Epidermal Growth Factor Induces Internalization But Not Degradation of the Epidermal Growth Factor Receptor in a Human Breast Cancer Cell Line

Abstract

Metabolism of the epidermal growth factor (EGF) receptor was studied in the MDA-MB-231 human breast cancer cell line. As in normal fibroblasts the EGF receptor from MDA-MB-231 cells was synthesized from a M_r =160,000 precursor and tunicamycin treatment of cells resulted in accumulation of a M_r =130,000 polypeptide. Unlike normal fibroblasts in which a M_r =170,000 mature form of the EGF receptor was found, MDA-MB-231 cells contained a M_r =172,000 mature form. Addition of EGF to MDA-MB-231 cells led to rapid internalization of EGF receptors, however, internalization did not affect receptor half-life and receptors did not recycle to the cell surface. EGF receptors could be visualized by immunofluorescence and remained sequestered in intracellular membranous structures following internalization. EGF was degraded slowly by MDA-MB-231 cells relative to degradation of EGF by normal cells. A high endogenous level of in vivo phosphorylation of threonine 654 of the EGF receptor was found in MDA-MB-231 cells and treatment of cells with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further stimulated phosphorylation of this residue. EGF induced receptor internalization resulted in dephosphorylation of threonine 654. The significance of these unusual properties of EGF receptor metabolism in MDA-MB-231 cells is discussed.     

Biotin-mediated delivery of exogenous macromolecules into soybean cells

We have demonstrated that attachment of biotin to a variety of macromolecules allows the uptake of those macromolecules into cultured soybean cells (Glycine max Merr cv Kent). Macromolecules that were nondestructively delivered into intact cells in large numbers (>10⁶/cell) by this technique include bovine insulin (M_r about 5,700), bovine ribonuclease (M_r about 14,000), human hemoglobin (M_r about 64,000), and bovine serum albumin (M_r about 68,000). It is hypothesized that this methodology may be useful for delivering antibodies, toxins, enzymes, and genetic material into living plant cells without requiring prior removal of the cell wall or infection with Agrobacterium.     

Identification and Purification of a Derepressible Alkaline Phosphatase from Anacystis nidulans R2

We have examined the increase in alkaline phosphatase activity in the cyanobacterium Anacystis nidulans R2 upon phosphate deprivation. Much of the activity is released into the medium when A. nidulans is osmotically shocked, indicating that the enzyme is located either in the periplasmic space or is loosely bound to the cell wall. The polypeptide associated with phosphatase activity has been identified as a single species of M_r 160,000. Several lines of evidence demonstrate that this polypeptide is responsible for alkaline phosphatase activity: (a) It is absent when cells are grown in the presence of phosphate and specifically accumulates during phosphate deprivation. (b) It is the major periplasmic polypeptide extracted by osmotic shock. (c) It represents over 90% of the protein in a fraction of periplasmic polypeptides enriched for phosphatase activity. (d) Antibodies raised against the purified species of M_r 160,000 inhibit phosphatase activity by approximately 70%.     

Identification of the cell surface and nuclear receptors for NGF in a breast carcinoma cell line

¹²⁵I-nerve growth factor (NGF) was found to be internalized and translocated to the nucleus of SKBr5 breast carcinoma cells. The cytoplasm and chromatin isolated from nonmitotic cells accumulated two-and five-fold, respectively, more of ¹²⁵I-NGF than the cells undergoing mitosis. MAb 20.4 developed against the NGF cell surface receptor immunoprecipitated the 80,000 M_r receptor from plasma membrane and two protein species from the chromatin; 90,000 M_r (major band) and 200,000 M_r (minor band). In SKBr5 cells, binding of NGF to the chromatin did not affect synthesis of rRNA. Proliferation of SKBr5 cells was slightly stimulated by NGF. In control melanoma A875 cells, which express the 230,000 M_r chromatin receptor, NGF inhibited both rRNA synthesis and cell proliferation. We suggest that the 90,000 M_r chromatin receptor expressed by SKBr5 cells represents a “nonactive”, ligand-binding subunit of the high molecular weight receptor for NGF. The critical role of the chromatin receptor for NGF in rRNA-dependent cell proliferation is discussed. © 1993 Wiley-Liss, Inc.     

