Specific functions of lysosomal proteases in endocytic and autophagic pathways

Endolysosomal vesicles form a highly dynamic multifunctional cellular compartment that contains multiple highly potent proteolytic enzymes. Originally these proteases have been assigned to cooperate solely in executing the unselective ‘bulk proteolysis’ within the acidic milieu of the lysosome. Although to some degree this notion still holds true, evidence is accumulating for specific and regulatory functions of individual ‘acidic’ proteases in many cellular processes linked to the endosomal/lysosomal compartment. Here we summarize and discuss the functions of individual endolysosomal proteases in such diverse processes as the termination of growth factor signaling, lipoprotein particle degradation, infection, antigen presentation, and autophagy. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Convergence of the endocytic and lysosomal pathways in soybean protoplasts

Ultrastructural analysis of endocytosis of cationized ferritin (CF) has been combined with ultrastructural localization of acid phosphatases (AcPase) in soybean (Glycine max (L.) Merr.) protoplasts. While CF is an electron-dense marker of organelles of the endocytic pathway, ultrastructural histochemistry of AcPase identifies the organelles involved in the synthesis, transport, and storage of lytic-compartment enzymes, i.e. the lysosomal pathway. Acid phosphatases have been localized using both lead- and cerium-precipitation techniques. Protoplasts have been exposed to CF for 5 min, 30 min, or 3 h and processed for AcPase localization. At 5 min, smooth vesicles contain both CF and AcPase. By 30 min, Golgi cisternae and multivesicular bodies contain both labels. By 3 h, vacuoles become labelled with both CF and AcPase. The large central vacuoles contain intraluminal membranes which are associated with both AcPase and CF. These observations extend the analogy between plant vacuoles and animal lysosomes and demonstrate the points at which the endocytic pathway of plants converges with the lysosomal pathway.Abbreviations AcPase acid phosphatase - CF cationized ferritin - ER endoplasmic reticulum - MVB multivesicular body - PCR partially coated reticulum - PM plasma membrane     

Vacuolar proteases from Candida glabrata: Acid aspartic protease PrA,neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression

BackgroundThe Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata.AimsThe present paper is the first report on proteolytic activity in the C. glabrata vacuole.MethodsBiochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA.ResultsOur results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source.ConclusionsThe proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen.     

The convergent point of the endocytic and autophagic pathways in leydig cells

Endocytic tracers and marker enzyme of lysosomes were used in the present study to analyze the processes of autophagocytosis and endocytosis,and the convergent point of these two pathways in Leydig cells.The endocytic and autophagic compartments can be easily identified in Leydig cells,which makes easier to difine the stages of two pathways than was possible before.The evidences indicated that late endosomes (dense MVBs) deliver their endocytosed gold tracers together with lysosomal enzymes to the early autophagosomes and they are the convergent point of the two pathways.During this convergent process,the early autophadosomes transform into late autophagosomes and the late endosomes transform into mature lysosomes.     

Vacuole import and degradation pathway: Insights into a specialized autophagy pathway

Glucose deprivation induces the synthesis of pivotagluconeogenic enzymes such as fructose-1,6-bisphos-phatase, malate dehydrogenase, phosphoenolpyruvatecarboxykinase and isocitrate lyase in Saccharomycescerevisiae. However, following glucose replenishment,these gluconeogenic enzymes are inactivated and de-graded. Studies have characterized the mechanismsby which these enzymes are inactivated in response toglucose. The site of degradation of these proteins hasalso been ascertained to be dependent on the dura-tion of starvation. Glucose replenishment of short-termstarved cells results in these proteins being degradedin the proteasome. In contrast, addition of glucose tocells starved for a prolonged period results in theseproteins being degraded in the vacuole. In the vacuoledependent pathway, these proteins are sequestered inspecialized vesicles termed vacuole import and degra-dation (Vid). These vesicles converge with the endo-cytic pathway and deliver their cargo to the vacuolefor degradation. Recent studies have identified thatinternalization, as mediated by actin polymerization, isessential for delivery of cargo proteins to the vacuolefor degradation. In addition, components of the targetof rapamycin complex 1 interact with cargo proteins during glucose starvation. Furthermore, Tor1p dissoci-ates from cargo proteins following glucose replenish-ment. Future studies will be needed to elaborate on the importance of internalization at the plasma membrane and the subsequent import of cargo proteins into Vid vesicles in the vacuole dependent degradation pathway.     

