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Tissue remodelling is dependent on the integration of signals that control turnover of ECM (extracellular matrix). Breakdown and endocytosis of collagen, a major component of the ECM, is central to this process. Whereas controlled secretion of matrix-degrading enzymes (such as matrix metalloproteinases) has long been known to mediate ECM breakdown, it is becoming clear that uPARAP/Endo180 (where uPARAP stands for urokinase plasminogen activator receptor-associated protein) serves as a receptor that mediates endocytosis of collagen by several types of cells. In the liver, the stellate cells play a major role in turnover of ECM including collagens. These cells synthesize various collagens and also produce matrix metalloproteinases. In the present study, we investigated the capacity of rat hepatic stellate cells to endocytose and degrade 125I-labelled heat-denatured collagen I. It was found that the collagen is efficiently taken up and degraded by these cells. Degradation was inhibited by inhibitors of lysosomal proteases (leupeptin and E-64d) and the vacuolar proton pump (concanamycin A), indicating that it takes place in lysosomes. Furthermore, endocytosed FITC-labelled collagen was shown to reach late endocytic compartments in which it colocalized with LysoTracker (a marker of late endocytic compartments). Competition experiments showed that uPA and unlabelled collagen are capable of inhibiting binding and uptake of [125I]collagen in a dose-dependent manner. Moreover, Western-blot analysis of cell lysate (using a polyclonal rabbit human-Endo180 antiserum) revealed a single band at 180 kDa. In addition, the antiserum was capable of reducing [125I]collagen binding to the cell surface. Finally, using two primers designed from the human uPARAP/Endo180 mRNA sequence, the expression of uPARAP/Endo180 mRNA was detected by reverse transcriptase-PCR. These results together suggest that uPARAP/Endo180 mediates endocytosis of collagen in rat liver stellate cells.

Human LAMAN (lysosomal a-mannosidase) was synthesized as a 120 kDa precursor in transfected COS cells [African-green-monkey kidney cells], which was partly secreted as a single-chain form and partly sorted to the lysosomes being subsequently cleaved into three peptides of 70, 40 and 15 kDa respectively. Both the secreted and the lysosomal forms contained endo H (endoglucosidase H)-resistant glycans, suggesting a common pathway through the trans-Golgi network. A fraction of LAMAN was retained intracellularly as a single-chain endo H-sensitive form, probably in the ER (endoplasmic reticulum). The inherited lack of LAMAN causes the autosomal recessive storage disease a-mannosidosis. To understand the biochemical consequences of the disease-causing mutations, 11 missense mutations and two in-frame deletions were introduced into human LAMAN cDNA by in vitro mutagenesis and the resulting proteins were expressed in COS cells. Some selected mutants were also expressed in Chinese-hamster ovary cells. T355P (Thr355Pro), P356R, W714R, R750W and L809P LAMANs as well as both deletion mutants were misfolded and arrested in the ER as inactive single-chain forms. Six of the mutants were transported to the lysosomes, either with less than 5% of normal specific activity (H72L, D196E/N and R220H LAMANs) or with more than 30% of normal specific activity (E402K LAMAN). F320L LAMAN resulted in much lower activity in Chinese-hamster ovary cells when compared with COS cells. Modelling into the three-dimensional structure revealed that the mutants with highly reduced specific activities contained substitutions of amino acids involved in the catalysis, either co-ordinating Zn2+ (His72 and Asp196), stabilizing the active-site nucleophile (Arg220) or positioning the active-site residue Asp319 (Phe320).

The process by which clathrin-coated vesicles are produced involves interactions of multifunctional adaptor proteins with the plasma membrane, as well as with clathrin and several accessory proteins and phosphoinositides. Here we review recent findings highlighting new insights into mechanisms underlying clathrin-dependent endocytosis.

To obtain information about the role of phosphoinositide 3-kinase (PI3K) in the endocytic pathway in hepatocytes, the uptake and intracellular transport of asialo-orosomucoid (ASOR) was followed in cells treated with wortmannin or LY294002. The two inhibitors, at concentrations known to inhibit the enzyme, did not affect internalization or the number of surface asialoglycoprotein receptors, but they caused a paradoxical increase (approx. 50% above control values) in the degradation of ASOR labelled with [(125)I]tyramine cellobiose ([(125)I]TC). Wortmannin or LY204002 inhibited the autophagic sequestration of lactate dehydrogenase very effectively, and the enhanced degradation of [(125)I]TC-ASOR could be an indirect effect of reduced autophagy, as an amino acid mixture known to inhibit autophagy also caused increased degradation of [(125)I]TC-ASOR, and its effect was not additive to that of wortmannin or LY294002. Wortmannin or LY294002 had pronounced effects on the late parts of the endocytic pathway in the hepatocytes: first, dense lysosomes disappeared and were replaced by swollen vesicles; secondly, degradation of [(125)I]TC-ASOR took place in an organelle of lower buoyant density (in a sucrose gradient) than the bulk of lysosomes (identified in the gradient by lysosomal marker enzymes). With increasing length of incubation with wortmannin or LY294002, the density distributions of the lysosomal markers also shifted to lower density and gradually approached that of the labelled degradation products. The labelled degradation products formed from [(125)I]TC-labelled proteins were trapped at the site of formation, because they did not penetrate the vesicle membranes. The results obtained indicate that internalization and intracellular transport of ASOR to lysomes may take place in the absence of PI3K activity in rat hepatocytes. On the other hand, fusion of late endosomes with lysosomes seems to produce 'hybrid organelles' (active lysosomes) that are unable to mature into dense lysosomes.

