Role of clathrin- and actin-dependent endocytotic pathways in lung phospholipid uptake

We evaluated the contribution of endocytotic pathways to pulmonary uptake of surfactant lipids from the alveolar space. Resting and stimulated 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) uptake of unilamellar liposomes labeled with either [(3)H]dipalmitoylphosphatidylcholine ([(3)H]DPPC) or 1-palmitoyl-2-[12-(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl-phosphatidylcholine (NBD-PC) was studied in isolated perfused rat lungs and isolated type II cells. Amantadine and phenylarsine oxide, inhibitors of clathrin-mediated endocytosis, each decreased [(3)H]DPPC uptake under resting conditions by approximately 40%; their combination had no additional effect. Cytochalasin D, an inhibitor of actin-dependent processes, reduced liposome uptake by 55% and potentiated the effect of either clathrin inhibitor alone. Relative inhibition for all agents was higher in the presence of 8-Br-cAMP. The effect of inhibitors was similar for liposomes labeled with [(3)H]DPPC or NBD-PC. By fluorescence microscopy, NBD-PC taken up by lungs was localized primarily to alveolar type II cells and was localized to lamellar bodies in both lungs and isolated cells. These studies indicate that both clathrin-mediated and actin-mediated pathways are responsible for endocytosis of DPPC-labeled liposomes by alveolar type II cells in the intact lung.     

Effect of metabolic alterations on the density and the contents of cathepsins B, H and L of lysosomes in rat macrophages

Crude lysosomal preparations from non-cultured peritoneal rat macrophages were shown to separate into high-density fractions rich in cathepsin B and H and low-density fractions rich in cathepsin L when layered on Percoll density gradients. Morphologically, the heavy lysosome fractions were found to consist mainly of lysosomes labeled with gold particles for anti-(cathepsin B, H and L). The light lysosome fractions contained lysosomes labeled with anti-(cathepsin B, H and L) and many other contaminants. In addition, small vesicles labeled by anti-(cathepsin L) were detected in these fractions. Addition of calf serum to the cultured macrophages induced an increase in the density of lysosomes in both dose-dependent and time-dependent fashions. Cathepsins B, H and L all shifted to the heavy lysosome fractions following the addition of serum. Progressive increase in fluorescence-labeled calf IgG in the heavy lysosome fractions after its addition suggests that the continuous entrance of excess proteins to lysosomes causes an increase in their density. This idea is supported by the fact that the density of lysosomes increased in parallel with the accumulation of horseradish peroxidase taken up in the heavy lysosome fractions. Increase in the density of lysosomes after treatment with ethyl(2S,3S)-3[(S)-3-methyl-1-(3-methyl-butylcarbamoyl)]oxirane-2- carboxylate (E-64-d) was marked in the cells cultured with serum-containing medium but slight in serum-deprived cells. However, the level of pyruvate kinase, an autophagic sequestration marker in heavy autolysosomes from E-64-d-treated cells, was much higher in serum-deprived cells, indicating that the contribution of heterophagic sequestration towards an increase in the density of lysosomes is much greater than that of autophagy.     

Macrophage and type II cell catabolism of SP-A and saturated phosphatidylcholine in mouse lungs

Type II cells and macrophages are the major cells involved in the alveolar clearance and catabolism of surfactant. We measured type II cell and macrophage contributions to the catabolism of saturated phosphatidylcholine and surfactant protein A (SP-A) in mice. We used intratracheally administered SP-A labeled with residualizing (125)I-dilactitol-tyramine, radiolabeled dipalmitoylphosphatidylcholine ([(3)H]DPPC), and its degradation-resistant analog [(14)C]DPPC-ether. At 15 min and 7, 19, 29, and 48 h after intratracheal injection, the mice were killed; alveolar lavage was then performed to recover macrophages and surfactant. Type II cells and macrophages not recovered by the lavage were subsequently isolated by enzymatic digestion of the lung. Radioactivity was measured in total lung, lavage fluid macrophages, alveolar washes, type II cells, and lung digest macrophages. Approximately equal amounts of (125)I-dilactitol-tyramine-SP-A and [(14)C]DPPC-ether associated with the macrophages (lavage fluid plus lung digest) and type II cells when corrected for the efficiency of type II cell isolation. Eighty percent of the macrophage-associated radiolabel was recovered from lung digest macrophages. We conclude that macrophages and type II cells contribute equally to saturated phosphatidylcholine and SP-A catabolism in mice.     

