Studies on the mechanisms of autophagy: maturation of the autophagic vacuole

Data presented in the accompanying paper suggests nascent autophagic vacuoles are formed from RER (Dunn, W. A. 1990. J. Cell Biol. 110:1923-1933). In the present report, the maturation of newly formed or nascent autophagic vacuoles into degradative vacuoles was examined using morphological and biochemical methods combined with immunological probes. Within 15 min of formation, autophagic vacuoles acquired acid hydrolases and lysosomal membrane proteins, thus becoming degradative vacuoles. A previously undescribed type of autophagic vacuole was also identified having characteristics of both nascent and degradative vacuoles, but was different from lysosomes. This intermediate compartment contained only small amounts of cathepsin L in comparison to lysosomes and was bound by a double membrane, typical of nascent vacuoles. However, unlike nascent vacuoles vet comparable to degradative vacuoles, these vacuoles were acidic and contained the lysosomal membrane protein, lgp120, at the outer limiting membrane. The results were consistent with the stepwise acquisition of lysosomal membrane proteins and hydrolases. The presence of mannose-6-phosphate receptor in autophagic vacuoles suggested a possible role of this receptor in the delivery of newly synthesized hydrolases from the Golgi apparatus. However, tunicamycin had no significant effect on the amount of mature acid hydrolases present in a preparation of autophagic vacuoles isolated from a metrizamide gradient. Combined, the results suggested nascent autophagic vacuoles mature into degradative vacuoles in a stepwise fashion: (a) acquisition of lysosomal membrane proteins by fusing with a vesicle deficient in hydrolytic enzymes (e.g., prelysosome); (b) vacuole acidification; and (c) acquisition of hydrolases by fusing with preexisting lysosomes or Golgi apparatus-derived vesicles.     

Origin of melanosome structures and cytochemical localizations of tyrosinase activity in differentiating epidermal melanocytes of newborn mouse skin

The mode of differentiation of epidermal melanocytes was studied by ultrastructural cytochemistry in the skin of newborn mice of strain C57BL/10J. From observations of epidermal melanoblasts and melanocytes, stage I melanosomes, including both unit membranes and inner matrices, appear to be formed from Golgi vacuoles or rough endoplasmic reticulum (RER). Stage I melanosomes were positive to ammoniacal silver-nitrate reaction in the melanoblasts of 1-day-old mice. All stages of melanosomes were similarly positive in the differentiating melanocytes of 2-day-old mice. However, Golgi apparatus, RER, and vesicles were negative. Therefore, it is conceivable that structural proteins, originated from Golgi vacuoles or RER, are developed into specialized proteins and are detected by this reaction in stage I melanosomes. Stage I melanosomes were dopa-negative in the melanoblasts. Stage I and II melanosomes were similarly negative in the differentiating melanocytes. Thus, the melanoblasts are thought to begin production of stage I melanosomes prior to the onset of tyrosinase activity. In the differentiating melanocytes, dopa-melanin depositions were observed in stage III and IV melanosomes, trans Golgi saccules, and small vesicles derived from these saccules, but not in RER. These vesicles were in contact with, or fused to, melanosomes. These findings suggest that tyrosinase may be transferred by Golgi vesicles into stage I and II melanosomes originating from Golgi vacuoles or RER.     

Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver

Summary Morphologically detectable protein (intramembrane particles) and cholesterol (filipin labelling) in the membranes of autophagic vacuoles and lysosomes were studied in mouse hepatocytes using thin-section and freeze-fracture electron microscopy. Both isolated autophagic vacuoles and lysosomes, and intact tissue blocks were used due to the facts (i) that lysosomes are difficult to recognize in freeze-fracture replicas of intact hepatocytes, and (i) that filipin penetration into the tissue blocks is unsatisfactory. Intramembrane particle density was low in the membranes of early autophagic vacuoles (defined as round-shaped vacuoles in which an inner membrane parallel with the outer limiting membrane was clearly visible). The lysosomal membranes contained considerably more intramembrane particles. Particle-rich lysosomes or other vesicles were observed to fuse with the early autophagic vacuoles. The membranes of nascent autophagic vacuoles with morphologically intact contents were usually not labelled by filipin, whereas the membranes of all other autophagic vacuoles and lysosomes were heavily labelled. The increased cholesterol in the membranes of slightly older autophagic vacuoles is presumably derived from cholesterol-rich lysosomes or other vesicles fusing with the vacuoles and from the degrading organelles inside the autophagic vacuoles.     

