scholarly journals Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. II. Internalization of proteins.

1979 ◽  
Vol 82 (1) ◽  
pp. 45-56 ◽  
Author(s):  
M Willinger ◽  
N Gonatas ◽  
F R Frankel
Keyword(s):  
Plasma Membrane ◽  
Polymorphonuclear Leukocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Proteins ◽  
Cell Protein ◽  
Resting Cells ◽  
Experimental Conditions ◽  
Particle Uptake ◽  
Differentiated Cells ◽  
Free Iodine

The distribution of surface proteins during phagocytosis by rabbit peritoneal polymorphonuclear leukocytes was studied to determine whether the proteins of the phagocytic vesicles of these differentiated cells were representative of the entire set of plasma membrane proteins. Phagocytosis of bovine serum albumin-diisodecylphthalate emulsion by lactoperoxidase-iodinated rabbit neutrophils was linear over 15-20 min at a rate of 96 microgram oil/min/mg cell protein. This rate was similar to that of unlabeled cells. Incorporation of cell-associated free iodine by endogenous myeloperoxidase during phagocytosis was inhibited by 1 mM cyanide, which had no effect on the rate of particle uptake. The surface of intact neutrophils contained at least 13 iodinated proteins distinguishable by polyacrylamide gel electrophoresis followed by autoradiography. Isolated phagosomes were deficient in six of these proteins. The plasma membrane fraction of these cells was missing five of these same proteins which, however, were enriched in a dense surface fraction (Willinger, M., and F. R. Frankel. J. Cell Biol. 82: 32-44). When experimental conditions were reversed, and the PMNs were labeled after phagocytosis, these five proteins remained on the cell surface, while at least three of the major proteins found on resting cells were depleted. Incubating the cells with colchicine, which has been shown to affect the distribution of some plasma membrane constituents during phagocytosis, had no effect on the distribution of surface proteins in our system. These results indicate that a nonrandom interiorization of lactoperoxidase-labeled surface proteins of polymorphonuclear leukocytes occurs during phagocytosis.

scholarly journals Quantitative proteomics of MDCK cells identify unrecognized roles of clathrin adaptor AP-1 in polarized distribution of surface proteins

2019 ◽  
pp. 201821076 ◽  
Author(s):  
Paulo S. Caceres ◽  
Diego Gravotta ◽  
Patrick J. Zager ◽  
Noah Dephoure ◽  
Enrique Rodriguez-Boulan
Keyword(s):  
Plasma Membrane ◽  
Isotope Labeling ◽  
Mdck Cells ◽  
Current Model ◽  
Large Population ◽  
Adaptor Proteins ◽  
Surface Proteins ◽  
Experimental Conditions ◽  
Large Populations ◽  
Clathrin Adaptor

The current model of polarized plasma membrane protein sorting in epithelial cells has been largely generated on the basis of experiments characterizing the polarized distribution of a relatively small number of overexpressed model proteins under various experimental conditions. Thus, the possibility exists that alternative roles of various types of sorting machinery may have been underestimated or missed. Here, we utilize domain-selective surface biotinylation combined with stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry to quantitatively define large populations of apical and basolateral surface proteins in Madin-Darby canine kidney (MDCK) cells. We identified 313 plasma membrane proteins, of which 38% were apical, 51% were basolateral, and 11% were nonpolar. Silencing of clathrin adaptor proteins (AP) AP-1A, AP-1B, or both caused redistribution of basolateral proteins as expected but also, of a large population of apical proteins. Consistent with their previously reported ability to compensate for one another, the strongest loss of polarity was observed when we silenced AP-1A and AP-1B simultaneously. We found stronger evidence of compensation in the apical pathway compared with the basolateral pathway. Surprisingly, we also found subgroups of proteins that were affected after silencing just one adaptor, indicating previously unrecognized independent roles for AP-1A and AP-1B. While AP-1B silencing mainly affected basolateral polarity, AP-1A silencing seemed to cause comparable loss of apical and basolateral polarity. Our results uncover previously overlooked roles of AP-1 in polarized distribution of apical and basolateral proteins and introduce surface proteomics as a method to examine mechanisms of polarization with a depth not possible until now.