SP-A is necessary for increased clearance of alveolar DPPC with hyperventilation or secretagogues

The role of surfactant protein-A (SP-A) in pulmonary uptake and metabolism of [(3)H]dipalmitoylphosphatidylcholine ([(3)H]DPPC) was studied in SP-A gene-targeted mice (SP-A -/-). Unilamellar liposomes were instilled into the trachea of anesthetized mice. Uptake was measured as dpm in lungs plus liver and kidney for in vivo experiments and in lungs and perfusate for isolated lung experiments. [(3)H]DPPC uptake increased with CO(2)-induced hyperventilation in wild-type mice (SP-A +/+) but was unchanged in SP-A -/-. Secretagogue treatment approximately doubled the uptake of [(3)H]DPPC in isolated lungs from SP-A +/+ but had no effect in SP-A -/-. Lungs degraded 23 +/- 1.2% of internalized [(3)H]DPPC in SP-A +/+ and 36 +/- 0.6% in SP-A -/-; degradation increased with 8-bromoadenosine 3',5'-cyclic monophosphate in SP-A +/+ but was unchanged in SP-A -/-. Activity of lysosomal-type phospholipase A(2) (PLA(2)) was significantly greater in lungs from SP-A -/- compared with SP-A +/+. Thus SP-A is necessary for lungs to respond to hyperventilation or secretagogues with increased DPPC uptake and also modulates the PLA(2)-mediated degradation of internalized DPPC.     

Surfactant protein-A plays an important role in lung surfactant clearance: evidence using the surfactant protein-A gene-targeted mouse

Previous studies with the isolated perfused rat lung showed that both clathrin- and actin-mediated pathways are responsible for endocytosis of dipalmitoylphosphatidylcholine (DPPC)-labeled liposomes by granular pneumocytes in the intact lung. Using surfactant protein-A (SP-A) gene-targeted mice, we examined the uptake of [(3)H]DPPC liposomes by isolated mouse lungs under basal and secretagogue-stimulated conditions. Unilamellar liposomes composed of [(3)H]DPPC: phosphatidylcholine:cholesterol:egg phosphatidylglycerol (10:5:3:2 mol fraction) were instilled into the trachea of anesthetized mice, and the lungs were perfused (2 h). Uptake was calculated as percentage of instilled disintegrations per minute in the postlavaged lung. Amantadine, an inhibitor of clathrin and, thus, receptor-mediated endocytosis via clathrin-coated pits, decreased basal [(3)H]DPPC uptake by 70% in SP-A +/+ but only by 20% in SP-A -/- lung, data compatible with an SP-A/receptor-regulated lipid clearance pathway in the SP-A +/+ mice. The nonclathrin, actin-dependent process was low in the SP-A +/+ lung but accounted for 55% of liposome endocytosis in the SP-A -/- mouse. With secretagogue (8-bromoadenosine 3',5'-cyclic monophosphate) treatment, both clathrin- and actin-dependent lipid clearance were elevated in the SP-A +/+ lungs while neither pathway responded in the SP-A -/- lungs. Binding of iodinated SP-A to type II cells isolated from both genotypes of mice was similar indicating a normal SP-A receptor status in the SP-A -/- lung. Inclusion of SP-A with instilled liposomes served to "rescue" the SP-A -/- lungs by reestablishing secretagogue-dependent enhancement of liposome uptake. These data are compatible with a major role for receptor-mediated endocytosis of DPPC by granular pneumocytes, a process critically dependent on SP-A.     