Cellular metabolic and autophagic pathways: Traffic control by redox signaling

It has been established that the key metabolic pathways of glycolysis and oxidative phosphorylation are intimately related to redox biology through control of cell signaling. Under physiological conditions glucose metabolism is linked to control of the NADH/NAD redox couple, as well as providing the major reductant, NADPH, for thiol-dependent antioxidant defenses. Retrograde signaling from the mitochondrion to the nucleus or cytosol controls cell growth and differentiation. Under pathological conditions mitochondria are targets for reactive oxygen and nitrogen species and are critical in controlling apoptotic cell death. At the interface of these metabolic pathways, the autophagy–lysosomal pathway functions to maintain mitochondrial quality and generally serves an important cytoprotective function. In this review we will discuss the autophagic response to reactive oxygen and nitrogen species that are generated from perturbations of cellular glucose metabolism and bioenergetic function.     

Vacuolar accumulation of acridine orange and neutral red in zygotic and somatic embryos of carrot (Daucus carota L.)

Summary The accumulation of neutral red and acridine orange, to indicate differences in vacuolar pH, was studied during embryogenesis of carrot. Neutral red accumulated barely in proembryogenic masses, but was present conspicuously in globular-shaped somatic embryos. From the late globular to the torpedo-shaped stage, it was mainly found in the root side of the somatic embryo. Here, neutral red was predominantly present in large dark-red to purple stained vesicles. In the cotyledons neutral red was found in small orange vesicles. In zygotic embryos of carrot, the dye was uniformly distributed with no specific localization in organelles. During germination, however, neutral red accumulated mainly in regions in the root side and the hypocotyl of the germling. Acridine orange was dispersed erratically in proembryogenic masses with a great variety in intensity. It was quite obviously present in early stages of somatic embryogenesis and restricted to the root side in late globular to torpedo-shaped embryos. Confocal images revealed the vacuolar presence of the fluorescence and the predominant presence in the protoderm. During germination of zygotic embryos the signal changed from uniform to localized, with sharp borders between fluorescent and non-fluorescent regions. Two to three days after the beginning of germination, acridine orange accumulated preferentially in the root tip of the germling. Differences between somatic and zygotic embryos and similarities between somatic embryogenesis and zygotic embryo germination are discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pH_c cytosolic pH - pH_e extracellular pH - pH_v vacuolar pH     

Down-regulating protein kinase C alpha: Functional cooperation between the proteasome and the endocytic system

Ubiquitination, proteasome, caveolae and endosomes have been implicated in controlling protein kinase Cα (PKCα) down-regulation. However, the molecular mechanism remained obscure. Here we show that endosomes and proteasome cooperate in phorbol ester 12-O-tetradecanoyl phorbol acetate (TPA)-induced down-regulation of PKCα. We show that following TPA treatment and translocation to the plasma membrane, PKCα undergoes multimonoubiquitination prior to its degradation by the proteasome. However, to reach the proteasome, PKCα must travel through the endocytic system from early to late endosomes. This route requires functional endosomes, whereby endosomal alkalinization, or ablation, abrogates completely PKCα degradation maintaining the enzyme at the plasma membrane. This route also depends on synaptotagmin (Syt) II and the Rab7 GTPase, whereby Syt II knock-down or expression of the GDP-locked Rab7 inactive mutant prevents PKCα degradation. We further show that proteasome plays a dual role, where an active proteasome is required for deubiquitination of PKCα, a step crucial to prevent PKCα targeting to the endocytic recycling compartment. Finally, we show that the association with rafts-localized cell surface proteins that internalize in a clathrin-independent fashion is necessary to allow the trafficking of PKCα from the plasma membrane to the proteasome, its ultimate degradation station.     

