Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane.     

Relations between plasma membrane and lysosomal membrane. 1. Fate of covalently labelled plasma membrane protein

To quantify the kinetics of the plasma membrane flow into lysosomes, we covalently labelled at 4 degrees C the pericellular membrane of rat fibroblasts and followed label redistribution to the lysosomal membrane using purified lysosomal preparations. The polypeptides were, either labelled with 125I by the lactoperoxidase procedure, or conjugated to [3H]peroxidase using bisdiazobenzidine as a bifunctional reagent. Both labels were initially bound to plasma membrane, as indicated by their equilibrium density in sucrose or Percoll gradients and their displacement by digitonin, as well as by electron microscopy. Upon cell incubation at 37 degrees C, both covalent labels were lost from cells with diphasic kinetics: a minor component (35% of cell-associated labels) was rapidly released (half-life less than 1 h), and most label (65%) was released slowly (half-life was 20 h for incorporated 125I and 27 h for 3H). Immediately after labelling up to 30 h after incubation at 37 degrees C, the patterns of 125I-polypeptides quantified by autoradiography after SDS-PAGE were indistinguishable, indicating no preferential turnover for the major plasma membrane polypeptides. The redistribution of both labels to lysosomes was next quantified by cell fractionation. At equilibrium (between 6 and 25 h of cell incubation) 2-4% of cell-associated 125I label was recovered with the purified lysosomal membranes. By contrast, when 3H-labelled cells were incubated for 16 h, most of the label codistributed with lysosomes. However, only 6% of cell-associated 3H was bound to lysosomal membrane. These results indicate that in cultured rat fibroblasts, a minor fraction of plasma membrane polypeptides becomes associated with the lysosomal membrane and is constantly equilibrated by membrane traffic.     

Turnover of the plasma membrane proteins of hepatoma tissue culture cells.

The turnover of the plasma membrane proteins of hepatoma tissue culture cells was examined by three different methods--loss of polypeptides labeled in situ by lactoperoxidase-catalyzed iodination, loss of membrane polypeptides labeled with amino acid precursors, and loss from the membrane of fucose-labeled polypeptides. In both logarithmically growing and density-inhibited cells the proteins of the membrane are degraded with a half-life of about 100 hours. This is longer than the half-life of total cell protein, 50 to 60 hours, and longer than the doubling time of the cells, about 30 hours. Similar values for the rate of degradation of the membrane proteins were obtained by each of the three techniques. The same fucose-labeled polypeptides are present in the microsomal and the plasma membrane fractions of hepatoma tissue culture cells as analyzed by electrophoresis in dodecyl sulfate-acrylamide gels. But the fucose-labeled polypeptides were lost from the microsomal fraction at a faster rate than from the plasma membrane. Autoradiographic and double labeling techniques using 125I and 131I, or [3H]leucine and [14C]leucine were used to measure the relative rates of degradation of the proteins in the plasma membrane. All of the leucine-labeled polypeptides and the iodinated polypeptides had similar rates of degradation. These results support a model for the biogenesis of the plasma membrane in which the proteins are incorporated and removed in large structural units.     

Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells

The fate of the L-cell plasma membrane proteins labeled by enzymatic iodination was studied. The disappearance of label from growing cells exhibits a biphasic behavior, with 5-20% lost rapidly (t1/2 similar to 2 h) and 80-90% lost relatively slowly (t1/2 similar to 25-33 h). The loss is temperature dependent and serum independent, and is accompanied by the appearance of 51% (125-I)monoiodotyrosine (MIT) in the medium by 47 h. A variable amount (1-14%) of acid-insoluble label can be recovered in the medium over 47 h. Sodium dodecyl sulfate (SDS)-polyacrylamide gel labeling patterns from cells cultured up to 48 h after iodination reveal no change in the relative distribution of radioactivity, indicating similar rates of degradation for most of the labeled membrane proteins. The fate of the labeled membrane proteins was studied at various times after phagocytosis of nondigestible polystyrene particles. Iodinated L cells phagocytose sufficient 1.1 mum latex beads in 60 min to interiorize 15-30% of the total cell surface area. Electron microscope autoradiography confirmed that labeled membrane is internalized during phagocytosis. The latex-containing phagocytic vacuoles are isolated by flotation in a discontinuous sucrose gradient. 15-30% of the total incorporated label and a comparable percentage of alkaline phosphodiesterase I activity (PDase, a plasma membrane enzyme marker) are recovered in the phagocytic vacuole fraction. Lysosomal enzyme activities are found in the latex vacuole fraction, indicating formation of phagolysosomes. SDS gel analyses reveal that all of the radioactive proteins initially present on the intact cell's surface are interiorized to the same relative extent. Incorporated label and PDase activity disappear much more rapidly from the phagolysosomes than from the whole cell. In the phagolysosomal compartment, greater than 70% of the TCA-precipitable labeled proteins and all of the PDase activity are lost rapidly (t1/2 equals 1-2 h) but similar 30% of the labeled proteins in this compartment are degraded with a 17-20 h half-life. The slowly degraded label is due to specific long-lived polypeptides, of 85,000 and 8,000-15,000 daltons, which remain in the phagolysosomal membrane up to 40 h after phagocytosis.     

Surface polypeptides of the cultured Chinese hamster ovary cell.

The organization of the plasma membrane of logarithmically growing Chinese hamster ovary (CHO) suspension cells has been probed using surface label techniques in conjunction with subcellular fractionation and sodium dodecyl sulfate gel electrophoresis. Five components of apparent molecular weights 137,000, 121,000, 97,000, 67,000, and 57,000 have been shown to be exposed at the outer surface of the cell. These components fully meet the criteria of being (a) reactive with two or more surface label reagents, (b) enriched in a purified plasma membrane fraction, and (c) sensitive to proteolytic digestion of intact cells. Three other components of molecular weights 200,000, 44,000 and 30,000 are also reactive with certain surface label reagents, but fail to meet other criteria for cell surface components. Two polypeptides of molecular weights 180,000 and 37,000 are substantially enriched in the plasma membrane fraction, but are unreactive with surface label reagents. The organization of the CHO cell membrane and the applicability of surface label techniques to cultured cell systems are discussed.     

Basolateral plasma membranes of intestinal epithelial cells. Identification by lactoperoxidase-catalysed iodination and isolation after density perturbation with digitonin.

Lactoperoxidase-catalysed iodination was used to label intestinal epithelial cell sheets with 125I. The iodination was carried out under conditions that allowed little penetration of lactoperoxidase into the cells and membrane-bound 125I therefore provided an effective marker for following plasma-membrane fragments through subcellular-fractionation procedures. 2. After homogenization and isopycnic zonal centrifugation through sucrose gradients two peaks of membrane-bound 125I were detected. One coincided with brush border enzymes such as alkaline phosphatase, disaccharidases and L-leucine B-naphthylamidase, whereas the other was coincident with the major peak of (Na++K+)-stimulated ATPase (adenosine triphosphatase), which has been thought to be concentrated in the basolateral plasma membranes of these cells. Neither peak of 125I reflected the distribution of any marker for an intracellular organelle. 3. A larger proportion of the (Na++K+)-stimulated ATPase, and thus of the basolateral plasma-membrane material, was found in a crude 'mitochondrial' fraction. It was not readiily separated from mitochondria by conventional techniques of subcellular fractionation. 4. Treatment of the 'mitochondrial' fraction with digitonin increased the density of basolateral plasma membrane but had little effect on mitochondrial density. A purified preparation of digitonin-loaded basolateral plasma membranes was isolated at a density of 1.20-1.22 by isopycnic centrifugation. 5. The enzymic composition of this preparation of basolateral plasma membranes is compared with previous preparations isolated from intestinal mucosal 'scrape' materials and from isolated cells.     

