Endocytosis, intracellular transport, and turnover of anionic and cationic proteins in cultured mouse peritoneal macrophages

It was previously shown that cultured mouse peritoneal macrophages ingest anionic derivatives of horseradish peroxidase (HRP) and ferritin by fluid-phase endocytosis and accumulate them in lysosomes. Cationic derivatives were taken up by adsorptive endocytosis and transported to lysosomes but subsequently appeared also in stacked cisternae, tubules, and vesicles of the Golgi complex. In the present investigation, the effect of molecular net charge on the rate of cellular inactivation of a protein was studied. The results demonstrate that anionized HRP was inactivated at a higher initial rate than cationized HRP. This is in agreement with the finding that the cationic protein partly escaped from the digestive compartment of the cells, that means the lysosomes. The effects of phorbol myristate acetate (PMA)--a diterpene ester and a tumor promoter--and monensin--a carboxylic ionophore and a perturbant of the Golgi complex--on fluid-phase endocytosis of HRP and intracellular transport of cationized ferritin (CF) were also studied. PMA stimulated fluid-phase endocytosis of HRP but did not interfere with transport of CF to the Golgi complex. Contrarily, monensin inhibited uptake of HRP and almost totally blocked transport of CF to the Golgi complex. The findings support the idea that membrane and content of endocytic vesicles are treated separately. The content is emptied into lysosomes where macromolecular material normally is degraded. The membrane becomes part of the lysosomal envelope in connection with endocytic vesicle-lysosome fusion. Subsequently, membrane patches are detached from the lysosomes and reutilized. This is at least partly mediated via the Golgi complex and particularly its tubular and vesicular parts. Since the cationic tracers do not bind to the membrane in a stable way, it is not possible to extend the conclusions to individual membrane constituents.     

Recovery of surface membrane in anterior pituitary cells. Variations in traffic detected with anionic and cationic ferritin

Cells dissociated from rat anterior pituitaries were incubated with native or cationized ferritin (CF) to trace the fate of surface membrane. Native ferritin, which did not bind to the cell surface, was taken up in small amounts by bulk-phase endocytosis and was found increasingly (over 1-2 h) concentrated in lysosomes. CF at 100-fold less concentrations bound rapidly to the cell membrane, was taken up by endocytosis in far greater amounts, and within 15-60 min was found increasingly within multiple stacked Golgi cisternae, around forming secretion granules, and within elements of GERL, as well as within lysosomes. The findings demonstrate that the fate of the tracer--and presumably also that of the surface membrane--varies with the same molecule differing only in net charge: vesicles carrying anionic ferritin (net negative charge) fuse only with elements of the lysosomal system whereas those carrying CF (net positive charge) can fuse not only with elements of the lysosomal system, but also with elements along the secretory pathway (Golgi cisternae and condensing granules) as well.     

Pathways of endocytosis in cultured macrophages. Electron microscopic autoradiographic tracing of iodinated plasma membrane proteins

Lactoperoxidase-mediated iodination at 4 degrees C--an established method for covalent labelling of plasma membrane proteins--and quantitative electron microscopic autoradiography were used to follow the pathways of endocytosis in mouse macrophages in vitro. Directly after the labelling, the autoradiographic grains were concentrated to the cell surface. After warming to 37 degrees C, radioactive material was rapidly internalized into cytoplasmic vesicles and subsequently transferred to lysosomes as well as to the Golgi complex. Maximum grain density (% grains/% volume) over the vesicles was observed after 15 min, over the lysosomes after 30 to 45 min and over the Golgi complex after 30 and 90 min. Throughout the experimental period (120 min), the vesicles showed the largest fraction of intracellular grains, but higher grain densities occurred in lysosomes as well as in stacked Golgi cisternae and Golgi-associated vesicles. In spite of the internalization process, the labelling of the cell surface came to a steady state already after 30 min and at all intervals more than 50% of the autoradiographic grains were localized to this compartment. About 25% of the cell-associated radioactivity was lost rapidly with a half-life of 20 to 25 min and the remaining 75% slowly with a half-life of 7 to 9 h. The results indicate that membrane internalized by endocytosis partly follows a route to the lysosomes and that, additionally, there exists a route to and through the Golgi complex. They further support earlier notions of a bidirectional traffic between the surface and interior of the cell and suggest that recycling of membrane components may take place from endocytic vesicles, lysosomes, as well as the Golgi complex.     