scholarly journals Molecular mechanism of Fast Endophilin-Mediated Endocytosis

2020 ◽  
Vol 477 (12) ◽  
pp. 2327-2345 ◽  
Author(s):  
Alessandra Casamento ◽  
Emmanuel Boucrot
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Molecular Mechanism ◽  
Receptor Activation ◽  
Membrane Curvature ◽  
Surface Proteins ◽  
Resting Cells ◽  
Cellular Functions ◽  
Surface Receptors ◽  
Key Steps

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Clathrin-mediated endocytosis (CME) is the housekeeping pathway in resting cells but additional Clathrin-independent endocytic (CIE) routes, including Fast Endophilin-Mediated Endocytosis (FEME), internalize specific cargoes and support diverse cellular functions. FEME is part of the Dynamin-dependent subgroup of CIE pathways. Here, we review our current understanding of the molecular mechanism of FEME. Key steps are: (i) priming, (ii) cargo selection, (iii) membrane curvature and carrier formation, (iv) membrane scission and (v) cytosolic transport. All steps are controlled by regulatory mechanisms mediated by phosphoinositides and by kinases such as Src, LRRK2, Cdk5 and GSK3β. A key feature of FEME is that it is not constitutively active but triggered upon the stimulation of selected cell surface receptors by their ligands. In resting cells, there is a priming cycle that concentrates Endophilin into clusters on discrete locations of the plasma membrane. In the absence of receptor activation, the patches quickly abort and new cycles are initiated nearby, constantly priming the plasma membrane for FEME. Upon activation, receptors are swiftly sorted into pre-existing Endophilin clusters, which then bud to form FEME carriers within 10 s. We summarize the hallmarks of FEME and the techniques and assays required to identify it. Next, we review similarities and differences with other CIE pathways and proposed cargoes that may use FEME to enter cells. Finally, we submit pending questions and future milestones and discuss the exciting perspectives that targeting FEME may boost treatments against cancer and neurodegenerative diseases.

scholarly journals Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. I. Identification of surface proteins.

1979 ◽  
Vol 82 (1) ◽  
pp. 32-44 ◽  
Author(s):  
M Willinger ◽  
F R Frankel
Keyword(s):  
Gel Electrophoresis ◽  
Polymorphonuclear Leukocytes ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Surface Proteins ◽  
Intact Cells ◽  
Dense Fraction ◽  
Protease Treatment ◽  
Neutrophil Cell

To study the fate of external membrane proteins during phagocytosis, rabbit peritoneal neutrophils were labeled by enzymatic iodination. Iodine was incorporated into at least 13 proteins ranging in size from approximately 250,000 to 18,000 daltons as judged from autoradiography of gels after SDS-polyacrylamide gel electrophoresis of labeled cells. The major contractile proteins of neutrophils, actin and myosin, were not labeled when intact cells were iodinated but were labeled when homogenates of these cells were iodinated. Nine of the iodinated proteins were released by mild protease treatment of intact cells. A plasma membrane-rich fraction was isolated by density centrifugation. This fraction was enriched at least 10-fold for lactoperoxidase-labeled acid-insoluble proteins. It was enriched to the same extent for the presence of iodinated wheat germ agglutinin that had been bound to intact cells at 4 degrees C before homogenization. Analysis of SDS-polyacrylamide gel electrophoresis revealed that the proteins of this fraction were predominantly of high molecular weight. However, only 8 of the 13 proteins iodinated on intact cells were found in this fraction. The remaining five were enriched in a dense fraction containing nuclei, intact cells, and membranous vesicles, and may represent a specialized segment of the neutrophil cell surface.

scholarly journals Increase in the concentration of major boar sperm surface proteins during maturation in the epididymis