Topography of the Dictyostelium discoideum plasma membrane: analysis of membrane asymmetry and intermolecular disulfide bonds

Through the application of a unique method for isolating plasma membranes, it was possible to specifically iodinate cytoplasm-exposed plasma membrane proteins in vegetative cells of the cellular slime mold Dictyostelium discoideum. The original procedure [Chaney, L. K., & Jacobson, B. S. (1983) J. Biol. Chem. 258, 10062] which involved coating cells with colloidal silica has been modified to yield a more pure preparation. The presence of the continuous and dense silica pellicle on the outside surface of the isolated plasma membrane permitted the specific labeling of cytoplasm-exposed membrane proteins. Lactoperoxidase-catalyzed iodination was employed to label cell-surface and cytoplasm-exposed membrane proteins. The isolated and radioiodinated membranes were then compared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cell-surface and cytoplasmic face labeling patterns were distinct. A total of 65 proteins were found to be accessible to at least one surface of the membrane. Sixteen intermolecular disulfide bond complexes were observed in the plasma membrane of Dictyostelium; most of these complexes involved glycoproteins and, hence, were exposed to the cell surface.     

Cell surface constituents of sarcoma 180 ascites tumor cells

The cell surface protein components of Sarcoma 180 ascites tumor cells have been investigated by a combination of plasma membrane isolation techniques and lactoperoxidase iodination. For plasma membrane isolation cells were homogenized in the presence or absence of Zn²⁺ and fractionated by sucrose density gradient centrifugation or a two-phase partition to give large membrane fragments or membrane envelopes. Membrane purification was monitored by phase contrast microscopy and chemical and enzyme marker assays. The membrane preparations were analyzed by acrylamide gel electrophoresis in sodium dodecylsulfate. Each preparation showed a common protein pattern of about 15 bands ranging in molecular weights from 33 000 to >300000. Two carbohydrate-containing bands were also present in all preparations. Membranes prepared with Zn²⁺ were much less fragmented and showed much greater amounts of three high molecular weight components than those prepared in the absence of Zn²⁺. This might suggest a role for these components in membrane stabilization.The tumor cells were also subjected to iodination with lactoperoxidase, followed by membrane isolation and acrylamide gel electrophoresis in sodium dodecylsulfate in order to identify polypeptides accessible to the cell surface. The major radioactive band coincided with the major carbohydrate-containing band, presumably a surface glycoprotein. A second carbohydrate-containing band showed variable labeling behavior between different cell preparations. This material had a high molecular weight, as indicated by both acrylamide gel electrophoresis and gel permeation chromatography in dodecylsulfate. Several other components are labeled to a lesser extent in the intact cell.     

Topography of Chlamydomonas: fine structure and polypeptide components of the gametic flagellar membrane surface and the cell wall

Surface polypeptide components of the flagellar membrane of Chlamydomonas reinhardi Dang. gametes are identified by their accessibility to in-vivo vectoral labeling by glucose oxidase-coupled lactoperoxidase-dependent ¹²⁵I iodination. Vectoral labeling is accomplished without observable adverse effects on cell viability or gametic function. Flagella isolated from labeled wild-type cells carry about 3% of the total incorporated label, which is found by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be distributed among 16 identifiable polypeptide bands. The most prominent surface-labeled species migrates in the M_r (relative molecular weight) 350 k region of the gel; each of the remaining iodinated polypeptides, which range in M_r from 25 k to 500 k, carries only a small proportion of incorporated label. To determine which polypeptides are unique to the flagellum and which are contaminants from the cell wall, wild-type profiles were compared with those of mutant strains and of mechanically isolated cell walls. Identification of contaminants was also facilitated by two-dimensional peptide mapping. We conclude that only 11 of the labeled bands are contributed by flagellar polypeptides; the remaining five bands are shown to be contaminants from the cell wall, and additional cell-wall polypeptides are found to co-migrate with flagellar species. A polypeptide designated as a possible membrane tubulin in preliminary studies is shown here to be different from tubulin in its peptide map. The 11 polypeptides assigned as specific flagellar surface components are candidate participants in such biological events as sexual adhesion, flagellar surface motility, and sensory signalling.     

