-time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.