Membrane retrieval in epithelial cells of isolated thyroid follicles.

Follicles from rat and pig thyroid glands were isolated by digestion with collagenase. The epithelial cells of isolated follicles maintain their structural and functional polarity as shown by incorporation of 3H-leucine and autoradiography. To trace the fate of surface membrane, isolated follicles were opened, stimulated with thyrotropin and incubated for various time intervals with cationized ferritin (CF), uncharged dextran, native ferritin (NF), and latex spheres (0.5 mum in diameter) which were either pre-coated with CF or added together with CF. Uncharged dextran and native ferritin did not bind to the luminal cell membrane, were taken up in small amounts and accumulated in lysosomes; anionic NF was not found in Golgi cisternae in contrast to uncharged dextran which occassionally reached a few Golgi stacks. CF bound rapidly and in clusters to the luminal plasmalemma, preferentially to coated pits, was taken up by endocytosis, accumulated in lysosomes after 5 min and reached the Golgi cisternae after 30 min. Latex spheres were taken up by engulfment through fusion of microvilli and reached the lysosomes. CF particles coating the latex spheres may detach at this station and reach the Golgi cisternae. The findings show that the route of small tracers depends on the charge of the tracer, in agreement with results obtained by Farquhar [8]. Vesicles carrying NF can be traced to lysosomes only, whereas vesicles containing uncharged dextran or - more conspicuously -CF also fuse with Golgi membranes. Large tracers (latex beads) reach only the lysosomes, but CF taken up with them may move to Golgi cisternae.     

Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.     

Immuno-electronmicroscopical localization of a microvillus membrane disaccharidase in the human small-intestinal epithelium with monoclonal antibodies

The cellular localization of the human intestinal disaccharidase, sucrase-isomaltase, was visualized in ultrathin cryosections by the use of specific monoclonal antibodies [25] followed by protein A-gold. The principle site of immunoreaction concerned the microvillus membrane, which supports current concepts of the localization of these hydrolases. One antibody against sucrase-isomaltase also showed labeling of the Golgi apparatus, apical vesicles, and lysosomes, but not of the basolateral membrane. The labeling of the Golgi complex was uniform, suggesting the absence of accumulation of sucrase-isomaltase in cisternae during its passage through this organelle. Absence of labeling of the basolateral membrane appears to support the view that newly synthesized sucrase-isomaltase is transferred directly from the Golgi complex to the microvillus membrane, bypassing the basolateral membrane. However, the results do not exclude the possibility of a very rapid passage through the basolateral membrane. A substantial fraction of the sucrase-isomaltase occurred in lysosomes, which indicates that this organelle plays a major role in the catabolism of microvillar hydrolases. Transport of sucrase-isomaltase to lysosomes might occur by endocytosis or via the crinophagic pathway. The latter was previously postulated to reflect a regulatory mechanism at the post-Golgi level for the surface expression of microvillar membrane proteins.     

Sorting nexin 17 accelerates internalization yet retards degradation of P-selectin

The transient appearance of P-selectin on the surface of endothelial cells helps recruit leukocytes into sites of inflammation. The tight control of cell surface P-selectin on these cells depends on regulated exocytosis of Weibel-Palade bodies where the protein is stored and on its rapid endocytosis. After endocytosis, P-selectin is either sorted via endosomes and the Golgi apparatus for storage in Weibel-Palade bodies or targeted to lysosomes for degradation. A potential player in this complex endocytic itinerary is SNX17, a member of the sorting nexin family, which has been shown in a yeast two-hybrid assay to bind P-selectin. Here, we show that overexpression of SNX17 in mammalian cells can influence two key steps in the endocytic trafficking of P-selectin. First, it promotes the endocytosis of P-selectin from the plasma membrane. Second, it inhibits the movement of P-selectin into lysosomes, thereby reducing its degradation.     