The membrane proteins of the vacuolar system I. Analysis of a novel method of intralysosomal iodination

A method has been developed to deliver an idoinating system into the confines of the phagolysosome, allowing us to study the nature of the phagolysosomal membrane. Lactoperoxidase (LPO) is covalently coupled to carboxylated latex spheres (LPO-latex) in a stable, enzymatically active form. The addition of LPO-latex to cultured macrophages leads to their rapid attachment, ingestion, and enclosure in a plasma membrane- derived phagocytic vacuole. These organelles rapidly fuse with preexisting lysosomes and are converted to phagolysosomes (PL) that demonstrates both acid phosphatase and lactoperoxidase activities. The exposure of LPO-latex containing cells to 125I- and an extracellular peroxide-generating system, glucose oxidase-glucose, at 4 degrees C leads to incorporation of label into TCA-precipitable material. The incorporated cel-associated label was present as monoiodotyrosine, and negligible amounts were found in lipids. Cell viability remained > 99%. Autoradiography at both the light and EM level revealed that > 97% of the cells were labeled, and quantitative analysis demonstrated the localization of grains to LPO-latex containing PL. PL were separated on sucrose gradients, and their radiolabel was confined almost exclusively to the membrane rather than soluble contents. SDS-polyacrylamide gel electrophoretic analysis of the peptides iodinated from within PL demonstrated at least 24 species with molecular weights ranging from 12,000 to 250,000. A very similar group of proteins was identified on the plasma membrane (PM) after surface iodination, and on latex phagosomes derived from iodinated PM. No novel proteins were detected in PL, either immediately after phagosome-lysosome fusion or after 1 h of intracytoplasmic residence. We conclude that the membrane proteins accessible to LPO-catalyzed iodination on the luminal surface of the PL and on the external face of the PM are similar, if not identical.     

Membrane receptors as general markers for plasma membrane isolation procedures. The use of 125-I-labeled wheat germ agglutinin, insulin, and cholera toxin.

Specific cell surface membrane receptors, labeled by forming a complex with low concentrations (about 10--9 M to 10--10 M) of a highly radioactive (125-I, carrier-free) ligand, can serve as simple, reliable, sensitive, and quantitative markers for plasma membranes in fractionation procedures. 125-I-Labeled insulin, cholera toxin and the plant lictins, wheat germ agglutinin (WGA), and concanavalin A are the receptor ligands used for labeling plasma membranes. Plasma membranes are labeled before homogenization by incubating intact cells briefly at 24 degrees or 4 degrees, or by very brief in situ perfusion of the organ, with the 125-I-Labeled marker. After removing the free 125-I-labeled ligand from the medium by washing (at 4 degrees), the membrane-marker complex remains intact over prolonged (days) periods of time at 4 degrees. Labeling occurs nearly exclusively on the cell surface, the specificity of this plasma membrane reaction is maintained through homogenization and fractionation, and little dissociation of the complex, detectable exchange of label, or aggregation occur even upon prolonged incubation of the homogenates. When desired, the complex can be dissociated deliberately by manipulating experimental conditions such as temperature or by adding specific simple sugars. The most generally suitable marker appears to be WGA. At least in certain tissues (e. g. fat cells) labeling of the plasma membrane with 125-I-WGA and 125-I-isnulin can be performed equally well and selectively in homogenates as in the intact cell. 125-I-Cholera toxin cannot be used in homogenates because of significant binding to nuclei. The use of 125-I-labeled WGA as a specific plasma membrane marker is illustrated in following the course of fractionations, and in quantitating the yield and purity, of plasma membranes from fat cells, lymphocytes, and liver. The results are compared with simultaneous measurements of the plasma membrane enzyme "markers," ATPase, 5-nucleotidase, and basal as well as hormone-stimulated adenylate cyclase activities. The fractionation of liver plasma membranes by aqueous dextran-polyethylene glycol two-phase polymer systems and by conventional differential centrifugation procedures arealso quantitated with the marker, 125I-WGA. Substantial quantities of plasma membrane material are no recovered in the interphase of the two-phase polymer system. Conventional liver fractionation procedures which retain, for further purification, only the readily sedimented pellet (2000 times g, 15 min) discard a very large (at least 70%) questenal hy     