Studies on vinblastine-induced autophagocytosis in mouse liver. II. Origin of membranes and acquisition of acid phosphatase

The origin of the membranes of autophagic vacuoles (AV) and acquisition of acid phosphatase into AV's were studied in vinblastine-induced autophagocytosis (VBL, 50 mg/kg, i.p.) in mouse hepatocytes. Using unbuffered OsO4, very intense staining was observed in the outer cisternae of the Golgi apparatus and also frequently in the cavity between the double membranes obviously destined to form AV's as well as in the cavity between the double membranes of newly formed AV's. There may occur a transformation process in the membranes limiting an AV analogous to that observed at the Golgi cisternae. The transformation of the outer AV membrane occurs independently of fusion with lysosomes. Inosine diphosphatase activity was localized within the cisternae and on the membranes of the endoplasmic recticulum and occasionally within the innermost cisterna of the Golgi apparatus. The results together with the unbuffered OsO4-staining pattern suggest that the membranes of most AV's are derived from the transformed smooth surfaced cisternae of the endoplasmic reticulum which do not have inosine diphosphatase activity. Acid phosphatase activity was localized in lysosomes, occasionally within the innermost cisternae of the Golgi apparatus, between the double membranes of a few newly formed AV's and within most older single membranes of a few newly formed AV's and within most older single membrane-limited AV's. VBL did not prevent the fusion of lysosomes with AV's.     

Herpes simplex virus 1 envelopment follows two diverse pathways

Herpesvirus envelopment is assumed to follow an uneconomical pathway including primary envelopment at the inner nuclear membrane, de-envelopment at the outer nuclear membrane, and reenvelopment at the trans-Golgi network. In contrast to the hypothesis of de-envelopment by fusion of the primary envelope with the outer nuclear membrane, virions were demonstrated to be transported from the perinuclear space to rough endoplasmic reticulum (RER) cisternae. Here we show by high-resolution microscopy that herpes simplex virus 1 envelopment follows two diverse pathways. First, nuclear envelopment includes budding of capsids at the inner nuclear membrane into the perinuclear space whereby tegument and a thick electron dense envelope are acquired. The substance responsible for the dense envelope is speculated to enable intraluminal transportation of virions via RER into Golgi cisternae. Within Golgi cisternae, virions are packaged into transport vacuoles containing one or several virions. Second, for cytoplasmic envelopment, capsids gain direct access from the nucleus to the cytoplasm via impaired nuclear pores. Cytoplasmic capsids could bud at the outer nuclear membrane, at membranes of RER, Golgi cisternae, and large vacuoles, and at banana-shaped membranous entities that were found to continue into Golgi membranes. Envelopes originating by budding at the outer nuclear membrane and RER membrane also acquire a dense substance. Budding at Golgi stacks, designated wrapping, results in single virions within small vacuoles that contain electron-dense substances between envelope and vacuolar membranes.     

The relative thickness of intracellular membranes in epithelial cells of the ventral lobe of the rat prostate

Synopsis The relative thickness of intracellular membranes of epithelial cells in the ventral lobe of the rat prostrate was measured by a densitometric method. Glutaraldehyde perfusion followed by ruthenium tetroxide immersion fixation appeared to be the most suitable method for membrane thickness measurements. By thickness, the membranes could be roughly subdivided into three groups. The inner and outer membranes of the mitochondrion made up the thinnest membranes of the cell. The second group of membranes consisted of the membranes of the rough-surfaced endoplasmic reticulum and the Golgi apparatus, the different faces of the latter organelle, and the Golgi vesicles. The thickest group of membranes included those of the cell membrane, secretory granules, condensing vacuoles, lysosomes, autophagic vacuoles and multivesicular bodies. The differences in thickness of the membranes are probably due to the varying protein/lipid ratio, and the qualities and proportions of the different lipids in the membranes.     

