Effect of glycyl-L-phenylalanine 2-naphthylamide on invertase endocytosed by rat liver.

The release by glycyl-L-phenylalanine 2-naphthylamide (Gly-L-Phe-2-NNap) of endocytosed invertase associated with the MLP fraction (sum of the M, L and P fractions [de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955) Biochem. J. 63, 604-617]) of rat liver was investigated and compared with the release of cathepsin C. The percentage of invertase released increases with time after the enzyme injection, whereas the release of cathepsin C is not influenced by this treatment and corresponds to 85-90% of the total activity of the enzyme. It takes about 2h to attain a similar release of both enzymes. The quantity of invertase releasable or not by Gly-L-Phe-2-NNap was plotted against the time after the injection. Results agree well with the hypothesis that unreleasable invertase is associated with a pre-lysosomal compartment, whereas releasable invertase is present in lysosomes. A kinetic analysis indicates that invertase enters the pre-lysosomal compartment with a zero-order rate constant of 0.48 unit/min per g fresh wt., and leaves this compartment with a first-order rate constant of 0.042 min-1.     

Intracellular transport of endocytosed proteins in rat liver endothelial cells.

1. Receptor-mediated endocytosis of mannose-terminated glycoproteins in rat liver endothelial cells has been followed by means of subcellular fractionation and by immunocytochemical labelling of ultrathin cryosections after intravenous injection of ovalbumin. For subcellular-fractionation studies the ligand was labelled with 125-tyramine-cellobiose adduct, which leads to labelled degradation products being trapped intracellularly in the organelle where the degradation takes place. 2. Isopycnic centrifugation in sucrose gradients of a whole liver homogenate showed that the ligand is sequentially associated with three organelles with increasing buoyant densities. The ligand was, 1 min after injection, recovered in a light, slowly sedimenting vesicle and subsequently (6 min) in larger endosomes. After 24 min the ligand was recovered in dense organelles, where also acid-soluble degradation products accumulated. 3. Immunocytochemical labelling of ultrathin cryosections showed that the ligand appeared rapidly after internalization in coated vesicles and subsequently in two larger types of endosomes. In the 'early' endosomes (1 min after injection) the labelling was seen closely associated with the membrane of the vesicle; after 6 min the ligand was evenly distributed in the lumen. At 24 min after injection the ligand was found in the lysosomes. 4. A bimodal distribution of endothelial cell lysosomes with different buoyant densities was revealed by centrifugation in iso-osmotic Nycodenz gradients, suggesting that two types of lysosomes are involved in the degradation of mannose-terminated glycoproteins in liver endothelial cells. Two populations of lysosomes were also revealed by sucrose-density-gradient centrifugation after injection of large amounts of yeast invertase. 5. In conclusion, ovalbumin is transferred rapidly through three endosomal compartments before delivering to the lysosomes. The degradation seems to take place in two populations of lysosomes.     

Fate of asialofetuin endocytosed by rat liver

We have investigated the endocytosis by rat liver of asialofetuin coupled to [125I] tyramine cellobiose: [125I] TCASF. Subcellular distribution of radioactive compounds was established after differential and isopycnic centrifugation and by analysing the fractions by SDS electrophoresis. Labelling secondary lysosomes was performed by injecting rats with Triton WR 1339 four days before injecting the protein. Results show that after being associated with endosomes [125I] TCASF is recovered in organelles where they are subjected to a first degradation, the density of these organelles is practically not affected by Triton WR 1339 injection. Later the degradation products are associated with lysosomes whose density is markedly lowered by Triton WR 1339 treatment. These observations suggest that the first intracellular organelles where [125I] TCASF is subjected to digestion are distinct from the secondary lysosome population. This could be in agreement with the hypothesis that supposes that endosomes acquire enzymes from primary lysosomes before fusion with secondary lysosomes.     