Intracellular degradation of asialoglycoproteins in hepatocytes starts in a subgroup of lysosomes

Isolated rat hepatocytes take up and degrade [125I]tyramine-cellobiose-labelled asialofetuin [( 125I]TC-AF). The labelled degradation products are trapped at the site of degradation. The intracellular transport of [125I]TC-AF was studied by means of cell fractionation in Nycodenz gradients. The labelled ligand was kept in a small, slowly sedimenting vesicle during the first minutes after uptake in the cells, and was then transferred to a larger endosome. Labelled degradation products first appeared in an organelle with the same density distribution as the larger endosome and then in a denser organelle. These observations suggest that two types of lysosome, 'light' lysosomes and 'dense', are sequentially involved in the degradation of the asialoglycoprotein. The bulk of the lysosomal enzymes is associated with the dense lysosome.     

Characterization of rabbit lung lysosomes and their role in surfactant dipalmitoylphosphatidylcholine catabolism

Although alveolar surfactant is rapidly catabolized in adult rabbit lungs, the pathways have not been characterized. Pathways of surfactant secretion and recycling involve lamellar bodies and multivesicular bodies, organelles shown to be related to lysosomes by cytochemistry and autoradiography. Since lysosomes are central to intracellular catabolic events, it is possible that lysosomes are involved in intrapulmonary surfactant catabolism. Lysosomes relatively free of contaminating organelles (as determined morphologically and by marker enzymes for mitochondria, endoplasmic reticulum, peroxisomes, and plasma membranes) were obtained from post-lavage lung homogenates of 1-kg rabbits by differential centrifugation in buffered sucrose and gradient separation in percoll (density, 1.075-1.165). The role of lung lysosomes in catabolism of dipalmitoylphosphatidylcholine (DPC) was then studied in rabbits killed 4, 12, and 24 h following intratracheal injection of [3H]DPC and [14C] dihexadecyl phosphatidylcholine (DPC-ether). While equal amounts of label were in the lamellar body containing fractions at 4 h, nearly 6-fold more DPC-ether label than DPC label was recovered in the lysosomal fractions. By 24 h, there was 15-fold more DPC-ether in the lysosomes. This is the first report of successful isolation of lysosomes relatively free of other organelles from rabbit lungs. The tracer studies indicate DPC and DPC-ether follow similar intracellular processing after alveolar uptake. The subsequent accumulation of the ether analog in the lysosomal fractions supports a role for these organelles in surfactant DPC catabolism.     

Lysosomal phospholipids from rat liver after treatment with different drugs

Rats were treated with 5 different drugs p-ethoxyacetanilide (I), indometacin (II) and nor-amidopyrine-methanesulfonate (III), O,O'-bis(diethylaminoethyl)hexestrol(IV) and choloroquine (V) for 3 - 4 weeks. Liver cell fractions were isolated by discontinuous gradient centrifugation and the specific activity of acid phosphatase was determined in each. Lysosomal fractions contained widely varying amounts of this marker enzyme, indicating that the concentration of lysosomes within these fractions differed. The amounts and patterns of phospholipids reflected this fact. Since we assumed bis(monoacylglycero)phosphate [(MAG)2-P; synonym:lysobisphosphatidic acid] is a marker lipid for secondary lysosomes, we expected and found significant quantities of this acidic phospholipid only in those lysosomal fractions which were also rich in acid phosphatase activity. 12% of the lysosomal phospholipids from animals receiving the hexestrol derivative (IV), and 19% of those from the chloroquine (V) experiment were present as (MAG)2P. The fatty acid compositions of this lysosomal phospholipid were not the same in all lysosome fractions. The more (MAG)2P present in the lysosomes, the more unsaturated are the fatty acids. Thus, after treatment with chloroquine, more than 90% of the fatty acids from (MAG)2P are unsaturated; C22:6 represents about 70% of the total.     