Internalization of the Hyaluronan Receptor CD44 by Chondrocytes

Chondrocytes express CD44 as a primary receptor for the matrix macromolecule hyaluronan. Hyaluronan is responsible for the retention and organization of proteoglycan within cartilage, and hyaluronan-chondrocyte interactions are important for the assembly and maintenance of the cartilage matrix. Bovine articular chondrocytes were used to study the endocytosis and turnover of CD44 and the effects of receptor occupancy on this turnover. Matrix-intact chondrocytes exhibit approximately a 6% internalization of cell surface CD44 by 4 h. Treatment with Streptomyces hyaluronidase to remove endogenous pericellular matrix increased internalization to approximately 20% of cell surface CD44 at 4 h. This turnover could be partially inhibited by the addition of exogenous hyaluronan to these matrix-depleted chondrocytes. Cell surface biotin-labeled CD44 was internalized by chondrocytes and this internalization was decreased in the presence of hyaluronan. Colocalization of internalized CD44 and fluorescein-labeled hyaluronan in intracellular vesicles correlates with the previous results of receptor-mediated endocytosis pathway for the degradation of hyaluronan by acid hydrolases. Taken together, our results indicate that CD44 is internalized by chondrocytes and that CD44 turnover is modulated by occupancy with hyaluronan.     

Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes

Epimastigote forms of Trypanosoma cruzi (the etiologic agent of Chagas disease) internalize and store extracellular macromolecules in lysosome-related organelles (LROs) called reservosomes, which are positive for the cysteine protease cruzipain. Despite the importance of endocytosis for cell proliferation, macromolecule internalization remains poorly understood in the most clinically relevant proliferative form, the intracellular amastigotes found in mammalian hosts. The main obstacle was the lack of a simple method to isolate viable intracellular amastigotes from host cells. In this work we describe the fast and efficient isolation of viable intracellular amastigotes by nitrogen decompression (cavitation), which allowed the analysis of amastigote endocytosis, with direct visualization of internalized cargo inside the cells. The method routinely yielded 5x10⁷ amastigotes—with typical shape and positive for the amastigote marker Ssp4—from 5x10⁶ infected Vero cells (48h post-infection). We could visualize the endocytosis of fluorescently-labeled transferrin and albumin by isolated intracellular amastigotes using immunofluorescence microscopy; however, only transferrin endocytosis was detected by flow cytometry (and was also analyzed by western blotting), suggesting that amastigotes internalized relatively low levels of albumin. Transferrin binding to the surface of amastigotes (at 4°C) and its uptake (at 37°C) were confirmed by binding dissociation assays using acetic acid. Importantly, both transferrin and albumin co-localized with cruzipain in amastigote LROs. Our data show that isolated T. cruzi intracellular amastigotes actively ingest macromolecules from the environment and store them in cruzipain-positive LROs functionally related to epimastigote reservosomes.     

Formation of dissociated enzyme subunits by chemical treatment during renaturation

When iodoacetate is added to denatured muscle aldolase undergoing renaturation, a major portion of the activity in the resulting enzyme remains in the monomeric form (of about 37,000 M_r). In the absence of iodoacetate, the renatured enzyme exists entirely as the tetramer. Iodoacetate treatment of native aldolase tetramer (M_r = 160,000) does not lead to dissociation. The stabilization of the monomer by iodoacetate treatment is presumably due to modification of a group at the intersubunit region. Active monomers of aldolase could be distinguished from native or renatured aldolase tetramer by gel-filtration and by the sensitivity of the monomer to inactivation in 2.3 m-urea.     