Effect on lysosomes of invertase endocytosed by rat-liver

The intracellular localization of invertase endocytosed by rat liver was investigated by analytical centrifugation in sucrose and Percoll gradients of mitochondrial fractions originating from rats killed 15 h after injection. After isopycnic centrifugation in a sucrose gradient, invertase is located in higher density zones than acid hydrolases. The difference between the distribution of invertase and that of acid hydrolases increases with the amount of invertase injected. When the invertase dose is sufficiently high, a change of lysosomal enzyme distribution is clearly visible. It consists in the shift of a proportion of these enzymes to higher density regions where invertase is located. The proportion of hydrolase activity affected by invertase is different for each enzyme measured; it is the least pronounced for acid phosphatase, and most for acid deoxyribonuclease and arylsulfatase. A pretreatment of the rat with Triton WR 1339 considerably decreases the equilibrium density of structures bearing invertase. Nevertheless invertase distribution is quite distinct from that of the bulk of lysosomal enzymes that are recovered in lower density zones of the gradient; on the other hand the invertase injection to rats treated with Triton WR 1339 causes a spreading of the acid hydrolase distribution towards higher density zones. The distribution of acid hydrolases and invertase in a Percoll gradient depends on the sucrose concentration of the solvent. It is shifted towards higher densities when the sucrose concentration increases. The phenomenon is more important for invertase. These results are best explained by supposing that invertase accumulates in a distinct population of lysosomes that can be individualized as a result of the density increase they are subjected to by the invertase they accumulate. It is proposed that these lysosomes mainly originate from non-parenchymal cells of the liver.     

Intracellular transport and degradation of 125I-tyramine cellobiose-labelled low-density lipoprotein endocytosed in vivo in rat liver cells studied by means of subcellular fractionation

The intracellular transport and degradation of in vivo endocytosed 125I-tyramine cellobiose-labelled low density lipoprotein (125I-TC-LDL) in rat liver cells were studied by means of subcellular fractionation in Nycodenz, sucrose and Percoll density gradients, as well as by means of analytical differential centrifugation. Initially, labelled LDL was located in endocytic vesicles of low densities. Subsequently, acid-soluble and acid-precipitable radioactivities were found in organelles with buoyant densities distinctly lower than that of the main peaks of the lysosomal marker enzymes acid phosphatase and N-acetyl-beta-glucosaminidase. These prelysosomal organelles may represent multivesicular bodies (MVBs). Finally, 6 h after injection and onwards, the acid-soluble radioactivity cosegregated completely with the two lysosomal marker enzymes, suggesting that the degradation products were in secondary lysosomes. The rate of intracellular processing of LDL was very slow compared to that of asialoglycoproteins, suggesting that LDL followed a unique intracellular pathway, that may be specific for this type of ligand.     

Degradation of endocytosed insulin in rat liver is mediated by low-density vesicles.

Uptake of 125I-insulin by the liver of intact rats is followed by rapid translocation of label to low-density vesicles. Subcellular-fractionation studies indicate that, although the 125I associated with these vesicles is predominantly trichloroacetic acid-precipitable, there is an acid-soluble component arising from processing of the hormone in vivo. H.p.l.c. analysis indicates that the acid-precipitable 125I is not intact iodoinsulin, but may correspond to 'clipped insulin'. Isolated low-density vesicles degrade the acid-precipitable iodopeptide intravesicularly when incubated at 37 degrees C in iso-osmotic medium at pH7. The rate constant for intravesicular degradation is consistent with the rate of insulin clearance by the liver in vivo. Pretreatment of the rats with chloroquine resulted in a decrease in proteolysis of the iodopeptide within the isolated vesicles.     

Complexes of rat alpha 1-macroglobulin and subtilisin are endocytosed by parenchymal liver cells.

Rat alpha 1-macroglobulin was isolated from plasma. Gel electrophoresis of the denatured and reduced protein showed two bands, with Mr values of 163 000 and 37 000. The large subunit contained an autolytic site. This subunit was also split after reaction of the macroglobulin with trypsin. Electron microscopy showed that the macroglobulin changed towards a more compact conformation after reaction with this proteinase. Subtilisin, or alpha 1-macroglobulin, was labelled with a sucrose-containing radio-iodinated group that stays in lysosomes after endocytosis and breakdown of the tagged protein. After intravenous injection into rats, alpha 1-macroglobulin was cleared from plasma with first-order kinetics, showing a half-life of about 9 h, whereas complexes of alpha 1-macroglobulin and subtilisin were cleared with half-lives of only 3 min. Liver contained about 60% of the label at 30 min after injection of complexes. About 90% of the liver radioactivity was found in parenchymal cells isolated after perfusion of the liver with a collagenase solution. Subcellular fractionation indicated a lysosomal localization of the complexes. We conclude that endocytosis by parenchymal liver cells is the major cause of the rapid clearance of alpha 1-macroglobulin-proteinase complexes from plasma.     

Uptake of yeast invertase by rat liver cells in vivo and in vitro.