Membrane proteins synthesized by rabbit reticulocytes

Intact rabbit reticulocyte cells synthesize two predominant species of polypeptides which are components of the cell plasma membrane. Previous work (Lodish, H. F. 1973. Proc. Natl. Acad. Sci. U. S. A. 70:1526- 1530.) showed that these proteins were synthesized by polyribosomes not attached to membranes. We show here that both polypeptides are confined to the cytoplasmic surface of the cell membrane. These studies utilized iodination of whole cells and of membranes with lactoperoxidase, and digestion of whole cells and membranes with chymotrypsin, One of these proteins is synthesized as a precursor, and about 20-40 amino acids are removed after it is incorporated into the membrane, We discuss the probable sites of synthesis of these and other classes of membrane proteins.     

Plant Plasma Membrane Proteins : II. Biotinylation of Daucus Carota Protoplasts and Detection of Plasma Membrane Polypeptides after Sds-Page

The ability of two biotinylating reagents, sulfosuccinimidobiotin and sulfosuccinimidyl 2-(biotinamido)ethyl-1,3′-dithiopropionate, to label plasma membrane proteins was examined. These compounds form covalent bonds with the free amino groups of proteins and label the proteins with biotin. Biotinylated proteins can be detected with avidin-peroxidase staining. Protoplasts isolated from embryogenic Daucus carota suspension cells were labeled with biotin and the membranes were separated on linear sucrose gradients. The conditions used for labeling the protoplasts did not cause protoplast rupture or loss of viability. The distribution of the biotin label in these linear sucrose gradients was analyzed and compared to the distribution of vanadate-sensitive ATPase activity, a marker for the plasma membrane. Both the biotin label and the vanadate-sensitive ATPase activity were strongly localized in the gradient at peak density of 1.16 gram per cubic centimeter. When the protoplast surface was labeled, biotinylated polypeptides were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polypeptides of 153, 94, 51, 30, 20, 17, and 14 kilodaltons were shown to be plasma membrane in origin. When a crude membrane pellet was labeled, numerous biotinylated polypeptides were distributed throughout the gradient. Because the position of the biotin label in the gradient is strongly correlated with the distribution of vanadate-sensitive ATPase, it is concluded that these biotinylating reagents are effective and reliable labels for proteins of the plant plasma membrane. Furthermore, these labels permit the positive identification of plasma membrane proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and can serve as convenient markers for solubilization and purification of these proteins.     

Surface peptides on intact Ehrlich ascites tumor cells : Identification by a method not requiring subcellular fractionation

Lactoperoxidase catalysed iodination of tyrosyl residues was used to label the exposed plasma membrane proteins in intact Ehrlich ascites tumor cells. Autoradiography of ¹²⁵I-labeled intact cells revealed that the label was predominantly associated with the plasma membrane. When whole cells were solubilized and subjected to gel electrophoresis, two major labeled peptide classes of 100 000 and 80 000 D along with 4 minor labeled classes were found. An identical labeling pattern was obtained when plasma membranes isolated from labeled cells were solubilized and subjected to gel electrophoresis. These results demonstrate that the number of exposed plasma membrane peptides and their molecular weights can be determined without first isolating the membrane by subcellular fractionation procedures, a standard approach in most studies.     