The effect of staurosporine, a protein kinase inhibitor, on asialoglycoprotein receptor endocytosis

Receptor-mediated endocytosis via coated pits is modulated by the activity of protein kinases and protein phosphorylation. We examined the effects of the potent protein kinase inhibitor staurosporine (SSP) on endocytosis of the asialoglycoprotein (ASGP) receptor in HepG2 cells. Staurosporine caused a rapid (<2 min) inhibition of ligand internalization from the cell surface. In contrast the rate of receptor exocytosis from intracellular compartments to the cell surface was not altered (t_1/2 = 8 min). This resulted in increased ASGP receptors at the plasma membrane (140% of control) while the total number of receptors per cell was unchanged. Receptor up-regulation was half-maximal at 30 nM SSP. At this concentration staurosporine also inhibited the internalization of iodinated transferrin by HepG2 cells and SK Hep-1 cells, another human hepatoma-derived cell line. Staurosporine was without effect on the non-receptor-mediated uptake of Lucifer yellow by pinocytosis. We investigated the possible involvement of protein kinase C in the inhibitory effects of staurosporine on receptor endocytosis. The active protein kinase C inhibitor H7 did not inhibit ASGP receptor internalization. Furthermore depletion of cellular protein kinase C by overnight incubation with 1 μM phorbol myristate acetate did not abrogate the SSP effect. Together these data suggest that the mechanism of SSP action is independent of the inhibition of protein kinase C. In conclusion staurosporine is a potent and rapid inhibitor of receptor trafficking which is specific for receptor internalization from the plasma membrane.     

Streptococcus pneumoniae Invades Endothelial Host Cells via Multiple Pathways and Is Killed in a Lysosome Dependent Manner

Streptococcus pneumoniae is one of the major causative agents of pneumonia, sepsis, meningitis and other morbidities. In spite of its heavy disease burden, surprisingly little is known about the mechanisms involved in the switch of life style, from commensal colonizer of the nasopharynx to invasive pathogen. In vitro experiments, and mouse models have shown that S. pneumoniae can be internalized by host cells, which coupled with intracellular vesicle transport through the cells, i.e. transcytosis, is suggested to be the first step of invasive disease. To further dissect the process of S. pneumoniae internalization, we chemically inhibited discrete parts of the cellular uptake system. We show that this invasion of the host cells was facilitated via both clathrin- and caveolae-mediated endocytosis. After internalization we demonstrated that the bulk of the internalized S. pneumoniae was killed in the lysosome. Interestingly, inhibition of the lysosome altered transcytosis dynamics as it resulted in an increase in the transport of the internalized bacteria out of the cells via the basal side. These results show that uptake of S. pneumoniae into host cells occurs via multiple pathways, as opposed to the often proposed view of invasion being dependent on specific, and singular receptor-mediated endocytosis. This indicates that the endothelium not only has a critical role as a physical barrier against S. pneumoniae in the blood stream, but also in degrading S. pneumonia cells that have adhered to, and invaded the endothelial cells.     

Lysosomal Trafficking of TGFBIp via Caveolae-Mediated Endocytosis

Transforming growth factor-beta-induced protein (TGFBIp) is ubiquitously expressed in the extracellular matrix (ECM) of various tissues and cell lines. Progressive accumulation of mutant TGFBIp is directly involved in the pathogenesis of TGFBI-linked corneal dystrophy. Recent studies reported that mutant TGFBIp accumulates in cells; however, the trafficking of TGFBIp is poorly understood. Therefore, we investigated TGFBIp trafficking to determine the route of its internalization and secretion and to elucidate its roles in the pathogenesis of granular corneal dystrophy type 2 (GCD2). Our data indicate that newly synthesized TGFBIp was secreted via the endoplasmic reticulum/Golgi-dependent secretory pathway, and this secretion was delayed in the corneal fibroblasts of patients with GCD2. We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation. In addition, the proteasome inhibitor MG132 inhibits the endocytosis of TGFBIp. Co-immunoprecipitation revealed that TGFBIp interacted with integrin α_Vβ₃. Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp. These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy.     