Yeast invertase injected intravenously in rats is rapidly taken up by the liver, reaching levels in that organ of 20% or more of the injected dose in about 12 h. At early time points, the bulk of the liver invertase appears in the sedimentable homogenates but, with time, there is a progressive increase in the fraction in the soluble phase, which remains at a constant proportion as the total hepatic invertase declines. The uptake of polyvinylpyrrolidone by the liver is much slower, as is its redistribution to the soluble fraction of homogenates. Separation of cell types from livers containing the markers revealed that the invertase was almost exclusively in the nonparenchymal cell population, while polyvinylpyrrolidone was distributed relatively indiscriminately between parenchymal and nonparenchymal cells. Measurements of uptake of invertase by liver cell preparations in vitro confirmed that nonparenchymal cells were much more active than parenchymal cells in this regard. Furthermore, the process was saturable with the former cell types and inhibitable by α-methylmannoside. Thus, it may be concluded that the uptake of invertase is via fluid pinocytosis in parenchymal cells and adsorptive pinocytosis in the nonparenchymal cells.     

Intracellular pathway followed by the insulin receptor covalently coupled to 125I-photoreactive insulin during internalization and recycling

After it interacts with a specific receptor on the cell surface, insulin is internalized in its target cell by an adsorptive endocytotic process and eventually degraded in lysosomes. It was also recently shown that the initial surface interaction between the hormone and its receptor is followed by an internalization of the receptor, which later is recycled back to the cell surface. In the present study the insulin receptor was tagged with a 125I-photoreactive insulin analogue that can be covalently coupled to the insulin receptor by ultraviolet irradiation. Using this tool we could trace by quantitative electron microscope autoradiography the intracellular pathway followed by this labeled receptor. The quantitative analysis of the intracellular distribution of the labeled material as a function of incubation time at 37 degrees C supports the following sequence of events: association first with clear vesicles, second with multivesicular bodies, third with dense bodies, and fourth, a return to the cell surface via clear vesicles. This insulin receptor recycling process is inhibited by monensin but unaffected by cycloheximide.     

The metabotropic glutamate receptor mGluR5 is endocytosed by a clathrin-independent pathway

Metabotropic glutamate receptors 5 (mGluR5) are members of the growing group C G protein-coupled receptor family. Widely expressed in mammalian brain, they are involved in modulation of the glutamate transmission. By means of transfection of mGluR5 receptors in COS-7 cells and primary hippocampal neurons in culture followed by immunocytochemistry and quantitative image analysis and by a biochemical assay, we have studied the internalization of mGluR5 splice variants. mGluR5a and -5b were endocytosed in COS-7 cells as well as in axons and dendrites of cultured neurons. Endocytosis occurred even in the absence of receptor activity, because receptors mutated in the glutamate binding site were still internalized as well as receptors in which endogenous activity had been inhibited by an inverse agonist. We have measured a constitutive rate of endocytosis of 11.7%/min for mGluR5a. We report for the first time the endocytosis pathway of mGluR5. Internalization of mGluR5 is not mediated by clathrin-coated pits. Indeed, inhibition of this pathway by Eps15 dominant negative mutants did not disturb their endocytosis. However, the large GTPase dynamin 2 is implicated in the endocytosis of mGluR5 in COS-7. mGluR5 is the first shown member of the group C G-protein coupled receptor family internalized by a nonconventional pathway.     

Intracellular pathways of endocytosed transferrin and non-specific tracers in epithelial cells lining the rete testis of the rat