Is dipalmitoylphosphatidylcholine a substrate for convertase?

Convertase has homology with carboxylesterases, but its substrate(s) is not known. Accordingly, we determined whether dipalmitoylphosphatidylcholine (DPPC), the major phospholipid in surfactant, was a substrate for convertase. We measured [(3)H]choline release during cycling of the heavy subtype containing [(3)H]choline-labeled DPPC with convertase, phospholipases A(2), B, C, and D, liver esterase, and elastase. Cycling with liver esterase or peanut or cabbage phospholipase D produced the characteristic profile of heavy and light peaks observed on cycling with convertase. In contrast, phospholipases A(2), B, and C and yeast phospholipase D produced a broad band of radioactivity across the gradient without distinct peaks. [(3)H]choline was released when natural surfactant containing [(3)H]choline-labeled DPPC was cycled with yeast phospholipase D but not with convertase or peanut and cabbage phospholipases D. Similarly, yeast phospholipase D hydrolyzed [(3)H]choline from [(3)H]choline-labeled DPPC after incubation in vitro, whereas convertase, liver esterase, or peanut and cabbage phospholipases D did not. Thus convertase, liver esterase, and plant phospholipases D did not hydrolyze choline from DPPC either on cycling or during incubation with enzyme in vitro. In conclusion, conversion of heavy to light subtype of surfactant by convertase may require a phospholipase D type hydrolysis of phospholipids, but the substrate in this reaction is not DPPC.     

Reduction of disulfide bonds within lysosomes is a key step in antigen processing.

Reduction of disulfide bonds is a key step in antigen processing both to allow the unfolding of protein antigens, increasing the access of proteolytic processing enzymes, and to expose free Cys residues within linear peptide epitopes recognized by T cells. We show here that reduction and alkylation of Ag (hen egg lysozyme and ribonuclease A) vastly increased their proteolysis (by specific enzymes or lysosomal fractions) and the production of specific immunogenic peptides that bound to class II MHC molecules recognized by T hybridoma cells. We also show that the lysosome is the vesicular compartment that mediates protein disulfide reduction. We coupled [125I]tyrosine to 131I-alpha 2-macroglobulin or [131I] transferrin via a reducible disulfide linker. Removal of [125I]tyrosine from the alpha 2-macroglobulin conjugate was initiated only after 15 to 20 min of uptake by macrophages, suggesting that reduction occurred late in the endocytic pathway. No reduction of transferrin conjugates was seen, indicating that early, recycling endosomes did not contain reducing activity. Subcellular fractionation showed that the disulfide bonds were reduced only in heavy density (lysosome) fractions and remained intact in fractions of light density (endosomes and plasma membrane). These results indicate the importance of lysosomes in the biochemical processing of protein Ag presented to T cells.     

GM-CSF regulates protein and lipid catabolism by alveolar macrophages

Metabolism of surfactant protein (SP) A and dipalmitoylphosphatidylcholine (DPPC) was assessed in alveolar macrophages isolated from granulocyte-macrophage colony-stimulated factor (GM-CSF) gene-targeted [GM(-/-)] mice, wild-type mice, and GM(-/-) mice expressing GM-CSF under control of the SP-C promoter element (SP-C-GM). Although binding and uptake of (125)I-SP-A were significantly increased in alveolar macrophages from GM(-/-) compared with wild type or SP-C-GM mice, catabolism of (125)I-SP-A was markedly decreased in GM(-/-) mice. Association of [(3)H]DPPC with alveolar macrophages from GM(-/-), wild-type, and SP-C-GM mice was similar; however, catabolism of DPPC was markedly reduced in cells from GM(-/-) mice. Fluorescence-activated cell sorter analysis demonstrated decreased catabolism of rhodamine-labeled dipalmitoylphosphatidylethanolamine by alveolar macrophages from GM(-/-) mice. GM-CSF deficiency was associated with increased SP-A uptake by alveolar macrophages but with impaired surfactant lipid and SP-A degradation. These findings demonstrate the important role of GM-CSF in the regulation of alveolar macrophage lipid and SP-A catabolism.     