Turnover of the surface proteins and the receptor for serum asialoglycoproteins in primary cultures of rat hepatocytes

The turnover of plasma membrane proteins in primary rat hepatocyte cultures was examined by following the loss of polypeptides labeled in situ by lactoperoxidase-catalyzed iodination using 125I and 131I. Most plasma membrane proteins had similar rates of degradation, having a half-life of approximately 85 h. By in situ labeling via lactoperoxidase-catalyzed iodination, as well as metabolically labeling cells with L-[35S]methionine, the asialoglycoprotein receptor, a plasma membrane constituent, was identified and shown to exist in three forms which were structurally related. The turnover of receptor on the cell surface was examined by following the loss of iodinated cell surface receptor, while the turnover of total cellular receptor, including both surface and internally localized receptor was assayed by following the loss of receptor labeled metabolically with [35S]methionine. The turnover rate in both cases was approximately 20 h. Receptor-mediated endocytosis of asialoglycoproteins had no effect on the turnover of the plasma membrane proteins or receptor. Based on estimates of the rate of metabolism of the asialoglycoprotein ligand relative to the turnover rate of the receptor, we conclude each molecule of receptor can deliver about 1,000 molecules of ligand to the lysosome to be degraded.     

Membrane proteins of the vacuolar system. III. Further studies on the composition and recycling of endocytic vacuole membrane in cultured macrophages

In previous publications (Muller, W.A., R.M. Steinman, Z.A. Cohn. 1980, J.Cell Biol. 86:292-314), we found that the membrane of macrophage phagolysosomes could be selectively radioiodinated in living cells, The technique required phagocytosis of lactoperoxidase covalently coupled to latex spheres (LPO-latex), followed by iodination on ice with Na(125)I and hydrogen peroxide. In this paper, we use the LPO-latex system to further analyze the composition and recycling of phagocytic vacuole membrane. Three approaches were employed to examine the polypeptide composition of the phagolysosome (PL) and plasma membranes (PM). (a) The efficiency of intracellular iodination was increased by increasing lysosomal pH with chloroquine. By one-dimensional SDS PAGE, the heavily labeled chloroquine-treated PL exhibited the same labeled polypeptides as PM iodinated extracellularly with LPO-latex. (b) Iodinated PL and PM were compared by two-dimensional gel electrophoresis. No differences in the isoelectric point and molecular weight of the major iodinated species were detected. (c) Quantitative immune precipitation was performed with five specific antibodies directed against cell surface antigens. Four antibodies precipitated similar relative amounts of labeled antigen on the cell surface and endocytic vacuole. One antibody, secreted by hybridoma 2.6, detected a 21-kdalton polypeptide that was enriched sevenfold in PL membrane. This enrichment was cell surface-derived, since the amount of labeled 2.6 was increased sevenfold when iodinated PM was driven into the cell during latex uptake. Therefore, intracellular iodination primarily detects PL proteins that are identical to their PM counterparts. Additional studies employed electron microscope autoradiography to monitor the centrifugal flow of radiolabeled polypeptides from PL to PM. Cells were iodinated intralysosomally and returned to culture for only 5-10 min at 37 degrees C. Most of the cell-associated label then redistributed to the cell surface or its adjacent area. Significant movement out of the lysosome compartment occurred even at 2 degrees C and 22 degrees C. Extensive and rapid membrane flow through the secondary lysosome presumably contributes to the great similarity between PM and PL membrane polypeptides.     