Summary The uptake and pathway of different markers and ligands for fluid-phase, adsorptive and receptor mediated endocytosis were analyzed in the epithelial cells lining the rete testis after their infusion into the lumen of these anastomotic channels. At 2 min after injection, diferric transferrin bound to colloidal gold was seen attached to the apical plasma membrane and to the membrane of endocytic coated and uncoated pits and vesicles. The injection of transferrin-gold in the presence of a 100-fold excess of unconjugated diferric transferrin revealed no binding or internalization of transferrin-gold. Similarly, apotransferrin-gold was neither bound to the apical plasma membrane nor internalized by these cells. These results thus indicate the presence of specific binding sites for diferric transferrin. At 5 min, internalized diferric transferrin-gold reached endosomes. At 15 and 30 min, the endosomes were still labeled but at these time intervals the transferrin-gold also appeared in tubular elements connected to or associated with these bodies or seen in close proximity to the apical plasma membrane. At 60 and 90 min, most of the transferrin-gold was no longer present in these organelles and was seen only exceptionally in secondary lysosomes. These results thus suggest that the tubular elements may be involved in the recycling of transferrin back to the lumen of the rete testis. The coinjection of transferrin-gold and the fluid-phase marker native ferritin revealed that both proteins were often internalized in the same endocytic pit and vesicle and shared the same endosome. However, unlike transferrin, native ferritin at the late time intervals appeared in dense multivesicular bodies and secondary lysosomes. When the adsorptive marker cationic ferritin and the fluid-phase marker albumin-gold were coinjected, again both proteins often shared the same endocytic pit and vesicle, endosome, pale and dense multivesicular body and secondary lysosomes. However, several endocytic vesicles labeled only with cationic ferritin appeared to bypass the endosomal and lysosomal compartments and to reach the lateral intercellular space and areas of the basement membrane. The rete epithelial cells, therefore, appear to be internalizing proteins and ligands by receptor-mediated and non-specific endocytosis which, after having shared the same endocytic vesicle and endosome, appear to be capable of being segregated and routed to different destinations.     

Intracellular localization of aldehyde dehydrogenase in rat liver

1. Distribution of aldehyde dehydrogenase activity in rat liver was studied by measuring the rate of disappearance of acetaldehyde in the presence of each of the subcellular fractions. These were obtained by rough separation of particulate fractions from the soluble portion of the cell, by differential centrifugation, and by isopycnic gradient centrifugation. 2. The maximal rate of acetaldehyde oxidation was 3.7 mumol/min per g, with an apparent K(m) value below 10(-5)m. The highest rate of activity was observed in phosphate buffers of high P(i) concentration (above 60mm). 3. The activity measured was completely dependent on NAD(+). 4. The microsomal fraction and the nuclei were inactive in the assay. Of the total activity 80% was found in the mitochondrial fraction and the remaining 20% in the cytoplasm. 5. The distribution pattern is important from the point of view of acetaldehyde oxidation during ethanol metabolism. The apparent discrepancy of the results obtained by different workers and the localization of acetaldehyde oxidation in vivo is discussed.     

Asymmetric synthesis followed by transmembrane movement of phosphatidylethanolamine in rat liver endoplasmic reticulum

Studies with phospholipase C have indicated that two-thirds of the phosphatidylethanolamine of rat liver endoplasmic reticulum is located in the inner leaflet of the membrane bilayer. Phosphatidyl[¹⁴C]ethanolamine is synthesised in microsomes incubated with CDP[¹⁴C]ethanolamine. Using phospholipase C as a probe we have observed that the labelled phospholipid is initially (1–2 min) concentrated in the ‘outer leaflet’ of the membrane bilayer. The specific activity of this pool of phosphatidylethanolamine was 3.5 times that of the inner leaflet. If, however, the microsomes were opened with 0.4% taurocholate or the French pressure cell to make both sides of the bilayer available to phospholipase C, the phosphatidylethanolamine behaves as a single pool for hydrolysis. On longer incubation, up to 30 min, with CDP[¹⁴C]ethanolamine the specific activity of the outer leaflet phosphatidylethanolamine becomes close to that of the inner leaflet. In chase experiments, in which microsomal phosphatidylethanolamine was labelled by incubation with CDP[¹⁴C]ethanolamine for 1 min, the reaction stopped by addition of calcium, and the microsomes isolated by centrifugation and reincubated, labelled phosphatidylethanolamine was transferred from the ‘outer leaflet’ to the ‘inner leaflet’, so that both were equally labelled. These observations suggest that phosphatidylethanolamine is synthesised at the cytoplasmic leaflet of the endoplasmic reticulum and subsequently transferred across the membrane to the cisternal leaflet of the bilayer. Transmembrane movement is apparently temperature-dependent and independent of continued synthesis of phosphatidylethanolamine.     

Intracellular distribution of 5' nucleotidase in rat liver

The intracellular distribution of 5' nucleotidase was investigated in rat liver by biochemical analysis of cell fractions obtained by differential centrifugation. The enzymatic activity was measured by determination of the inorganic phosphorus liberated from 5' nucleotides. The 5' nucleotidase activity was mainly found in the nuclear and microsomal fractions. An attempt to extract the enzyme from these fractions with Mg(++) ion solutions was unsuccessful. It is concluded that 5' nucleotidase is actually present in the nuclear and microsomal fractions of rat liver cells.