The effects of chronic ethanol administration on the rates of internalization of various ligands during hepatic endocytosis.

The purpose of the present study was to further characterize the ethanol-induced impairments in hepatic endocytosis. Specifically, we examined the effects of ethanol treatment on receptor-ligand internalization via the coated and noncoated pit pathways. Insulin, epidermal growth factor (EGF) and asialoorosomucoid (ASOR) were used as model ligands to study internalization by isolated hepatocytes. ASOR and EGF are thought to be internalized strictly in coated pit regions of the cell membrane, while insulin may be internalized in both coated and uncoated membrane regions. Ethanol administration for 5-7 weeks decreased internalization of ASOR and EGF while internalization of insulin was unchanged during a single round of endocytosis of surface-bound ligand. Similarly, a more quantitative measure of endocytosis, the endocytic rate constant, was decreased for EGF and ASOR but not for insulin in livers of experimental rats. When endocytosis of Lucifer yellow, a fluorescent dye known to be internalized in the cell by fluid-phase endocytosis was examined, the initial rates of dye uptake were not significantly altered by alcohol administration. These results indicate that ethanol may selectively impair internalization occurring by coated pits while it has a minimal effect on initial uptake of molecules which are internalized by noncoated membrane regions.     

Systems Biological Analysis of Epidermal Growth Factor Receptor Internalization Dynamics for Altered Receptor Levels