Properties of freshly isolated type II alveolar epithelial cells

The biochemical characteristics of type II alveolar epithelial cells dissociated from adult rabbit lung by instillation of low concentrations of an elastase trypsin mixture are reported. Cells studied immediately (within 4 h) after isolation were found to incorporate the radioactively labelled precursors [U-¹⁴C]glucose, [methyl-³H]choline and [³H]palmitate into cellular phosphatidylcholine at rates 2–10-fold higher than previously reported for cells not subject to short-term cell culture. Secretion of phosphatidylcholine was stimulated by beta-adrenergic agonists. Measurement of specific activities of enzymes of phospholipid biosynthesis in subcellular fractions of isolated lung cells showed a significant enrichment of acyl coenzyme A-lysophosphatidylcholine acyltransferase, an enzyme believed to be involved in pulmonary surfactant phosphatidylcholine remodeling, in the endoplasmic reticulum of type II cells. These observations support the utility of freshly isolated type II cells as a model system for the study of the functions of the alveolar epithelium.     

Amiodarone inhibits lung degradation of SP-A and perturbs the distribution of lysosomal enzymes

Amiodarone may induce lung damage by direct toxicity or indirectly through inflammation. To clarify the mechanism of direct toxicity, we briefly exposed rabbit alveolar macrophages to amiodarone and analyzed their morphology, synthesis, and degradation of dipalmitoylphosphatidylcholine (DPPC); distribution of lysosomal enzymes; and uptake of diphtheria toxin and surfactant protein (SP) A used as tracers of the endocytic pathway. Furthermore, in newborn rabbits, we studied the clearance of DPPC and SP-A instilled into the trachea together with increasing amounts of amiodarone. We found that in vitro amiodarone decreases the surface density of mitochondria and lysosomes while increasing the surface density of inclusion bodies, increases the incorporation of choline into DPPC, modifies the distribution of lysosomal enzymes, and does not affect the uptake and processing of diphtheria toxin but inhibits the degradation of SP-A. In vivo amiodarone inhibits the degradation of SP-A but not of DPPC. We conclude that the acute exposure to amiodarone perturbs the endocytic pathway acting after the early endosomes, alters the traffic of lysosomal enzymes, and interferes with the turnover of SP-A.     

Uptake of a natural surfactant and increased delivery of small organic anions into type II pneumocytes

The uptake of natural lung surfactant into differentiated type II cells may be used for the targeted delivery of other molecules. The fluorescent anion pyranine [hydroxypyren-1,3,6-trisulfonic acid, sodium salt (HPTS)] was incorporated into a bovine surfactant labeled with [3H]dipalmitoylphosphatidylcholine ([3H]DPPC). The uptake of [3H]DPPC and of HPTS increased with time of incubation and concentration, decreased with the size of the vesicles used, and was stimulated by 8-bromo-cAMP and partially inhibited by hypertonic sucrose. However, the amount of HPTS uptake was approximately 100 times smaller than that of [3H]DPPC. This large difference was due to a more rapid regurgitation of some of the HPTS from the cells but not to leakage from the surfactant before uptake. The acidification of the internalized surfactant increased linearly over 90 min to 7.13, and after 24 h, a pH of 6.83 was measured. In conclusion, after internalization of a double-labeled natural surfactant, the lipid moieties were accumulated in relation to the anions, which were targeted to a compartment not very acidic and in part rapidly expelled from the cells.     