Ultrastructural enzyme-cytochemical study of the intrinsic glandular cells in the corpus cardiacum of Locusta migratoria: relation to the secretory and endocytotic pathways,and to the lysosomal system

Summary Ultrastructural aspects of the secretory and the endocytotic pathways and the lysosomal system of corpus cardiacum glandular cells (CCG cells) of migratory locusts were studied using morphological, marker enzyme, immunocytochemical and tracer techniques. It is concluded that (1) the distribution of marker enzymes of trans Golgi cisternae and trans Golgi network (TGN) in locust CCG cells corresponds to that in most non-stimulated vertebrate secretory cell types; (2) the acid phosphatase-positive TGN in CCG cells is involved in sorting and packaging of secretory material and lysosomal enzymes; (3) these latter substances are produced continuously; (4) at the same time, superfluous secretory granules and other old cell organelles are degraded; (5) the remarkable endocytotic activity in the cell bodies and the minor endocytotic activity in cell processes are coupled mainly to constitutive uptake of nutritional and/or regulatory (macro)molecules, rather than to exocytosis; (6) plasma membrane recycling occurs mainly by direct fusion of tubular endosomal structures with the plasma membrane and little traffic passes the Golgi/TGN; and (7) so-called cytosomes arise mainly from autophagocytotic vacuoles and represent a special kind of complex secondary lysosomes involved in the final degradation of endogenous (cell organelles) and exogenous material.     

Purification and characterization of a 40.8-kDa cutinase in ungerminated conidia of Botrytis cinerea Pers.: Fr

Cytoplasmic soluble proteins from ungerminated conidia of Botrytis cinerea exhibited cutinase activity. A 40.8-kDa cutinase was purified to homogeneity from this crude conidial protein extract. This cutinase does not correspond either to constitutive or to induced lytic cutin enzymes already described by other authors. The possible role of this constitutive cutinase in the induction of other cutinolytic proteins in the early stages of infection of plants by B. cinerea is discussed.     

Polarized epithelial cells of the hamster seminal vesicle in a serum-free bicameral culture system: evidence of secretory and endocytic activities

Primary cultures of epithelial cells from the hamster seminal vesicle were established in a chemically defined medium supplemented with hormones and growth factors. Epithelial cell clusters were prepared combining enzymatic dissociation and mechanical disaggregation and then seeded in bicameral systems equipped with collagen-membrane inserts. A growth curve was generated and the cells were characterized morphologically and morphometrically by light and electron microscopy. The immunocytochemical detection of cytokeratins and the measurement of transepithelial electrical resistance were also performed. The secretory activity was studied by fluorography using L-[³⁵S]methionine as a precursor, and endocytosis was approached using horseradish peroxidase as a tracer. Our results show that epithelial cells of the seminal vesicle can be grown as a monolayer of morphological and functionally polarized cells which retain secretory and endocytic activities. These cell cultures might therefore prove useful to investigate further the regulation of secretion and endocytosis in the seminal vesicle and are a promising model to approach, in a broader scope, cell polarity and protein sorting and targeting.     

Entamoeba histolytica: comparison of the role of receptors and filamentous actin among various endocytic processes

Entamoeba histolytica is the causative agent of amoebic dysentery. Uptake of iron is critical for E. histolytica growth and iron-bound human transferrin (holo-transferrin) has been shown to serve as an iron source in vitro. Although a transferrin-binding protein has been identified in E. histolytica, the mechanism by which this iron source is taken up by this pathogen is not well understood. To gain insight into this process, the uptake of fluorescent-dextran, -holo-transferrin, and human red blood cells (hRBCs) was compared. Both dextran and transferrin were taken up in an apparent receptor-independent fashion as compared to hRBCs, which were taken up in a receptor-mediated fashion. Interestingly, the uptake of FITC-dextran and FITC-holo-transferrin differentially relied on an intact actin cytoskeleton suggesting that their internalization routes may be regulated independently.     

A comparison between the responses of neutral red and acridine orange: acridine orange should be preferential and alternative to neutral red as a dye for the monitoring of contaminants by means of biological sensors

The acridine orange (AO) and neutral red (NR) dyes, commonly used as probes to measure the internal pH in acidic vesicles, are compared in this article. The comparison between the two dyes (arising from calculations taking into account their analytical constants) illustrated that the use of AO is preferential to that of NR because the AO response is sensitive over the whole pH range between 4.0 and 7.4, whereas the NR response is effective only between pHs 4.0 and 6.0. In addition, it became evident from the mitochondrial respiration response that NR, unlike AO, is a protonophore. When taken into consideration, these two properties suggest that AO is more suitable than NR as an indicator of toxicity measurements in water samples because the environmental toxic compounds induce pH changes in the acidic vesicles of biological structures that are used as environmental biosensors.     

