Use of [3H]raffinose as a specific probe of autophagic sequestration

The trisaccharide [3H]raffinose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, was sequestered autophagically and accumulated in lysosomes and pre-lysosomal vacuoles. In contrast to the disaccharide [14C]sucrose previously used as a sequestration probe, raffinose was not taken up by the mitochondria. The sequestration of raffinose was completely inhibited by the autophagy suppressor 3-methyladenine, stressing its potential utility as a specific probe of hepatocytic autophagy.     

Autophagy, cathepsin L transport, and acidification in cultured rat fibroblasts.

The mechanisms of enzyme delivery to and acidification of early autophagic vacuoles in cultured fibroblasts were elucidated by cryoimmunoelectron microscopic methods. The cation-independent mannose-6-phosphate receptor (MPR) was used as a marker of the pre-lysosomal compartment, and cathepsin L and an acidotropic amine (3-(2,4-dinitroanilino)-3'-amino-N-methyl-dipropylamine (DAMP), a cytochemical probe for low-pH organelles) as markers of both pre-lysosomal and lysosomal compartments. In addition, cationized ferritin was used as an endocytic marker. In ultrastructural double labeling experiments, the bulk of all the antigens was found in vesicles containing tightly packed membrane material. These vesicles also contained small amounts of endocytosed ferritin and probably correspond to the MPR-enriched pre-lysosomal compartment. Some immunolabeling was also visible in the trans-Golgi network. In addition, cathepsin L, DAMP, and large amounts of ferritin were found in smaller vesicles which can be classified as mature lysosomes. Early autophagic vacuoles were defined as vesicles containing recognizable cytoplasm. MPR, cathepsin L, and DAMP, but not ferritin, were detected in the early vacuoles. Inhibition of the acidification in the early vacuoles by monensin did not prevent the delivery of MPR and cathepsin L. The presence of MPR in the vacuoles suggests that cathepsin L is not delivered to early autophagic vacuoles solely by fusion with mature, MPR-deficient lysosomes. Furthermore, although lysosomes were loaded with endocytosed ferritin, it was not detected in autophagic vacuoles. Either the trans-Golgi network or the MPR-enriched pre-lysosomes may be the main source of enzymes and acidification machinery for the autophagic vacuoles in fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy dependence of different steps in the autophagic-lysosomal pathway

The energy dependence of the autophagic-lysosomal pathway was investigated in isolated rat hepatocytes, using electroinjected [14C]lactose as an autophagy probe and atractyloside to alter intracellular ATP levels. Since autophagocytosed lactose is hydrolyzed in lysosomes, several steps in the pathway could be analyzed. The following observations were made. 1) The overall autophagic degradation of electroinjected [14C]lactose was strongly energy-dependent. More than 85% inhibition was obtained when the ATP content decreased from the control value of 10 mumol/g dry weight to 4 mumol/g dry weight. 2) The initial step, i.e. the autophagic sequestration of [14C]lactose, measured in the presence of vinblastine to prevent transfer of lactose to lysosomes, was as sensitive to small changes in ATP as was the overall lactose degradation. 3) The steady state level of sequestered [14C]lactose remained constant as ATP decreased from 10 to 4 mumol/g dry weight, indicating that the sequestration step and some postsequestrational process were inhibited to a similar extent by ATP depletion. 4) The final step in the pathway, intralysosomal hydrolysis, was measured by allowing [14C]lactose to preaccumulate intralysosomally in the presence of the reversible lysosome inhibitor propylamine. Following propylamine removal and inhibition of further sequestration by 3-methyladenine, ATP-dependent hydrolysis of the intralysosomal [14C]lactose could be demonstrated. However, this hydrolysis step was not as sensitive to small changes in ATP as was the sequestration step or the overall autophagic lactose degradation. Control of the autophagic-lysosomal pathway in response to energy deprivation would therefore not seem to occur at the lysosomal level, but may be exerted both at the sequestration step and at a postsequestrational, prelysosomal step.     

Use of digitonin extraction to distinguish between autophagic-lysosomal sequestration and mitochondrial uptake of [14C]sucrose in hepatocytes.