SCAMP1 function in endocytosis

Secretory carrier membrane proteins (SCAMPs) are ubiquitous components of recycling vesicles that shuttle between the plasma membrane, endosomes, and the trans-Golgi complex. SCAMPs contain multiple N-terminal NPF repeats and four highly conserved transmembrane regions. NPF repeats often interact with EH domain proteins that function in budding of transport vesicles from the plasma membrane or the Golgi complex. We now show that the NPF repeats of SCAMP1 bind to two EH domain proteins, intersectin 1, which is involved in endocytic budding at the plasma membrane, and gamma-synergin, which may mediate the budding of vesicles in the trans-Golgi complex. Expression of SCAMP1 lacking the N-terminal NPF repeats potently inhibited transferrin uptake by endocytosis. Our data suggest that one of the functions of SCAMPs is to participate in endocytosis via a mechanism which may involve the recruitment of clathrin coats to the plasma membrane and the trans-Golgi network.     

Receptor mediated endocytosis of hemoglobin-haptoglobin, galactosylated serum albumin and polymeric IgA by the liver

Labelled hemoglobin-haptoglobin (ham-hap), galactosylated serum albumin (gal-SA) and polymeric immunoglobulin A (p IgA) were injected intravenously to rats or mice. The labels disappeared from the plasma with a half-time of about 5 min and were almost entirely found associated with the liver where degradation products progressively appear. The uptake of hem-hap and gal-SA are partially saturable as a function of the plasmatic concentration and the uptake of gal-SA can be completely inhibited by the simultaneous injection of asialofetuin. About 45 min after injection to rats, labelled material appears in the bile in amounts corresponding to 3.9% of the injected dose (hem-hap), 2.8% (gal-SA) and 60.1% (p IgA). The molecular weight of the labelled material transferred into the bile has been characterized: it consists almost entirely of intact IgA and for about 60% of intact hem-hap and gal-SA. Cell fractionation experiments indicate that 4 min after injection, the label is associated with components which equilibrate around a density of 1.13 g/cm3 and which dissociate from marker enzymes of Golgi complex, plasma membrane and lysosomes. Longer times after injection (from 20 min for hem-hap and gal-SA to 1 h for p IgA) labelled material appears, within lysosomes. To explain all these data, we suggest that after binding to plasma membrane receptors, the ligands are rapidly interiorized into pinocytic vesicles which fuse with lysosomes. Most of the hem-hap and gal-SA molecules but only part of p IgA would be released and subsequently digested; these vesicles would dissociate from lysosomes and fuse with the biliary membrane where the molecules still bound to the membrane of the vesicles would be detached and excreted into the bile.     

The charge distribution in the rough endoplasmic reticulum/golgi complex transitional area investigated by microinjection of charged tracers

The distribution of microinjected ferritin, ranging in charge from anionic to highly cationic, has been used to indicate differences in surface charge on the rough endoplasmic reticulum and the Golgi complex of intact cells. Highly cationic ferritins (HCF)(HCF1, pI 7.9-9.1; HCF2, pI 8.5-9.4; and HCF3.pI 9.5-10.1) were mostly bound and caused swelling of the rough endoplasmic reticulum. Cationic ferritin (CF) (pI 7.0-8.0) and anionic ferritin (AF) (pI 4.0-4.4) caused no changes in morphology. The distribution of these ferritins in the cytoplasmic space varied with their charge. Significantly more CF was bound to surfaces than was found in the free cytoplasmic space. Conversely, there was significantly more AF in the free cytoplasmic space than close to surfaces. Therefore, the intracellular surfaces are negatively charged. Comparison of the structures in the secretory pathway showed no differences in ferritin binding to transition vesicles, rough endoplasmic reticulum, Golgi saccules or secretory vesicles. The Golgi complex beads are not distinguished by their charge. It is therefore unlikely that charge differences play a role in regulating membrane-membrane interactions in this region of the secretory pathway.     