Epidermal growth factor (EGF) receptor (EGFR) overexpression is a hallmark of many cancers. EGFR endocytosis is a critical step in signal attenuation, raising the question of how receptor expression levels affect the internalization process. Here we combined quantitative experimental and mathematical modeling approaches to investigate the role of the EGFR expression level on the rate of receptor internalization. Using tetramethylrhodamine-labeled EGF, we established assays for quantifying EGF-triggered EGFR internalization by both high resolution confocal microscopy and flow cytometry. We determined that the flow cytometry approach was more sensitive for examining large populations of cells. Mathematical modeling was used to investigate the relationship between EGF internalization kinetics, EGFR expression, and internalization machinery. We predicted that the standard parameter used to assess internalization kinetics, the temporal evolution r(t) of the ratio of internalized versus surface-located ligand·receptor complexes, does not describe a straight line, as proposed previously. Instead, a convex or concave curve occurs depending on whether initial receptor numbers or internalization adaptors are limiting the uptake reaction, respectively. To test model predictions, we measured EGF-EGFR binding and internalization in cells expressing different levels of green fluorescent protein-EGFR. As expected, surface binding of rhodamine-labeled EGF increased with green fluorescent protein-EGFR expression level. Unexpectedly, internalization of ligand· receptor complexes increased linearly with increasing receptor expression level, suggesting that receptors and not internalization adaptors were limiting the uptake in our experimental model. Finally, determining the ratio of internalized versus surface-located ligand·receptor complexes for this cell line confirmed that it follows a convex curve, supporting our model predictions.The epidermal growth factor receptor (EGFR)³ belongs to the family of transmembrane receptor tyrosine kinases and mediates diverse actions, including proliferation, differentiation, and apoptosis (1, 2). Overexpression and/or mutations of the EGFR occur in ∼40% of neoblastomas (3) and correlate with poor prognosis (4–6). Unstimulated EGFR is located at the plasma membrane as a monomer and pre-formed dimer (7). Upon ligand binding, EGFR forms a dimer, and trans-phosphorylation occurs at specific residues of the cytoplasmic domain (8). Phosphorylated EGFR recruits adaptor proteins from which different conserved signaling pathways are activated, namely the MAPK (9), phosphatidylinositol 3-kinase, and protein kinase C pathways (10).Furthermore, activated EGFR recruits various adaptor proteins that mediate receptor internalization by endocytosis (2). Endocytosis occurs via the recruitment of adaptor proteins to phosphorylated tyrosine residues of the receptor and formation of membrane invaginations, which eventually pinch off to form internalized early endosomes (2, 11) (see Fig. 1). Both constitutive endocytosis and ligand-induced EGFR endocytosis are critical events in EGF signal regulation (2, 12). Endosomal EGFR can be transited back to the plasma membrane or to the late endosome/lysosome for degradation (2). As the majority of internalized receptors are targeted for lysosomal degradation upon EGF stimulation (13), endocytic entry of active EGFR is a crucial step for signal attenuation, which is also highlighted by the findings that impaired or delayed internalization is highly oncogenic (14, 15).Open in a separate windowFIGURE 1.Scheme of ligand-induced internalization. EGF binds membrane-located EGFR to give rise to surface-bound EGF·EGFR complex RE_s. Via diffusion events, the activated receptor binds internalization adaptors IC, which leads to internalized receptors R_i.In light of the role of endocytosis in EGFR signal attenuation and the oncogenicity of EGFR overexpression, it is important to elucidate the relationship between high receptor expression levels relative to internalization pathway capacity and their effect on internalization dynamics.Mathematical modeling is an important tool in elucidating EGFR signaling, at the level of EGFR internalization (16–19) and, more recently, at the level of the integration of input signals into signaling events downstream of the EGFR, such as the MAPK cascade (20, 21). In earlier models, pioneering concepts such as the nonlinearity of the uptake reaction, because of the existence of alternative pathways that are entered with different affinities, were developed (16, 19). Also, the notion of saturability of the EGFR endocytosis system, in contrast to internalization of the transferrin receptor, for example, was introduced (18).Importantly, in mathematical formulations of EGFR endocytosis, the standard parameter used to estimate the rate of the internalization step (16) and to assess the effect of certain perturbations on internalization (22–24) is the temporal evolution of the ratio of internalized versus surface-located ligand·receptor complexes r(t). In Refs. 16, 17, it was mathematically determined that, under certain assumptions, this ratio describes a straight line with the slope corresponding to the rate of the internalization step. These assumptions were as follows: (i) that the number of surface-bound ligand·receptor complexes (RE_s) remains approximately constant during the measurements, and (ii) that the internalization step is a first-order process, i.e. it is directly proportional to RE_s and independent of a potentially limiting availability of internalization adaptors.The presence of multiple endocytotic routes (23, 25) and different EGFR affinities for EGF (26) argue against first-order kinetics. Moreover, the possible limited capacity of internalization adaptors may restrict EGFR internalization in cells expressing abnormally high numbers of EGFR (18). In this work we investigated the potential of EGFR internalization to occur as a nonlinear process by combining mathematical modeling with novel quantitative, live cell measurements of EGF internalization.We extended the previous derivation of the ratio of internalized versus surface-located ligand·receptor complexes r(t) (16, 17, 19) by eliminating above assumptions i and ii, which allowed us to investigate in silico different scenarios for the shape of r(t) as a function of the relative concentrations of EGFR and internalization adaptors. We predicted that r(t) is not a straight line as derived previously but is a convex or concave curve depending on whether receptors or internalization components are limiting the reaction, respectively.In earlier studies, quantitative measurements of parameters of EGFR endocytosis have been performed using classical biochemical techniques to detect cellular ligand uptake using radioactively labeled EGF (16, 24, 27) or biotin-labeled EGF (28). Importantly, both methods do not reach single cell precision and instead yield an integrated signal over a population of cells. To test our mathematical predictions we combined the following: (i) quantitative laser scanning confocal microscopy, and (ii) multiple parametric flow cytometry, using a custom Beckman Coulter FC500 equipped with a 488 and 561 nm laser excitation, to quantitatively measure the temporal and spatial dynamics of EGFR endocytosis using tetramethylrhodamine-tagged EGF (Rh-EGF) and GFP-EGFR. We show that both quantitative imaging and flow cytometry measurements were highly sensitive, allowing for live cell investigations and confirmation of the mathematical predictions.     