1986 ◽  
Vol 82 (1) ◽  
pp. 295-308
Author(s):  
N.K. Saxena ◽  
N. Saxena ◽  
W. Hunt ◽  
R.N. Peterson ◽  
L. Henry ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Protein ◽  
Surface Proteins ◽  
Secretory Cells ◽  
Boar Sperm ◽  
Sperm Plasma Membrane ◽  
Antigen Localization ◽  
Polyclonal Antisera ◽  
Narrow Area

High-resolution two-dimensional polyacrylamide gel electrophoresis analyses have indicated that several major boar sperm plasma membrane polypeptides (PMPs) increased in concentration during maturation in the epididymis. To investigate this further, monoclonal antibodies (MAbs) to two of these PMPs/glycoproteins referenced as 4.85 and 5.0 and polyclonal antisera (PCA) raised against PMP 5.0 were used to quantify these changes. ELISA assays for both PMPs, using solubilized plasma membrane (PM) and intact PM as antigens, indicated that both PMPs were present in greater concentration in cauda PM than in caput PM. ELISA assay using PCA against 5.0 and whole sperm from the cauda and caput epididymis also showed an increase in the concentration of this protein during transit through the epididymis. Indirect FITC fluorescence microscopy showed that MAbs reacted specifically with the luminal aspect of secretory cells of the corpus epididymis and, with greater intensity, with secretory cells of the cauda segment. When MAb tags were used the fluorescence technique indicated that PMP 5.0 was restricted to a narrow area of PM overlying the principal segment of the head of caput sperm but fluorescence extended further into the principal segment and into the flagellum of cauda PM. Since MAb binding sites appear to be partially masked, PCA were also used to localize antigenic sites. PCA to PMP 5.0 showed antigen localization in both head and flagellar PM of cauda and caput sperm. The fluorescence of cauda sperm PM, however, was significantly more intense than that of caput sperm PM. MAb to PMP 4.85 also bound to antigen more extensively on cauda sperm. The sum of these analyses suggests that major sperm PMPs are secreted by the epididymis and absorbed by sperm. The magnitude of surface protein changes occurring during epididymal transit in boar sperm appears to be greater than heretofore recognized.

scholarly journals High-throughput living-cell protein crosslinking analysis uncovers the physiological relevance of forming the “inserted” state of the ATP synthase ε-subunit

2019 ◽  
Author(s):  
Yang Liu ◽  
Jiayu Yu ◽  
Mengyuan Wang ◽  
Qingfang Zeng ◽  
Xinmiao Fu ◽  
...  
Keyword(s):  
Atp Synthase ◽  
High Throughput ◽  
Living Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Unnatural Amino Acid ◽  
Fine Tuning ◽  
Cell Protein ◽  
Experimental Conditions ◽  
Physiological Relevance ◽  
In Cells

AbstractATP synthase, a highly conserved multi-subunit enzyme complex having a common stoichiometry of α3β3γδεab2c8-15, functions to supply ATP as the universal energy currency for cells. It comprises of the peripheral F1 sector (α3β3γδε) and the membrane-integrated Fo sector (ab2c8-15). In vitro structural analyses revealed that the C-terminal domain of the ε-subunit could adopt either an “inserted” or “non-inserted” state (with or without interacting with the α/β-subunits), with the former being viewed as inhibitory for the ATP hydrolysis activity of ATP synthase. Nevertheless, as common in current protein researches, the physiological relevance of such an “inserted” state for ATP synthase functioning is hardly known. To decipher this, designed an unnatural amino acid-mediated living-cell protein photocrosslinking analysis pipeline by developing the scarless genome-targeted site-directed mutagenesis and the high-throughput gel polyacrylamide gel electrophoresis (HT-PAGE) techniques. Employing this powerful approach, we systematically examined the interactions involving the C-terminal helix of the ε-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the “inserted” and “non-inserted” states of the ε-subunit exist as an equilibrium in cells cultured under common experimental conditions, shifting to the former upon the appearance of unfavorable conditions, acting as a low-gear state to strengthen the ATP synthesis function. Such a fine-tuning mechanism allows the ATP synthase to reversibly and instantly switch between two functional states. Further, the two powerful techniques that we developed here might be applied to many aspects of protein researches.

scholarly journals Analysis with specific polyclonal antiserum indicates that the E1A-associated 300-kDa product is a stable nuclear phosphoprotein that undergoes cell cycle phase-specific modification.