Radio-iodination of plasma membrane polypeptides from isolated mouse spermatogenic cells

Cell surface polypeptides of mouse pachytene spermatocytes and round spermatids (steps 1–8) have been iodinated using 1,2,3,6,tetracholoro-3α, 6α-diphenylglycouril (IODOGEN). Labeled proteins have been assayed using two-dimensional polyacrylamide electrophoresis and radioautography. Purified plasma membranes, prepared from both spermatocytes and spermatids after the iodination of intact cells, exhibit 25–30 polypeptides which label reproducibly. No significant qualitative differences are noted in the labeled polypeptide map obtained from each of the purified cell types. Iodinated proteins range in molecular weight from greater than 100k daltons to approximately 40k daltons. The isoelectric points of labeled constituents range from pI 5.7 to 7.2. Three polypeptides represent the major iodinated species: p 94/5.8, p 75/5.9, and p 53/7.1. Comparison with total plasma membrane constituents assayed using Coomassie brilliant blue indicates that many of the radioactively labeled proteins are not present in quantities sufficient to allow ready detection without isotopic techniques. As a result, many of the proteins identified autoradiographically represent newly described surface components of mouse pachytene spermatocytes and round spermatids. The preparation of purified plasma membrane fractions prior to electrophoresis ensures that all iodinated species are in fact cell surface components. Furthermore, experiments designed to assess the vectorial nature of the IODOGEN-catalyzed labeling procedure suggest that most, if not all, of the iodinated species are exposed on the external side of the cell plasma membrane. Therefore, these studies have (1) identified hitherto unrecognized plasma membrane components of mouse pachytene spermatocytes and round spermatids and (2) provided the first available biochemical data concerning the molecular orientation of particular proteins in the surface membranes of developing mouse spermatogenic cells.     

Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. II. Cell surface localization of granule membrane and content proteins before and after degranulation

The compositional relationship between the cell surface of rabbit polymorphonuclear leukocytes (PMNs) and the membranes of PMN cytoplasmic granules has been investigated. Heterophilic PMNs obtained from peritoneal exudates contained 13 cell surface polypeptides ranging in molecular weight from 220,000 to 12,000 daltons as determined by lactoperoxidase-catalyzed protein iodination and gel electrophoresis. Of these, four polypeptides co-migrated with proteins identified as the major constituents of specific (SpG) and azurophilic (AzG) granule membranes. The most notable of these were cell surface proteins of 145,000 and 96,000 daltons that co-migrated with proteins identified as granule content proteins released from PMNs during exocytosis. Extensive washing did not remove these proteins from the cell surface. Iodination of PMNs after the release of SpG and AzG contents by calcium ionophore- induced exocytosis revealed that there was not a dramatic quantitative change in the proteins on the cell surface. Instead, there were large, quantitative increases in the relative amounts of (125)I that were incorporated into several pre-existing cell surface proteins; all of these cell surface proteins co-migrated as a set with those polypeptides identified as either granule membrane or content proteins. Although nearly all of the major polypeptides of SpG and AzG had counterparts on the cell surface of freshly isolated peritoneal exudates PMNs, there were several polypeptides that were unique to the cell surface. Thus, the PMN has at least three membrane compartments with strikingly different protein compositions.     

Yeast plasma membrane ghosts. An analysis of proteins by two-dimensional gel electrophoresis

We have examined yeast cell ghost preparations to assess their value in obtaining plasma membrane proteins. Ghosts prepared by two methods involving stabilization of spheroplast envelopes had similar protein patterns by two-dimensional gel electrophoresis, and approximately 200 proteins were resolved. Spheroplasts were lactoperoxidase iodinated, and recovery of label in ghost preparations was greater than 60%. Spheroplasts appeared to be impermeable to the lactoperoxidase reagents as judged by an examination of two-dimensional gel electrophoretic patterns of ghost proteins that had been iodinated in spheroplasts or in unsealed ghosts. Spheroplasts were also impermeable to pronase proteases. Surface iodination and surface proteolysis allowed us to identify exposed ghost proteins; the major ghost glycoprotein was exposed in spheroplasts.Two-dimensional patterns of ghost proteins were not heavily contaminated (?25% of all proteins) by proteins present in soluble or promitochondrial fractions, and estimates of surface label and total cell protein recovery suggested that the ghost fraction represents a cell envelope enrichment of 8–10 fold over whole cells.Resolution of ghost proteins by two-dimensional gel electrophoresis appears to be a powerful aid toward identifying membrane proteins.