Internalization of α-synuclein oligomers into SH-SY5Y cells

Aggregates of misfolded α-synuclein are a distinctive feature of Parkinson’s disease. Small oligomers of α-synuclein are thought to be an important neurotoxic agent, and α-synuclein aggregates exhibit prion-like behavior, propagating misfolding between cells. α-Synuclein is internalized by both passive diffusion and active uptake mechanisms, but how uptake varies with the size of the oligomer is less clear. We explored how α-synuclein internalization into live SH-SY5Y cells varied with oligomer size by comparing the uptake of fluorescently labeled monomers to that of engineered tandem dimers and tetramers. We found that these α-synuclein constructs were internalized primarily through endocytosis. Oligomer size had little effect on their internalization pathway, whether they were added individually or together. Measurements of co-localization of the α-synuclein constructs with fluorescent markers for early endosomes and lysosomes showed that most of the α-synuclein entered endocytic compartments, in which they were probably degraded. Treatment of the cells with the Pitstop inhibitor suggested that most of the oligomers were internalized by the clathrin-mediated pathway.     

P. Falciparum infected erythrocytes are capable of endocytosis

Summary P. falciparum, an intraerythrocytic parasite, obtains nourishment primarily through phagocytosis of the host cytosol but also through the incorporation of extracellular small molecules which enter through the parasitized red cell's membrane via pores. Normal mature erythrocytes are incapable of endocytosis. Several lines of evidence suggest that extracellular large molecules may be taken up when the mature red cell is parasitized byP. falciparum, but direct evidence has been lacking. We now report the use of ferritin, an electron dense protein, to demonstrate endocytosis inP. falciparum infected red cells. Parasitized red cells incubated with ferritin internalize that macromolecule as demonstrated by electron microscopy. While normal red cells incubated with ferritin took up none of the tracer molecule, parasitized red cells internalized substantial amounts. In addition both ferritin and apoferritin inhibited the growth ofP. falciparum in a dose dependent fashion, again indicating endocytosis of a macromolecule. These data indicate thatP. falciparum can somehow stimulate the mature erythrocyte to engage in endocytosis. We also note that both infected and non-infected red cells in a culture in whichP. falciparum is growing become abnormally sticky for ferritin. Moreover, parasitized red cells bind I¹²⁵-transferrin while non-parasitized erythrocytes do not. These observations suggest that a soluble parasite product alters the red cell membrane in a non-global manner, causing selective effects in relation to different proteins.     

Drug Uptake,Lipid Rafts,and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast

The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane.     

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLa^K44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.     

Subunit structure of three glyceraldehyde 3-phosphate dehydrogenases of some flowering plants.

A procedure is described for the purification of three glyceraldehyde phosphate dehydrogenases from a batch of beet leaves. Glyceraldehyde 3-phosphate:NADP⁺ reductase, nonphosphorylating (EC 1.2.1.9) has been purified over 1500-fold. The M_r of this enzyme is 190,000 and its subunits have an M_r of 53,000, suggesting a tetramer as the active form. Its pI is 6.0. Cytosolic glyceraldehyde 3-phosphate dehydrogenase, NAD dependent (EC 1.2.1.12), has an M_r of 145,000 and subunits of M_r 37,000. It is dissociated to inactive dimers by ATP, whereas NAD⁺ in the presence of reductant promotes its reactivation. The amino acid composition is related to glyceraldehyde 3-phosphate dehydrogenases from animal sources and is most similar to pea seed glyceraldehyde 3-phosphate dehydrogenase. The enzyme exhibits a range of pI values from 5 to 7, but a second electrofocusing in the presence of dithioerythritol results in a single main form with pI 5.33, consistent with the behavior in polyacrylamide and cellulose acetate gel electrophoresis. Chloroplast NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) has been obtained from beet, pea, Ranunculus, Arum, and maize leaves. The stable form is an oligomer of about 800,000 M_r (±10%), while a minor, possibly damaged fraction elutes as a retarded peak from agarose columns. The M_r 800,000 form is reversibly dissociated to protomers of M_r 160,000 by NADP⁺, with increase of apparent NADP-dependent activity. Two subunits are present in similar amounts in all association states and after all treatments: α with M_r 36,000, and β with M_r 41,000. The form found in density gradient ultracentrifugation has an M_r of 390,000. Isoelectric points of the various forms lie between pH 4.1 and 4.7 for all species, with a main peak usually at pI 4.45. The amino acid composition of beet chloroplast glyceraldehyde phosphate dehydrogenase is not closely related to that of beet leaf NAD-dependent glyceraldehyde 3-phosphate dehydrogenase.     