Patterning of endocytic vesicles and its control by voltage-gated Na<Superscript>+</Superscript> channel activity in rat prostate cancer cells: fractal analyses

Fractal methods were used to analyze quantitative differences in secretory membrane activities of two rat prostate cancer cell lines (Mat-LyLu and AT-2) of strong and weak metastatic potential, respectively. Each cells endocytic activity was determined by horseradish peroxidase uptake. Digital images of the patterns of vesicular staining were evaluated by multifractal analyses: generalized fractal dimension (D_q) and its Legendre transform f(), as well as partitioned iterated function system – semifractal (PIFS-SF) analysis. These approaches revealed consistently that, under control conditions, all multifractal parameters and PIFS-SF codes determined had values greater for Mat-LyLu compared with AT-2 cells. This would agree generally with the endocytic/vesicular activity of the strongly metastatic Mat-LyLu cells being more developed than the corresponding weakly metastatic AT-2 cells. All the parameters studied were sensitive to tetrodotoxin (TTX) pre-treatment of the cells, which blocked voltage-gated Na⁺ channels (VGSCs). Some of the parameters had a simple dependence on VGSC activity, whereby pre-treatment with TTX reduced the values for the MAT-LyLu cells and eliminated the differences between the two cell lines. For other parameters, however, there was a complex dependence on VGSC activity. The possible physical/physiological meaning of the mathematical parameters studied and the nature of involvement of VGSC activity in control of endocytosis/secretion are discussed.     

The vacuole as central element of the lytic system and sink for lipid droplets in maturing appressoria ofMagnaporthe grisea

Summary Histochemical and ultrastructural studies were carried out on a wild-type strain (Guyll) and a melanin-deficient mutant(büβ) of the rice-blast pathogen,Magnaporthe grisea (=Pyricularia oryzae), in order to investigate the destination of lipid storage reserves during appressorium development. Lipid droplets were abundant in conidia and were mobilised upon germination, accumulating in the appressorial hook which developed at the tip of each germ tube. Following the formation of a septum at the base of the nascent appressorium, one or a few closely appressed central vacuoles became established and were observed to enlarge in the course of appressorium maturation. On unyielding artificial surfaces such as glass or plastic, appressoria matured to completion within 36–48 h, by which time the enlarged vacuole filled most of the inside volume of the appressorium. Light and transmission electron microscopical observations revealed that the lipid droplets entered the vacuole by autophagocytosis and were degraded therein. Histochemical approaches confirmed the vacuole as the key lytic element in maturing appressoria. Endocytosis of a vital dye, Neutral Red, progressed via endosomes which migrated into the vacuole and lysed there, releasing their dye content into the vacuolar lumen. Furthermore, activity of the lysosomal marker enzyme, acid phospho-monoesterase, was strongly localised in the vacuole at all stages of appressorium maturation. It is therefore envisaged that vacuoles are involved in the degradation of lipid storage reserves which may act as sources of energy and/or osmotically active metabolites such as glycerol, which generate the very high turgor pressure known to be crucial for penetration of hard surfaces. On softer surfaces such as onion epidermis, appressoria ofM. grisea were able to penetrate before degradation of lipid droplets had been completed.     

The convergence of endosomal and autophagosomal pathways: Implications for APP-CTF degradation

We have reported previously that autophagy is responsible for amyloid precursor protein-C-terminal fragment (APP-CTF) degradation and therefore Aβ clearance. To elucidate the underlying mechanism, using LC3 affinity purification and mass spectrometry analysis, immunoprecipitation (IP), as well as live imaging analysis, we identified and demonstrated that the adaptor-related protein complex 2 (AP2) and PICALM (phosphatidylinositol binding clathrin assembly protein) are in a complex with LC3 and APP-CTF. Taken together, this new set of data suggests that the AP2-PICALM complex functions as an autophagic cargo receptor for the recognition and shipment of APP-CTF from the endocytic pathway to the LC3-dependent autophagic degradation pathway. Interestingly this AP2-LC3 connection seems to be involved in chemically-induced APP-CTF clearance as we observed using the small compound SMER28. The effect observed following SMER28 was significantly reduced after silencing AP2. While more work is required to elucidate the detailed molecular mechanisms involved, our actual data suggest that there is some level of specificity in the steps mentioned above.     