The dependence of enhanced lysosomal activity on the cellular aging of bovine aortic endothelial cells

Lysosomes are a cell organelle type processing antimicrobial activity. Here, we investigate the lysosomal activity in a primary cell line, bovine aortic endothelial cells (BAECs), during cellular aging, based on the antimicrobial activity of lysosomes isolated from BAECs at cell passages 4, 6, 8, and 10. Cytochemical analysis of lysosomes with LysoTracker reagent revealed the number of lysosome-like organelles surrounding the nucleus initially increased drastically in the BAECs and continued increasing gradually until passage 10. The lysosomes isolated from each successive passage of BAECs exhibited increased antimicrobial activity against Escherichia coli, and, in addition, an age-dependent increase in lysosome intensity coincided with increased lysosomal antimicrobial activity.     

Culture characteristics of cells derived from type II pneumocyte enriched fractions from rabbit and rat.

Type II cell enriched fractions were isolated from rabbit and rat lungs using density gradient centrifugation. Cultures established from these fractions contained predominantly cells similar in most morphological respects to type II pneumocytes. These were in continuous replicating culture for 1 year and still exhibited contact inhibition. Membrane-bound structures reminiscent of, but no longer strictly identical to, type II cell lamellar cytosomes were seen in cells from these long-term cultures although their numbers were reduced in comparison to lamellar bodies in freshly isolated cells. Mitochondrial numbers and sizes, determined morphometrically, were reduced after culture in comparison to freshly isolated type II cells and those in situ. Phosphatidylcholine was synthesized by these cells and released into the extracellular medium. Application of laser activated electronic sizing data, confirmed by direct micrometry, demonstrated a significant increase in cell size as a function of culture. This sizing data, after prior confirmation by electron microscopy, was used as an aid in identifying type II cells and macrophages in dispersion, especially with those cells derived from rabbit lungs.     

Culture characteristics of cells derived from type II pneumocyte enriched fractions from rabbit and rat

Summary Type II cell enriched fractions were isolated from rabbit and rat lungs using density gradient centrifugation. Cultures established from these fractions contained predominantly cells similar in most morphological respects to type II pneumocytes. These were in continuous replicating culture for 1 year and still exhibited contact inhibition. Membrane-bound structures reminiscent of, but no longer strictly identical to, type II cell lamellar cytosomes were seen in cells from these long-term cultures although their numbers were reduced in comparison to lamellar bodies in freshly isolated cells. Mitochondrial numbers and sizes, determined morphometrically, were reduced after culture in comparison to freshly isolated type II cells and those in situ. Phosphatidylcholine was synthesized by these cells and released into the extracellular medium. Application of laser activated electronic sizing data, confirmed by direct micrometry, demonstrated a significant increase in cell size as a function of culture. This sizing data, after prior confirmation by electron microscopy, was used as an aid in identifying type II cells and macrophages in dispersion, especially with those cells derived from rabbit lungs.     

Studies on the Secretion of Maize Root Cap Slime: IV. Evidence for the Involvement of Dictyosomes

The involvement of dictyosomes and their vesicles in secretion of slime by maize root cap cells is demonstrated by kinetic and organelle fractionation experiments using l-fucose as a specific marker for the secreted slime. Pulse-chase experiments show that l-[1-(3)H]fucose is incorporated into two distinct fractions of root cap cells. Incorporation into a water-soluble, ethyl alcohol-insoluble fraction of the homogenate has a peak at 20 minutes of chasing followed by rapid loss of label. Seventy per cent of the radioactivity in this fraction is secreted from the tissue during a 2-hour chase period. Incorporation of label from [(3)H]fucose into a water-insoluble fraction is kinetically different suggesting that in situ incorporation of label is occurring into the cell wall. Labeling of the water-soluble, ethyl alcohol-insoluble fraction with an (14)C-amino acid mixture differs from that of [(3)H]fucose. Thus, while release of the [(3)H]fucose-containing polymer begins after 10 to 15 minutes of chasing, the release of the (14)C-amino acid polymer is delayed an additional 5 to 10 minutes and occurs at a lower rate. Cesium chloride density gradient centrifugation of secreted material labeled with radioactivity from [(3)H]fucose indicates the presence of only one major component having a buoyant density similar to that of purified root cap slime (1.63 g cm(-3)). Sucrose density gradient centrifugation of homogenates of [(3)H]fucose-labeled root cap tissue shows that radioactivity in nondialyzable material occurs as a broad band between densities 1.12 and 1.18 g cm(-3) with a peak at density 1.15 g cm(-3), the same density at which dictyosomes were localized by electron microscopy. Autoradiography of organelle fractions shows that radioactivity was associated almost exclusively with dictyosomes.     