Liver endothelial cells (LECs) play an important homoeostatic role by removing potentially harmful macromolecules from blood. The extremely efficient endocytosis in LECs makes these cells an interesting model for the study of the involvement of phosphoinositides in the different steps of the endocytic process. In the present investigation we have studied the effect of wortmannin, an inhibitor of phosphatidylinositol kinases, on uptake, recycling and intracellular transport of (125)I-labelled ovalbumin, which is taken up in LECs via mannose-receptor-mediated endocytosis. Wortmannin was found to inhibit both uptake and degradation of ovalbumin. Further studies indicated that the reduced uptake via the mannose receptor was due both to a reduction of the number of surface receptors and a reduction in the rate of receptor-ligand internalization. Transport of ligand from endosomes to lysosomes was prevented, leading to increased recycling of internalized ligand. Wortmannin treatment released the Rab5 effector EEA1 from the endosomes and caused reduced size of early endosomes.

In a search for autophagosome-associated proteins, two-dimensional gel separations of proteins from purified autophagosomes, postnuclear supernatant, cytosol, lysosomes, mitochondria, endosomes and a cytomembrane fraction (mostly endoplasmic reticulum) were compared. Three proteins, with monomeric molecular masses of 43, 35 and 31 kDa, were enriched in total or sedimentable fractions of autophagosomes relative to the corresponding fractions of postnuclear supernatant, suggesting an association with the autophagosomal delimiting membrane. These proteins were also present on lysosomal membranes, but they were absent from mitochondria, and detected only in small amounts in the cytomembrane fraction and in endosomes, indicating that they were not associated with organelles sequestered by autophagy. However, all three proteins were present in the cytosol, suggesting that they were cytosolic proteins binding peripherally to the delimiting membrane of autophagosomes, probably to its innermost surface as indicated by their resistance to treatment of intact autophagosomes with proteinase or protein-stripping agents. Amino acid sequencing identified these proteins as an isoform of argininosuccinate synthase, an N-truncated variant of glyceraldehyde-3-phosphate dehydrogenase, and a sequence variant of short-chain 2-enoyl-CoA hydratase.

To investigate the properties and intracellular origin of autophagosomes, a procedure for the purification and isolation of these organelles from rat liver has been developed. Isolated hepatocytes were incubated with vinblastine to induce autophagosome accumulation; the cells were then homogenized and treated with the cathepsin C substrate glycyl-l-phenylalanine 2-naphthylamide to cause osmotic disruption of the lysosomes. Nuclei were removed by differential centrifugation, and the postnuclear supernatant was fractionated on a discontinuous Nycodenz density gradient. The autophagosomes, recognized by their content of autophagocytosed lactate dehydrogenase (LDH), could be recovered in an intermediate-density fraction, free from cytosol and mitochondria. Finally, the autophagosomes were separated from the endoplasmic reticulum and other membranous elements by centrifugation in a Percoll colloidal density gradient, followed by flotation in iodixanol to remove the Percoll particles. The final autophagosome preparation represented a 24-fold purification of autophagocytosed LDH relative to intact cells, with a 12% recovery. The purified autophagosomes contained sequestered cytoplasm with a normal ultrastructure, including mitochondria, peroxisomes and endoplasmic reticulum in the same proportions as in intact cells. However, immunoblotting indicated a relative absence of cytoskeletal elements (tubulin, actin and cytokeratin), which may evade autophagic sequestration. The autophagosomes showed no enrichment in protein markers typical of lysosomes (acid phosphatase, cathepsin B, lysosomal glycoprotein of 120 kDa), endosomes (early-endosome-associated protein 1, cation-independent mannose 6-phosphate receptor, asialoglycoprotein receptor) or endoplasmic reticulum (esterase, glucose-regulated protein of 78 kDa, protein disulphide isomerase), suggesting that the sequestering membranes are not derived directly from any of these organelles, but rather represent unique organelles (phagophores).