In isolated rat hepatocytes, electroinjected [14C]sucrose is sequestered both by mitochondria and by autophagosomes/lysosomes. Radioactivity can be selectively extracted from the latter organelles by low concentrations of digitonin, thereby providing a specific bioassay for autophagic sequestration. By including a digitonin extraction step in the assay procedure, autophagic [14C]sucrose sequestration could be shown to be virtually completely (greater than 90%) suppressed by the autophagy inhibitor 3-methyladenine (10 mM), whereas mitochondrial sugar uptake was unaffected. An amino acid mixture likewise suppressed autophagic sequestration very strongly, while having no detectable effect on the mitochondria.     

Amino acid control of autophagic sequestration and protein degradation in isolated rat hepatocytes

Sequestration of the inert cytosolic marker [14C]sucrose by sedimentable organelles was measured in isolated rat hepatocytes made transiently permeable to sucrose by means of electropermeabilization. Lysosomal integrity, protein degradation, autophagic sequestration, and other cellular functions were not significantly impaired by the electric treatment. Hepatocytes sequestered sucrose at an initial rate of approximately 10%/h, which is threefold higher than the estimated rate of autophagic-lysosomal protein degradation. Almost one-third would appear to represent mitochondrial fluid uptake; the rest was nearly completely and specifically inhibited by 3-methyladenine (3MA) and can be regarded as autophagic sequestration. A complete amino acid mixture was somewhat less inhibitory than 3MA, and partially antagonized the effect of the latter. This paradoxical effect, taken together with the high sequestration rate, may suggest heterogeneity as well as selectivity in autophagic sequestration. There was no detectable recycling of sequestered [14C]sucrose between organelles and cytosol. Studies of individual amino acids revealed histidine as the most effective sequestration inhibitor. Leucine may have a regulatory function, as indicated by its unique additive/synergistic effect, and a combination of Leu + His was as effective as the complete amino acid mixture. Asparagine inhibited sequestration only 20%, i.e., its very strong effect on overall (long-lived) protein degradation must partially be due to post-sequestrational inhibition. The lysosomal (amine-sensitive) degradation of short-lived protein was incompletely inhibited by 3MA, indicating a contribution from nonautophagic processes like crinophagy and endocytic membrane influx. The ability of an amino acid mixture to specifically antagonize the inhibition of short-lived protein degradation by AsN + GIN (but not by 3MA) may suggest complex amino acid interactions at the level of fusion between lysosomes and other vesicles in addition to the equally complex interactions at the level of autophagic sequestration.     

Inhibition of autophagic-lysosomal delivery and autophagic lactolysis by asparagine

Overall autophagy was measured in isolated hepatocytes as the sequestration and lysosomal hydrolysis of electroinjected [14C]lactose, using HPLC to separate the degradation product [14C]glucose from undegraded lactose. In addition, the sequestration step was measured separately as the transfer from cytosol to sedimentable cell structures of electroinjected [3H]raffinose or endogenous lactate dehydrogenase (LDH; in the presence of leupeptin to inhibit lysosomal proteolysis). Inhibitor effects at postsequestrational steps could be detected as the accumulation of autophaged lactose (which otherwise is degraded intralysosomally), or of LDH in the absence of leupeptin. Asparagine, previously shown to inhibit autophagic but not endocytic protein breakdown, strongly suppressed the autophagic hydrolysis of electroinjected lactose. Vinblastine, which inhibits both types of degradation, likewise suppressed lactose hydrolysis. Asparagine had little or no effect on sequestration, but caused an accumulation of autophaged LDH and lactose, indicating inhibition at a postsequestrational step. Neither asparagine nor vinblastine affected the degradation of intralysosomal lactose preaccumulated in the presence of the reversible lysosome inhibitor propylamine. However, if lactose was preaccumulated in the presence of asparagine, both asparagine and vinblastine suppressed its subsequent degradation. The data thus indicate that autophagic-lysosomal delivery, i.e., the transfer of autophaged material from prelysosomal vacuoles to lysosomes, is inhibited selectively by asparagine and non-selectively by vinblastine.     