Studies on vinblastine-induced autophagocytosis in mouse liver

Summary The origin of the membranes of autophagic vacuoles (AV) and acquisition of acid phosphatase into AV's were studied in vinblastine-induced autophagocytosis (VBL, 50 mg/kg, i.p.) in mouse hepatocytes. Using unbuffered OsO₄, very intense staining was observed in the outer cisternae of the Golgi apparatus and also frequently in the cavity between the double membranes obviously destined to form AV's as well as in the cavity between the double membranes of newly formed AV's. There may occur a transformation process in the membranes limiting an AV analogous to that observed at the Golgi cisternae. The transformation of the outer AV membrane occurs independently of fusion with lysosomes. Inosine diphosphatase activity was localized within the cisternae and on the membranes of the endoplasmic reticulum and occasionally within the innermost cisterna of the Golgi apparatus. The results together with the unbuffered OsO₄-staining pattern suggest that the membranes of most AV's are derived from the transformed smooth surfaced cisternae of the endoplasmic reticulum which do not have inosine diphosphatase activity. Acid phosphatase activity was localized in lysosomes, occasionally within the innermost cisternae of the Golgi apparatus, between the double membranes of a few newly formed AV's and within most older single membrane-limited AV's. VBL did not prevent the fusion of lysosomes with AV's.This research was supported by grants from the Ellen and Artturi Nyyssönen Foundation and the Heikki and Hilma Honkanen Foundation     

Filipin labelling and intramembrane particles on the membranes of early and later autophagic vacuoles in Ehrlich ascites cells

Cholesterol and intramembrane particle distribution on autophagic vacuole membranes was studied in Ehrlich ascites cells using filipin labelling and freeze-fracture electron microscopy. Unsaturated fatty acids were stained using imidazole-buffered osmium tetroxide. Autophagocytosis was induced with vinblastine, and early autophagic vacuoles were accumulated by lowering the ATP level in the cells with iodoacetate. Filipin labelling was observed in the limiting membranes of later, apparently hydrolase-containing autophagic vacuoles, whereas the most newly-formed, double-membrane limited vacuoles were not labelled. The limiting membranes of late, residual body-type vacuoles either showed patchy filipin-induced deformation or were completely smooth. Imidazole-buffered osmium tetroxide stained the membranes of newly-formed or developing autophagic vacuoles partly or entirely. The membranes of older vacuoles stained more weakly. Intramembrane particle density on the P-face of the outer limiting membranes of newly-formed autophagic vacuoles was similar to that on endoplasmic reticulum, and the density seemed to increase slightly later on. The size of the P-face particles increased when the vacuoles became older. The limiting membranes of late, residual body-type vacuoles were almost smooth. The inner limiting membranes and the membranes inside the autophagic were always almost particle-free. In conclusion, the amount of cholesterol, unsaturated fatty acids and protein in autophagic vacuole membranes changes during vacuole maturation.     

Immunocytochemical study of the surrounding envelope of autophagic vacuoles in cultured rat hepatocytes

By the use of electron immunoperoxidase cytochemistry at the ultrastructural level, the relationship of the surrounding sac of the autophagic vacuoles to the different cytomembranes was studied. When the endoplasmic reticulum was completely stained for microsomal carboxyesterase E1, the enzyme was not found to be labeled in the developed envelopes forming autophagic vacuoles. The autophagic envelope at the formative stages was also devoid of albumin which intensely stained Golgi cisternae. However, although it was rare, the endoplasmic reticulum showed an electron-lucent region like an early autophagic envelope in its cisternae which was lacking in carboxyesterase E1. In addition, deeply curving swelled cisternae where carboxyesterase E1 was found at the edges were occasionally encountered. These observations suggest that the segregating membranes arise from an endoplasmic reticulum and the structural characteristics of the endoplasmic membranes change at very early stages of formation of autophagic vacuoles. Acid phosphatase, a lysosomal marker enzyme, began to be localized on sections of the double membranes of newly created autophagic vacuoles. The enzyme spread all along the limiting membranes of the autophagic vacuoles, while, at the same time, the double membranes were converted into a single membrane. A lysosomal membrane glycoprotein (LGP107) was also localized on the surrounding envelope of autophagic vacuoles in a fashion similar to that of acid phosphatase. Lysosomal hydrolases seem to play some role in the conversion of double limiting membranes into a single limiting membrane.     