1991 ◽  
Vol 11 (11) ◽  
pp. 5389-5397 ◽  
Author(s):  
P Yaciuk ◽  
E Moran
Keyword(s):  
Cell Cycle ◽  
Cell Function ◽  
Rat Kidney ◽  
Sodium Dodecyl ◽  
Polyclonal Antiserum ◽  
Cell Protein ◽  
Cycle Phase ◽  
Resting Cells ◽  
Kidney Cells ◽  
Nuclear Phosphoprotein

Binding of a 300-kDa host cell protein (p300) is tightly correlated with the ability of the adenovirus E1A products to induce quiescent baby rat kidney cells to proliferate. We have generated rabbit polyclonal antibodies against p300 to characterize this protein further. We have found p300 to be a nuclear phosphoprotein that is actively synthesized in both quiescent and proliferating baby rat kidney cells. In partially purified mitotic cell populations, we observe a form of p300 with decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels that shares a nearly identical partial proteolytic digest pattern with p300. The slower-migrating form of p300 is greatly reduced by treating immune complexes with potato acid phosphatase. The relative stability and presence of p300 even in resting cells suggests that p300 has a basal cell function, but the appearance of differentially modified forms during the cell cycle suggests the possibility that p300 function is modulated specifically in growing cells.

scholarly journals Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles

1996 ◽  
Vol 316 (3) ◽  
pp. 999-1004 ◽  
Author(s):  
Lorella PASCOLO ◽  
Savino DEL VECCHIO ◽  
Ronald K. KOEHLER ◽  
J. Enrique BAYON ◽  
Cecile C. WEBSTER ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Experimental Conditions ◽  
Saturation Kinetics ◽  
Basolateral Plasma Membrane ◽  
Component C ◽  
Uptake Studies

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB–HSA affinity constants (K´f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 μM, K´f (litre/mol) was inversely related to [HSA], irrespective of the [Bt]/[HSA] ratio. K´f was 2.066×106+(3.258×108/[HSA]). When 50 mM KCl was iso-osmotically substituted for sucrose, the K´f value was significantly lower {2.077×106+(1.099×108/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] ⩽ 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with Km values of 28±7 and 57±8 nM respectively (mean±S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112±12 versus 45±4 pmol of UCB·mg-1 of protein·15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K´f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with Km values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K´f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.

scholarly journals Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

1998 ◽  
Vol 330 (2) ◽  
pp. 853-860 ◽  
Author(s):  
N. J. Silvia MORENO ◽  
Li ZHONG ◽  
Hong-Gang LU ◽  
Wanderley DE SOUZA ◽  
Marlene BENCHIMOL
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Laser Scanning ◽  
Membrane Surface ◽  
Surface Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Cytoplasmic Ph ◽  
Bafilomycin A1

Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07±0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.

scholarly journals Macromolecular properties and polymeric structure of canine tracheal mucins

1991 ◽  
Vol 276 (2) ◽  
pp. 525-532 ◽  
Author(s):  
V Shankar ◽  
A K Virmani ◽  
B Naziruddin ◽  
G P Sachdev
Keyword(s):  
Gel Electrophoresis ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Hydrophobic Properties ◽  
Experimental Conditions ◽  
Protein Core ◽  
Polymeric Structure ◽  
Composite Gel ◽  
Self Association ◽  
Static Laser Light Scattering