Autophagic-lysosomal and mitochondrial sequestration of [14C]sucrose. Density gradient distribution of sequestered radioactivity

[14C]Sucrose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, was sequestered by sedimentable subcellular particles during incubation of the cells at 37 degrees C. The sedimentation characteristics of particle-associated [14C]sucrose were different from the lysosomal marker enzyme acid phosphatase, suggesting an involvement of organelles of greater size than the average lysosome. Isopycnic banding in isotonic metrizamide/sucrose density gradients resolved two major peaks of radioactivity: a light peak (1.08-1.10 g/ml) coinciding with lysosomal marker enzymes, and a dense peak (1.15 g/ml), coinciding with a mitochondrial marker enzyme. The dense peak was preferentially associated with large-size particles having the sedimentation properties of mitochondria, and it was resistant to the detergent digitonin at a concentration which extracted all of the radioactivity in the light peak. Similarly the autophagy inhibitor 3-methyladenine prevented accumulation of [14C]sucrose in the light peak, while the radioactivity in the dense peak was unaffected. We therefore tentatively conclude that the light peak represents autophagic sequestration of [14C]sucrose into lysosomes (and probably autophagosomes) while the dense peak represents a mitochondrial uptake unrelated to autophagy.     

The effect of leupeptin on intracellular digestion of asialofetuin in rat hepatocytes

The effect of leupeptin on the intracellular distribution of asialofetuin, endocytosed by isolated rat hepatocytes, was studied. By means of sucrose gradient centrifugation it was found that leupeptin led to accumulation of undegraded ¹²⁵I-labeled asialofetuin both in lysosomes and in an organelle of lower density (probably an endocytic vesicle). To decide whether the protease inhibitor interfered with the uptake of asialofetuin into lysosomes we studied its effect on the intracellular distribution of [¹⁴C]sucrose-asialofetuin. Acid-soluble radioactivity formed from [¹⁴C]sucrose-asialofetuin is trapped within the lysosomes and the rate of uptake of this ligand in the lysosomes can therefore be studied. Using [¹⁴C]sucrose-asialofetuin it was found that leupeptin, in addition to inhibiting proteolysis inside the lysosomes, retards the transport of asialofetuin into these organelles. Reduced uptake of asialofetuin into lysosomes was seen only after incubating the cells with leupeptin for more than about 30 min. The leupeptin effect on the transport of asialofetuin may therefore be secondary to accumulation of undegraded substrates inside the lysosomes.     

Influence of starvation,Triton WR-1339 and [131I]-human serum albumin on rat liver lysosomes

The response of rat liver lysosomes to starvation and administration of lysosomotropic agentsviz. Triton WR-1339 and [¹³¹I]-human serum albumin, was assessed in terms of their distribution pattern after isopycnic sucrose density gradient centrifugation. Starvation induced changes in lysosomes appeared to be similar to that produced by the detergent uptake. Both the treatments caused a distinct decline in the equilibration densities of the organelles. On the other hand, injected labelled protein failed to comigrate with the lysosomal markers in starved as well as Triton treated rats and conspicuously remained in a region of high specific gravity in the gradient. These findings indicate retarded fusion between secondary lysosomes and [¹³¹I]-human serum albumin containing phagosomes in the livers of rats subjected to starvation or detergent treatment     

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.     

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.     

Autophagic sequestration of [14C]sucrose introduced into isolated rat hepatocytes by electrical and non-electrical methods

Isolated rat hepatocytes were found to become permeable to [14C]sucrose at 0 degree C under three different conditions: Immediately following their liberation from the collagenase-perfused liver. Following a short incubation under hypoxic conditions. After electropermeabilisation. All three conditions were characterised by the formation of small protuberances (blebs) indicative of localised cell surface damage, and it is possible that the stretched plasma membrane of such blebs acted as a high-permeability region. Disappearance of blebs and restoration of normal plasma membrane impermeability could be achieved by a short (15 min) incubation at 37 degrees C. It could be shown that [14C]sucrose introduced into rat hepatocytes by non-electrical means was autophagically sequestered at the same rate as [14C]sucrose introduced electrically. In both cases the sequestration was inhibited by the specific autophagy inhibitor 3-methyladenine to a similar extent. The subcellular distribution of sequestered isotope in metrizamide/sucrose density gradients was found to be independent of the conditions of its introduction into cells.     