The role of the Golgi complex in the isolation and digestion of organelles

The origin of the membranes and lytic enzymes involved in autophagy has been studied in metamorphosing insect fat body.The Golgi complex has two functions in the organelle destruction which takes place when fat body cells change their activities. (1) It gives rise to envelopes which externalize organelles scheduled for destruction. Microbodies, mitochondria and rough endoplasmic reticulum are sequentially removed from the cytoplasm by investment in isolation membranes. During the isolating phase, isolation membranes have the same osmiophilia as the outer saccular and microvesicular components of the Golgi complex, they do not contain lytic enzymes and they are specific in their adhesion to organelles scheduled for destruction. (2) The Golgi complex gives rise to lytic enzymes. Primary lysosomes which contain acid phosphatase fuse with the isolation bodies formed from invested organelles to become autophagic vacuoles. During this lytic phase, acid phosphatase is present in the inner saccules and microvesicular components of the Golgi complex, in the primary lysosomes seen fusing with isolation bodies and in autophagic vacuoles.     

Light and electron microscopic immunocytochemical localization of clathrin in rat cerebellum and kidney

The precise cellular and subcellular locations of coated vesicle protein, clathrin, in rat kidney and cerebellum have been visualized by immunocytochemical techniques. In the renal tubular epithelia, clathrin-positive products were found on both free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No clathrin was observed in the cisternae of RER or the Golgi apparatus. Clathrin-positive reaction products could also be seen on coated pits, coated vesicles, Golgi-associated vesicles, basolateral cell membrane, the ground substance, and in the autophagic vacuoles. In cerebellar Purkinje and granule cell bodies, reaction products were seen localized on coated vesicles, on the budding areas from the Golgi-associated membrane and Golgi-associated vesicles. Furthermore, the membrane of the multivesicular body, the bound-ribosomes, and the ground substance were also stained. In the myelinated axon, the clathrin appeared to be concentrated on certain segments and seemed to fill in the space between neurotubules and some vesicles. In certain synaptic terminals clathrin was often seen attached to presynaptic vesicles, presynaptic membrane, and post-synaptic membrane. However, in most mossy fibers, some synaptic vesicles were not stained. These observations suggest that clathrin is synthesized on bound and free ribosomes and discharged into the cytosol where it becomes associated with a variety of ground substances and assembles on coated pits, coated vesicles, Golgi-associated vesicles, presynaptic vesicles, and pre- and postsynaptic membranes. Clathrin may be finally degraded in autophagic vacuoles.     

Biosynthesis of lipoprotein: location of nascent apoAI and apoB in the rough endoplasmic reticulum of chicken hepatocytes

Our previous studies showed that in hepatic RER of young chickens, nascent apoAI is not associated with lipoprotein particles and only becomes part of these lipoprotein structures in the Golgi. In this study, we have used three different methodologies to determine the locations of apoAI and apoB in the RER and compared them to that of albumin. Immunoelectron microscopic examination of the RER cell fractions showed that both apoAI and apoB were associated only with the RER membrane whereas albumin was located both within the lumen and on the limiting membrane of the vesicles. To examine the possibility of membrane integration of nascent apoAI and apoB in the RER, we administered L-[3H]leucine to young chickens for 10 min, isolated RER, treated this cell fraction with buffers of varying pH, and measured the release of radioactive albumin, apoAI, and apoB. The majority of nascent apoAI (64%), nascent apoB (100%), and nascent albumin (97%) was released from RER vesicles at pH 11.2, suggesting that, like albumin, apolipoproteins are not integrated within the membrane. To determine if nascent apoproteins are exposed to the cytoplasmic surface, we administered L-[3H]leucine to young chickens and at various times isolated RER and Golgi cell fractions. Radioactive RER and Golgi cell fractions were treated with exogenous protease and the percent of nascent apoAI and apoB accessible to proteolysis was determined and compared to that of albumin. At 5, 10, and 20 min of labeling, 35-56% of nascent apoAI and 60-75% of apoB in RER were degraded, while albumin was refractive to this treatment. At all times both apolipoproteins and albumin present in Golgi cell fractions were protected from proteolysis. These biochemical and morphological findings indicate that apoAI and apoB are associated with the rough microsomal membrane and are partially exposed to the cytoplasmic surface at early stages of secretion. They may later enter the luminal side of the ER and, on entering the Golgi, form lipoprotein particles.     