Spatial organization of digestion,secretory mechanisms and digestive enzyme properties in Pheropsophus aequinoctialis (Coleoptera: Carabidae)

Aminopeptidase (soluble form M_r 110,000), carboxypeptidase A (soluble form M_r 47,000), maltase (a dimer composed of two identical M_r 60,000 subunits) and trypsin (two charge isomers with M_r 34,000) are found in major amounts in the crop and midgut tissue, whereas amylase (a trimer of three identical M_r 18,000 subunits) and cellobiase (a trimer of three identical M_r 27,000 subunits) occur mainly in the crop and midgut contents. Subcellular fractions of midgut cells were obtained by conventional homogenization, followed by differential centrifugation or differential calcium precipitation. The results suggest that part of the aminopeptidase and carboxypeptidase A activity is bound to microvilli, that major amounts of trypsin and maltase are trapped in the cell glycocalyx and finally that soluble aminopeptidase, amylase and cellobiase occur in intracellular vesicles. The data support the hypothesis that most protein and carbohydrate digestion takes place in the crop under the action of enzymes passed forward from the midgut, after being secreted by exocytosis. Nevertheless, part of the intermediate and final digestion occurs at the surface of the midgut cells. The peculiar features of the digestion of P. aequinoctialis beetles, including their partly fluid peritrophic membranes, are thought to be derived from putative Coleoptera ancestors.     

Cellular Distribution of Calmodulin and Calmodulin-Binding Proteins in Vicia faba L.

The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. ¹²⁵I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with M_r 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (M_r 39,000, 88,000), stems (M_r 45,000, 60,000, 64,000), and roots (M_r 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.     

The malonate decarboxylase enzyme system of Malonomonas rubra: evidence for the cytoplasmic location of the biotin-containing component

Malonate decarboxylase of Malonomonas rubra is a complex enzyme system involving cytoplasmic and membrane-bound components. One of these is a biotin-containing protein of M_r 120'000, the location of which in the cytoplasm was deduced from the following criteria: (i) If the cytoplasm was incubated with avidin and the malonate decarboxylase subsequently completed with the membrane fraction the decarboxylase activity was abolished. The corresponding incubation of the membrane with avidin, however, was without effect. (ii) Western blot analysis identified the single biotin-containing polypeptide of M_r 120'000 within the cytoplasm. (iii) Transmission electron micrographs of immuno-gold labeled M. rubra cells clearly showed the location of the biotinyl protein within the cytoplasm, whereas the same procedure with Propionigenium modestum cells indicated the location of the biotin enzyme methylmalonyl-CoA decarboxylase in the cell membrane. The biotin-containing protein of the M. rubra malonate decarboxylase enzyme system was not retained by monomeric avidin-Sepharose columns but could be isolated with this column in a catalytically inactive form in the presence of detergents. If the high binding affinity of tetrameric avidin towards biotin was reduced by destructing part of the tryptophan residues by irradiation or oxidation with periodate, the inhibition of malonate decarboxylase by the modified avidin was partially reversed with an excess of biotin. Attempts to purify the biotin protein in its catalytically active state using modified avidin columns were without success.