Transport of protein toxins into cells: pathways used by ricin,cholera toxin and Shiga toxin

Ricin, cholera, and Shiga toxin belong to a family of protein toxins that enter the cytosol to exert their action. Since all three toxins are routed from the cell surface through the Golgi apparatus and to the endoplasmic reticulum (ER) before translocation to the cytosol, the toxins are used to study different endocytic pathways as well as the retrograde transport to the Golgi and the ER. The toxins can also be used as vectors to carry other proteins into the cells. Studies with protein toxins reveal that there are more pathways along the plasma membrane to ER route than originally believed.     

Accelerated internalization of junctional membrane proteins (connexin 43, N-cadherin and ZO-1) within endocytic vacuoles: An early event of DDT carcinogenicity

Stability of cell-to-cell interactions and integrity of junctional membrane proteins are essential for biological processes including cancer prevention. The present study shows that DDT, a non-genomic carcinogen used at a non-cytotoxic dose (1 μM), rapidly disrupted the cell-cell contacts and concomitantly induced the formation of cytoplasmic vacuoles close to the plasma membrane in the SerW3 Sertoli cell line. High-resolution deconvolution microscopy reveals that this vacuolization process was clathrin-dependent since a hyperosmotic media (0.2 M sucrose) blocked rhodamine-dextran endocytosis. In response to DDT, junctional proteins such as Cx43, N-Cadherin and ZO-1 were internalized and present in vacuoles. In Cx43-GFP transfected cells, time lapse videomicroscopy demonstrates that DDT rapidly enhanced fragmentation of the gap junction plaques and abolished the gap junction coupling without major modification of Cx43 phosphorylation status. Repeated exposure to DDT resulted in chronic gap junction coupling injury. The present results demonstrate that one of the early effect of DDT is to interfere with the plasma membrane and to perturb its function, specifically its ability to establish cell-cell junctions that are essential for tissue homeostasis and control of cell proliferation and differentiation. Such an alteration may play a specific role during carcinogenesis.     

Vacuolar pH oscillations in mesophyll cells accompany oscillations of photosynthesis in leaves: Interdependence of cellular compartments,and regulation of electron flow in photosynthesis

Oscillations of photosynthesis induced in leaves of Vicia faba L. were accompanied by oscillations not only in the pH of the chloroplast stroma, but also by pH oscillations in the cytosol and in the vacuole of leaf mesophyll cells. Cytosolic pH oscillations were in phase with stromal oscillations, but antiparallel to vacuolar pH oscillations. During maxima of photosynthesis, the cytosolic pH exhibited maxima and the vacuolar pH minima. Vacuolar acidification is interpreted to be the result of energized proton transport from the cytosol into the vacuole. Since the ratio of dihydroxyacetone phosphate to phosphoglycerate is maximal during the peaks of photosynthesis (Stitt et al., 1988, J. Plant Physiol. 133, 133–143; Laisk et al., 1991, Planta 185, 554–562), while the activity of NADP-malic dehydrogenase is highest during minima of photosynthesis (Scheibe and Stitt, 1988, Plant Physiol. Biochem. 26, 473–481), the present data indicate in agreement with earlier observations (Yin et al., 1991, Planta 184, 30–34) that light-dependent cytosolic energization is brought about by the oxidation of dihydroxyacetone phosphate rather than of malate. They also indicate that the over-reduction of the electrontransport chain observed during minima of photosynthesis is relieved not predominantly by oxaloacetate reduction and export of the resulting malate from the chloroplasts but by another reaction, presumably oxygen reduction.Abbreviations CDCF 5-(and 6-)carboxy-2,7-dichlorofluorescein     

Effects of d-allose on the endocytic activity of dendritic cells and the subsequent stimulation of T cells

We examined the effects of a rare sugar, d-allose, which is 6-carbon monosaccharide, on endocytosis and T cell stimulation by dendritic cells (DCs). The endocytosis of BCG-anti-BCG immune complexes by DCs markedly decreased in d-allose-containing medium. Co-culture with T cells (mixed leukocyte reaction, MLR) of DCs, which had been exposed to BCG in d-allose-supplemented medium, induced apoptosis of CD4⁺ T cells in a manner dependent on d-allose concentration. After the MLR, DCs cultured in the medium with d-allose expressed less CD40 and more Fas ligands than those cultured without d-allose. It was suggested that the functions of DCs, internalization, processing and the subsequent antigen presentation to T cells, are down-regulated via the action of d-allose.