alpha-Mannosidosis is a lysosomal storage disorder that is caused by the deficiency of lysosomal alpha-mannosidase. Feline alpha-mannosidosis is a well-characterized animal model used for studying pathological and therapeutic aspects of lysosomal storage disorders. We here report the purification of feline liver lysosomal alpha-mannosidase and determination of its cDNA sequence. The active enzyme consisted of three polypeptides, with molecular masses of 72, 41 and 12 kDa, joined by non-covalent forces. The cDNA sequence of feline lysosomal alpha-mannosidase was determined from reverse transcriptase PCR products obtained from skin fibroblast mRNA. The deduced amino acid sequence contained the N-terminal sequences of the 72 and 41 kDa peptides. This indicated that the enzyme is synthesized as a single-chain precursor with a putative signal peptide of 50 amino acids followed by a polypeptide chain of 957 amino acids, which is cleaved into the three polypeptides of the mature enzyme. The deduced amino acid sequence was 81.1 and 83.2% identical with the human and bovine lysosomal alpha-mannosidases sequences respectively. A 4 bp deletion was identified in an alpha-mannosidosis-affected Persian cat by DNA sequencing of reverse transcriptase PCR products. The deletion resulted in a frame shift from codon 583 and premature termination at codon 645. No lysosomal alpha-mannosidase activity could be detected in the liver of this cat. A domestic long-haired cat expressing a milder alpha-mannosidosis phenotype than the Persian cat had a lysosomal alpha-mannosidase activity of 2% of normal. This domestic long-haired cat did not possess the 4 bp deletion, proving molecular heterogeneity for feline alpha-mannosidosis.

A photoactivatable porphyrin, tetra(4-sulphonatophenyl)porphine (TPPS4), was shown to accumulate in rat hepatocytes as a linear function of dose after intravenous injection, and to localize predominantly in hepatocytic lysosomes. A major fraction of the lysosomal enzymes acid phosphatase and N-acetyl-beta-D-glucosaminidase was inactivated by TPPS4 after 20 h of contact with the drug in vivo in the absence of photoactivation. On exposure of isolated hepatocytes to light, photoactivated TPPS4 caused additional inactivation of the lysosomal enzymes as well as inactivation of intralysosomal lactate dehydrogenase (LDH), a cytosolic enzyme that accumulated in lysosomes as a result of autophagy during a 2 h incubation of hepatocytes at 37 degrees C in the dark (in the presence of the proteinase inhibitor leupeptin to prevent degradation of intralysosomal LDH). Photoactivation of TPPS4 also induced lysosomal rupture, with a loss of lysosomal enzymes, autophagocytosed LDH, endocytosed 125I-tyramine-cellobiose-asialo-orosomucoid and TPPS4 from the lysosomes. However, LDH-containing autophagosomes, accumulated in the presence of vinblastine (a microtubule inhibitor used to prevent the fusion of lysosomes with autophagosomes or endosomes), were not affected by TPPS4. TPPS4 may thus be useful as a selective lysosomal (or endosomal) perturbant in the study of autophagic-endocytic-lysosomal interactions.

T-kininogen, the major kininogen in rat plasma, releases Ile-Ser-bradykinin (T-kinin) when incubated with trypsin, but is not a substrate for tissue kallikrein. Enzymes able to release T-kinins from T-kininogen have been found in the rat submandibular gland, but precise identification of these enzymes and their possible relationship to kallikrein-like enzymes has not been established. We studied T-kininogenase activity in fractionated submandibular gland homogenate. The main T-kininogen catalytic enzyme was purified and characterized, and found to be identical to antigen gamma, a kallikrein-like enzyme which we have previously characterized. Of other identified kallikrein-like enzymes only tonin showed weak T-kininogenase activity, which was about 0.25% of that of antigen gamma. No other T-kininogen catalytic enzymes were observed. Antigen gamma released a kinin which was identified as T-kinin by reverse-phase h.p.l.c. The T-kininogenase activity of antigen gamma had a Km of 29 +/- 4 microM and a kcat/Km of 140 M-1.s-1, and was comparable with its high and low molecular mass-kininogenase activity (7.4 and 10 micrograms of kinin/h per mg respectively). In contrast, tissue kallikrein released 0.2 and 42,200 micrograms of kinin/h per mg respectively. Thus antigen gamma is a weak kininogenase. The isoelectric point of antigen gamma, but not its molecular mass, differed from that of other kallikrein-like enzymes. Isoelectrofocusing in flat-bed gels combined with immunostaining was therefore a convenient method for identification. The kallikrein-like nature of antigen gamma was demonstrated by its immunological similarity to tissue kallikrein and tonin and by 91% and 87% amino acid sequence similarity with tonin and kallikrein respectively (67 amino acids sequenced). Complete identity was also not observed with other sequenced kallikrein genes, mRNAs or proteins.

1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of (14)C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [(14)C]-leucine, -proline and -phenylalanine over a period of 4hr. With [(14)C]leucine, incorporation into gramicidin S takes place in the range pH6-9 with maximum incorporation at pH7.0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [(14)C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.