Energy dependence of autophagic protein degradation in isolated rat hepatocytes

The effect of small changes in intracellular ATP on autophagic flux was studied in isolated rat hepatocytes by using inhibitors of ATP production or by varying the metabolic conditions. The following observations were made. There was a linear relationship between endogenous protein degradation and intracellular ATP, the rate of proteolysis declining with decreasing ATP concentrations. 15% of the maximal proteolysis is either independent of ATP or has a very high affinity for this metabolite. There was a linear relationship between the autophagic sequestration of cytosolic [14C]sucrose and intracellular ATP, the sequestration rate decreasing with decreasing ATP concentrations. ATP depletion did not cause release of [14C]sucrose previously sequestered in autophagosomes and lysosomes at high ATP levels. Intracellular accumulation of chloroquine, used as an indicator of the pH inside lysosomes and other acidic cell compartments, diminished with decreasing cellular ATP content. Amino acids inhibited proteolysis without affecting ATP levels or chloroquine accumulation. We conclude from the high sensitivity of autophagy towards relatively small changes in the concentration of intracellular ATP that, besides amino acids, ATP is a very important factor in controlling the rate of autophagy in rat hepatocytes.     

Translocation of photosynthates into vacuoles in spinach leaf protoplasts

A method was developed for the isolation of vacuoles from the mesophyll protoplasts of spinach leaf, employing the discontinuous Ficoll density gradient centrifugation technique. Isolated vacuole preparations were judged to be free from other organellar fractions based on the assays of marker enzyme activities of individual organelles.

Using this isolation method, a time-dependent translocation of ¹⁴C-labeled photosynthates into vacuoles was determined. In contrast to a significant transport of ¹⁴C organic acids such as malate and citrate within 10 to 15 minutes ¹⁴C neutral sugars and amino acids were barely transported into vacuoles during 40 minutes incubation, in spite of the fact that a relatively large amount of these compounds are found in the vacuoles. It was also found that a majority of [¹⁴C]sucrose remains in the cytosol, apparently not actively moving into the vacuoles. Overall results appear to suggest that vacuoles are not actively engaged in photosynthetic carbon metabolism in spinach leaf protoplasts.

     

Evidence that swainsonine pretreatment of rats leads to the formation of autophagic vacuoles and endosomes with decreased capacity to mature to, or fuse with, active lysosomes.

The plant toxin swainsonine causes a variety of biochemical and morphological changes in animal tissues. In rat liver there is an extensive vacuolization which is not accompanied by an accumulation of oligosaccharide. In investigating this proliferation of autophagic vacuoles we have found that swainsonine administration leads to a shift in the density of liver lysosomes as indicated by the distribution of several lysosomal glycosidases in sucrose gradients. Whereas most of these activities are normally found in low density fractions, only a minor portion occurring in high density fractions, the reverse distribution is observed after the administration of microgram doses of swainsonine. Two promoters of the accumulation of autophagic vacuoles, vinblastine and chloroquine, caused the expected increase in very light vacuoles as measured by localization of two acid hydrolases. However, this effect of the two agents was blocked by swainsonine pretreatment. Moreover, swainsonine decreased the degradation of endocytosed asialofetuin and increased the retention of the glycoprotein in very light fractions. These results suggest that vesicle movement and/or fusion is inhibited by the pretreatment with the toxin. That the effects are mediated by a change in vacuolar membrane is suggested by the finding that lysosomes prepared from the livers of swainsonine-fed rats are much more fragile than control lysosomes, more so in metrizamide solutions than in sucrose solutions. The swainsonine may exert its effect through its known ability to alter the biosynthesis of complex glycoproteins, which are abundant and distinctive in lysosomal membranes.     

The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that the NPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(-) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 degrees C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.     

Effect of culture duration on hepatocyte subcellular membranes involved in endocytosis.

The influence of culture duration on some characteristics of hepatocyte subcellular membranes involved in endocytosis was investigated. Activity of enzymes located in plasma membrane, Golgi apparatus and lysosomes increases with time. These modifications are accompanied with several changes in the sedimentation properties of these organelles. Endocytosis of [14C]sucrose and [14C]sucrose-LDL is not affected by culture age. On the contrary, [14C]sucrose-ASF endocytosis strongly decreases in these conditions. These modifications are delayed to some extent by lowering the temperature. Addition to the culture medium of 3-methyladenine (an inhibitor of autophagy), sodium butyrate, dimethylsulfoxide, phenobarbital or nicotinamide does not prevent the decrease of ASF endocytosis caused by culture duration. These results indicate that one must be cautious when extrapolating to liver in vivo, observations on endocytosis obtained with primary culture of hepatocytes.     