Two high-Mr mucus glycoproteins (mucins), CTM-A and CTM-B, were highly purified from canine tracheal pouch secretions, and their macromolecular properties as well as polymeric structure were investigated. On SDS/composite-gel electrophoresis, a diffuse band was observed for each mucin. Polyacrylamide-gel electrophoresis using 6% gels also showed the absence of low-Mr contaminants in the mucins. Comparison of chemical and amino acid compositions revealed significant differences between the two mucins. Using a static-laser-light-scattering technique, CTM-A and CTM-B were found to have weight-average Mr values of about 11.0 x 10(6) and 1.4 x 10(6) respectively. Both mucins showed concentration-dependent aggregation in buffer containing 6 M-guanidine hydrochloride. Under similar experimental conditions, reduced-alkylated CTM-A had an Mr of 5.48 x 10(6) and showed no concentration-dependent aggregation. Hydrophobic properties of the mucins, investigated by the fluorescent probe technique using mansylphenylalanine as the probe, showed the presence of a large number of low-affinity (KD approx. 10(5) M) binding sites. These sites appeared to be located on the non-glycosylated regions of the protein core, since Pronase digestion of the mucins almost completely eliminated probe binding. Reduction of disulphide bonds of CTM-A and CTM-B did not significantly alter the probe-binding properties. Also, addition of increasing NaCl concentrations (0.03-1.0 M) to the buffer caused only a small change in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable degradation, using a combination of chemical and enzymic methods. On SDS/PAGE the protein core was estimated to have an Mr of approx. 60,000. On the basis of the protein and carbohydrate contents of the major mucin CTM-A, the mucin monomer was calculated to have an Mr of approx. 140,000. The high Mr (11 x 10(6] observed by physical methods is therefore due to self-association of the mucin monomer subunits.

scholarly journals Urea transporters UT-A1 and UT-A3 accumulate in the plasma membrane in response to increased hypertonicity

2008 ◽  
Vol 295 (5) ◽  
pp. F1336-F1341 ◽  
Author(s):  
Nathan W. Blessing ◽  
Mitsi A. Blount ◽  
Jeff M. Sands ◽  
Christopher F. Martin ◽  
Janet D. Klein
Keyword(s):  
Plasma Membrane ◽  
Western Blotting ◽  
Collecting Duct ◽  
Surface Proteins ◽  
Direct Response ◽  
Urea Permeability ◽  
Protein Pool ◽  
Hyperosmolar Solutions ◽  
Medullary Collecting Duct ◽  
Osmotic Agents

The UT-A1 and UT-A3 urea transporters are expressed in the terminal inner medullary collecting duct (IMCD) and play an important role in the production of concentrated urine. We showed that both hyperosmolarity and vasopressin increase urea permeability in perfused rat terminal IMCDs and that UT-A1 and UT-A3 accumulate in the plasma membrane in response to vasopressin. In this study, we investigated whether hyperosmolarity causes UT-A1 and/or UT-A3 to accumulate in the plasma membrane or represents a complimentary stimulatory pathway. Rat IMCD suspensions were incubated in 450 vs. 900 mosM solutions. We biotinylated the IMCD surface proteins, collected, and analyzed them. Membrane accumulation was assessed by Western blotting of the biotinylated protein pool probed with anti-UT-A1 or anti-UT-A3. We studied the effect of NaCl, urea, and sucrose as osmotic agents. Membrane-associated UT-A1 and UT-A3 increased relative to control levels when either NaCl (UT-A1 increased 37 ± 6%; UT-A3 increased 46 ± 13%) or sucrose (UT-A1 increased 81 ± 13%; UT-A3 increased 60 ± 8%) was used to increase osmolarity. There was no increase in membrane UT-A1 or UT-A3 when urea was added. Analogously, UT-A1 phosphorylation was increased in NaCl- and sucrose- but not in urea-based hyperosmolar solutions. Hypertonicity also increased UT-A3 phosphorylation. We conclude that the increase in the urea permeability in response to hyperosmolarity reflects both UT-A1 and UT-A3 movement to the plasma membrane and may be a direct response to tonicity. Furthermore, this movement is accompanied by, and may require, increased phosphorylation in response to hypertonicity.

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