Novel biphasic traffic of endocytosed EGF to recycling and degradative compartments in lacrimal gland acinar cells

The purpose of this study was to delineate the traffic patterns of EGF and EGF receptors (EGFR) in primary cultured acinar epithelial cells from rabbit lacrimal glands. Uptake of [(125)I]-EGF exhibited saturable and non-saturable, temperature-dependent components, suggesting both receptor-mediated and fluid phase endocytosis. Accumulation of [(125)I] was time-dependent over a 120-min period, but the content of intact [(125)I]-EGF decreased after reaching a maximum at 20 min. Analytical fractionation by sorbitol density gradient centrifugation and phase partitioning indicated that within 20 min at 37 degrees C [(125)I] reached an early endosome, basal-lateral recycling endosome, pre-lysosome, and lysosome. Small components of the label also appeared to reach the Golgi complex and trans-Golgi network. Intact [(125)I]-EGF initially accumulated in the recycling endosome; the content in the recycling endosome subsequently decreased, and by 120 min increased amounts of [(125)I]-labeled degradation products appeared in the pre-lysosomes and lysosomes. Confocal microscopy imaging of FITC-EGF and LysoTrackerRed revealed FITC enriched in a dispersed system of non-acidic compartments at 20 min and in acidic compartments at 120 min. Both confocal immunofluorescence microscopy and analytical fractionation indicated that the intracellular EGFR pool was much larger than the plasma membrane-expressed pool at all times. Cells loaded with [(125)I]-EGF released a mixture of intact EGF and [(125)I]-labeled degradation products. The observations indicate that in lacrimal acinar cells, EGFR and EGF-EGFR complexes continually traffic between the plasma membranes and a system of endomembrane compartments; EGF-stimulation generates time-dependent signals that initially decrease, then increase, EGF-EGFR traffic to degradative compartments.     

Organelle proteomics: identification of the exocytic machinery associated with the natural killer cell secretory lysosome

Natural killer (NK) cells and cytotoxic T lymphocytes eliminate virally infected and transformed cells. Target cell killing is mediated by the regulated exocytosis of secretory lysosomes, which deliver perforin and proapoptotic granzymes to the infected or transformed cell. Yet despite the central role that secretory lysosome exocytosis plays in the immune response to viruses and tumors, little is known about the molecular machinery that regulates the docking and fusion of this organelle with the plasma membrane. To identify potential components of this exocytic machinery we used proteomics to define the protein composition of the NK cell secretory lysosome membrane. Secretory lysosomes were isolated from the NK cell line YTS by subcellular fractionation, integral membrane proteins and membrane-associated proteins were enriched using Triton X-114 and separated by SDS-PAGE, and tryptic peptides were identified by LC ESI-MS/MS. In total 221 proteins were identified unambiguously in the secretory lysosome membrane fraction of which 61% were predicted to be either integral membrane proteins or membrane-associated proteins. A significant proportion of the proteins identified play a role in vesicular trafficking, including members of both the Rab GTPase and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and protein families. These proteins include Rab27a and the SNARE vesicle-associated membrane protein-7, both of which were enriched in the secretory lysosome fraction and represent potential components of the machinery that regulates the exocytosis of this organelle in NK cells.