Shift of equilibrium density induced by 3,3''-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles

Galactosylated BSA (galBSA) and its conjugate to horseradish peroxidase (galBSA-HRP) enter the galactose-specific pathway of hepatocytes. 10 min after intravenous injection, structures containing either ligand sediment mostly between 33,000 and 3 X 10(6) g X min (LP fraction) and have an equilibrium density of 1.11-1.13 g/ml in sucrose gradients (Quintart, J., P. J. Courtoy, J. N. Limet, and P. Baudhuin, 1983, Eur. J. Biochem., 131:105-112). Such low density fractions, prepared from rats given galBSA-HRP, were incubated for 30 min at 25 degrees C in 5.5 mM 3,3'-diaminobenzidine (DAB) and 11 mM H2O2 in buffered sucrose. Upon equilibration in a second sucrose gradient, the galBSA-HRP distribution shifted towards higher (approximately 1.19 g/ml) density, but the bulk of protein remained at low density. In the absence of H2O2, galBSA-HRP distribution was also found at low density. As observed by electron microscopy, particles equilibrating at higher density after DAB cytochemistry were largely made of vesicles or tubules filled with DAB reaction product. The density shift of galBSA-HRP-containing organelles after incubation with DAB and H2O2 is attributed to the trapping of HRP- oxidized DAB inside the host organelles. If the low density fractions isolated from a rat injected with [3H]galBSA-HRP were mixed in vitro with similar fractions from another rat given [14C]galBSA, the 3H distribution shifted after DAB cytochemistry, but the 14C distribution was essentially unaffected. By contrast, if both derivatives were injected simultaneously, a concomitant density shift was observed. In conclusion, the DAB-induced density shift was specific to ligand-HRP- containing organelles. The potentials of the method include the purification of HRP-containing particles and the study of their association to ligands, fluid-phase tracers, or marker enzymes.     

Sucrose transport by the Escherichia coli lactose carrier.

Several lines of evidence suggest that sucrose is transported by the lactose carrier of Escherichia coli. Entry of sucrose was monitored by an osmotic method which involves exposure of cells to a hyperosmotic solution of disaccharide (250 mM). Such cells shrink (optical density rises), and if the solute enters the cell, there is a return toward initial values (optical density falls). By this technique sucrose was found to enter cells at a rate approximately one third that of lactose. In addition, the entry of [14C]sucrose was followed by direct analysis of cell contents after separation of cells from the medium by centrifugation. Sucrose accumulated within the cell to a concentration 160% of that in the external medium. The addition of sucrose to an anaerobic suspension of cells resulted in a small alkalinization of the external medium. These data are consistent with the view that the lactose carrier can accumulate sucrose by a proton cotransport system. The carrier exhibits a very low affinity for the disaccharide (150 mM) but a moderately rapid Vmax.     

Phycomyces: discovery of the aiming error in the avoidance response

Vacuoles were prepared from germinating castor bean endosperm (Ricinus communis var Hale) and purified by filtration through a cotton layer under physiological osmolarity. The purity of vacuoles prepared by this method was comparable with that prepared by a sucrose step gradient centrifugation reported in a previous paper (Nishimura, Beevers 1978 Plant Physiol 62: 44-48). It was shown by assays of marker enzymes that the final preparation contained trace contamination of other organelles (glyoxysomes, mitochondria, and endoplasmic reticulum) and the cytosol. The isolated vacuoles were stained with neutral red, indicating that the intravacuolar pH is acidic. Intravacuolar pH of isolated vacuoles was determined by measuring the distribution of [¹⁴C]methylamine in the vacuoles and by directly measuring the pH of vacuolar extracts. The pH of isolated vacuolar extracts was 5.7 to 5.9. Similar values were obtained by the methylamine method and it was shown that intravacuolar pH increased as the pH of the medium was increased.