Studies on vinblastine-induced autophagocytosis in mouse liver. IV. Origin of membranes

The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

Studies on vinblastine-induced autophagocytosis in mouse liver

Summary The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS : II. The Origin and Fate of Glycerol3H-Labeled Phospholipids of Cellular Membranes

The membranes of Acanthamoeba palestinensis were studied by examination in fixed cells, and then by following the movements of glycerol-³H-labeled phospholipids by cell fractionation. Two previously undescribed structures were observed: collapsed cytoplasmic vesicles of cup shape, and plaques in food vacuole and plasma membrane similar in size to the collapsed vesicles. It appeared that the plaques formed by insertion of collapsed vesicles into membranes and/or that collapsed vesicles formed by pinching off of plaques. Fractions were isolated, enriched with nuclei, rough endoplasmic reticulum (RER), plasma membrane, Golgi-like membranes, and collapsed vesicles. The changes in specific activity of glycerol-³H-labeled phospholipids in these membranes during incorporation, turnover, and after pulse-labeling indicated an ordered sequence of appearances of newly synthesized phospholipids, first in nuclei and RER, then successively in Golgi membranes, collapsed vesicles, and finally, plasma membrane. In previous work we had found no large nonmembranous phospholipid pool in A. palestinensis. These observations are consistent with the hypothesis that membrane phospholipids are synthesized, perhaps as integral parts of membranes, in RER and nuclei. Subsequently, some of the newly synthesized phospholipids are transported to the Golgi complex to become integrated into the membranes of collapsed vesicles, which are precursors of the plasma membrane. Collapsed vesicles from the plasma membrane by inserting into it as plaques. When portions of the plasmalemma from food vacuoles, collapsed vesicles pinch off from their membranes and are recycled back to the cell surface.     

On the heterogeneous glycosylation of the membranes of the trans Golgi network in rabbit luteal cells

In rabbit luteal cells the transmost element (G2) of the Golgi apparatus bears cytochemical resemblances to the limiting membrane of lysosomes and it was suggested that lysosomal membranes may originate from the above element. But in the normal Golgi apparatus it cannot be made out whether the considered molecules are indeed membrane bound. Perfusing the rabbit ovary with buffer containing monensin or ammonium chloride allowed to vesiculate the trans Golgi network (G2-G1) selectively. Controls showed a well-preserved ultrastructure. Parts of the limiting membrane of the vacuoles derived from the transmost reticulum (G2) were spiny coated and carried an osmiophilic inner layer. They also showed a heavy precipitate for acid phosphatase (AcPase) and were strongly stained with phosphotungstic acid (PTA) at low pH. By neutralizing the acidic groups, involved in the PTA-staining, it was possible to show that the same membranes were more heavily glycosylated. The MvB's and the limiting membrane of lysosomes showed the same staining characteristics. The other membrane domains revealed a gradient in PTA staining and in AcPase activity. It is concluded that the trans Golgi network (G2-G1) is an acidic compartment. The presence of differentially glycosylated membranes reveals a sorting mechanism for membranous components. The highly glycosylated membrane stretches seem to be involved in endocytosis and in the formation of lysosomal membranes.     

The function of the golgi complex in transitional epithelium. Synthesis of the thick cell membrane

The superficial squamous cells of rat transitional epithelium are limited, on their luminal face, by an asymmetrically thickened membrane. Patches of similar thick membrane are found in the walls of the Golgi cisternae and it is suggested that the Golgi system is the site of assembly of the thick plasma membrane. This implies membrane flow from the Golgi apparatus to the cell surface, and there is indirect evidence that the membrane is transported in the form of fusiform vacuoles, derived from the Golgi cisternae, which fuse with, and become part of, the free cell membrane. Uptake of injected Imferon shows that similar, large, thick-walled vacuoles may be formed by invagination of the free cell surface. Some of these vacuoles are subsequently transformed into multivesicular bodies and autophagic vacuoles. The formation of other large heterogeneous bodies is described, and some of these are shown to have acid phosphatase activity.     

Evidence for a novel route of wheat storage proteins to vacuoles

Wheat seed storage proteins are deposited in protein bodies (PB) inside vacuoles, but their subcellular site of aggregation and their route to vacuoles are still controversial. In the present work, an ultra structural analysis of developing wheat endosperm at early to mid maturation was performed to address these issues. Golgi complexes were rarely detected, indicating that their role in wheat storage protein transport is limited. In contrast, a considerable amount of PB was detected in the cytoplasm. Many of these PB were surrounded by RER membranes and were enlarged by fusion of smaller PB. Small, electron lucent vesicles were detected around the surfaces of the PB in the cytoplasm, or attached to them, suggesting that such attachments and subsequent fusion of the vesicles with each other lead to the formation of small vacuoles containing PB inclusions. Immunogold labeling with serum raised against yeast-BiP, an ER-localized protein, demonstrated that the wheat BiP homolog was present within the PB in the cytoplasm as well as inside vacuoles. This confirmed that the PB were formed within the RER and that the Golgi complex was not involved in their transport to vacuoles. It is concluded that a considerable part of the wheat storage proteins aggregate into PB within the RER and are then transported as intact PB to the vacuoles by a novel route that does not utilize the Golgi complex.     

Autophagy-related vacuoles in mouse gallbladder epithelium

The mouse gallbladder epithelial cells contain very heterogeneous vacuolar population. In an attempt to classify these vacuoles we identified NADPase and TPPase activity as well as the location of HRP which is used as the endocytotic marker. The results of the present study show that the vacuoles can be classified into three categories: (1) the vacuoles predominantly containing loose membrane coils related to the nascent autophagic vacuoles, (2) vacuoles containing densely packed membranes and exhibiting a positive HRP reaction, indicating the convergence of endocytotic and autophagic pathway, and (3) vacuoles composed of degraded membrane structures and containing the reaction product of NADPase activity, showing that the fusion of the lysosomes with the autophagosome-endosome took place. The highly developed cis, medial and trans Golgi compartments reflect the biosynthetic and endocytotic activity of the gallbladder epithelium.     

On the heterogeneous glycosylation of the membranes of the trans Golgi network in rabbit luteal cells

Summary In rabbit luteal cells the transmost element (G₂) of the Golgi apparatus bears cytochemical resemblances to the limiting membrane of lysosomes and its was suggested that lysosomal membranes may originate from the above element. But in the normal Golgi apparatus it cannot be made out whether the considered molecules are indeed membrane bound. Perfusing the rabbit ovary with buffer containing monensin or ammonium chloride allowed to vesiculate the trans Golgi network (G₂–G₁) selectively. Controls showed a well-preserved ultrastructure.Parts of the limiting membrane of the vacuoles derived from the transmost reticulum (G₂) were spiny coated and carried an osmiophilic inner layer. They also showed a heavy precipitate for acid phosphatase (AcPase) and were strongly stained with phosphotungstic acid (PTA) at low pH. By neutralizing the acidic groups, involved in the PTA-staining, it was possible to show that the same membranes were more heavily glycosylated. The MvB's and the limiting membrane of lysosomes showed the same staining characteristics. The other membrane domains revealed a gradient in PTA staining and in AcPase activity.It is concluded that the trans Golgi network (G₂–G₁) is an acidic compartment. The presence of differentially glycosylated membranes reveals a sorting mechanism for membranous components. The highly glycosylated membrane streches seem to be involved in endocytosis and in the formation of lysosomal membranes.