Cell-free systems to study vesicular transport along the secretory and endocytic pathways

Proteins bound for the cell surface, lysosomes, and secretory storage granules share a common pathway of intracellular transport. After their synthesis and translocation into the endoplasmic reticulum, these proteins traverse the secretory pathway by a series of vesicular transfers. Similarly, nutrient and signaling molecules enter cells by endocytosis, and move through the endocytic pathway by passage from one membrane-bound compartment to another. Little is known about the mechanisms by which proteins are collected into transport vesicles, or how these vesicles form, identify their targets, and subsequently fuse with their target membranes. An important advance toward our understanding these processes has come from the establishment of cell-free systems that reconstitute vesicular transfers in vitro. It is now possible to measure, in vitro, the transport of proteins from the endoplasmic reticulum to the Golgi, between Golgi cisternae, and the formation of transport vesicles en route from the trans Golgi network to the cell surface. Along the endocytic pathway, cell-free systems are available to study clathrin-coated vesicle formation, early endosome fusion, and the fusion of late endosomes with lysosomes. Moreover, the selective movement of receptors between late endosomes and the trans Golgi network has also been reconstituted. The molecular mechanisms of vesicular transport are now amenable to elucidation.     

Flow and shuttle of plasma membrane during endocytosis

A striking feature of endocytosis is the large amount of surface membrane that is brought into the cells through the formation of endocytic vesicles. Little is known about the fate of this membrane material. It is implausible that it would be destroyed in lysosomes, as the rate of turnover of the constituents of plasma membrane is much too low with respect to the rate of endocytosis in all cells studied so far. Conversely, plasma membrane fragments, internalized by endocytosis cannot merely be incorporated in lysosomes, as these organelles have been shown to maintain their size, despite continuous and active endocytosis. We present evidence that plasma membrane antigens, detected by means of specific antibodies, are internalized during endocytosis and reach lysosomes. They are thereafter returned back to cell surface. These results indicate the existence of a shuttle of membrane elements between the cell surface and lysosomes.     

Quantitative ultrastructural, autoradiographic evidence for the magnitude and early involvement of the Golgi apparatus complex in the endocytosis of wheat germ agglutinin by cultured neuroblastoma

Several ligands undergo endocytosis into the Golgi apparatus. We have examined with a quantitative ultrastructural, autoradiographic method the sequential endocytosis of tritiated wheat germ agglutinin (3H-WGA) by cultured murine neuroblastoma cells. Cells were incubated with 3H-WGA for 1 hour at 4 degrees C, washed, and incubated in complete medium without ligand at 37 degrees C for 5, 15, 30, and 120 minutes. At 5 minutes, the optimized sources/micron 2 of neuroblastoma cell area, which represented the grain density of each compartment, were as follows: smooth vesicles and tubules, 1.03 +/- 0.88; Golgi-associated vesicles, i.e., clusters of vesicles within a 1 micron radius of the Golgi cisterns, 1.03 +/- 0.31; Golgi cisterns, less than 0.01; and lysosomes, 0.26 +/- 0.16. At 15 minutes grain densities were: smooth vesicles and tubules, 0.9 +/- 0.34; Golgi-associated vesicles, 1.41 +/- 0.28; Golgi cisterns, 0.73 +/- 0.41; and lysosomes, 0.1 +/- 0.09. At 30 minutes grain densities were: smooth vesicles and tubules, 0.46 +/- 0.46; Golgi-associated vesicles, 1.78 +/- 0.34; Golgi cisterns, 0.89 +/- 0.78; and lysosomes, 0.39 +/- 0.14. At 2 hours, smooth vesicles, tubules, and Golgi cisterns were not labeled, Golgi-associated vesicles were still labeled (0.71 +/- 0.1), and lysosomes were heavily labeled (2.17 +/- 0.22). These results are consistent with the hypotheses that either the Golgi complex (cisterns and associated vesicles) is an early and intermediate step of the endocytosis of 3H-WGA into lysosomes or that it constitutes part of a separate and quantitatively significant pathway of endocytosis of this ligand.     

Intracellular trafficking of lysosomal membrane proteins

Lysosomes are the site of degradation of obsolete intracellular material during autophagy and of extracellular macromolecules following endocytosis and phagocytosis. The membrane of lysosomes and late endosomes is enriched in highly glycosylated transmembrane proteins of largely unknown function. Significant progress has been made in recent years towards elucidating the pathways by which these lysosomal membrane proteins are delivered to late endosomes and lysosomes. While some lysosomal membrane proteins follow the constitutive secretory pathway and reach lysosomes indirectly via the cell surface and endocytosis, others exit the trans-Golgi network in clathrin-coated vesicles for direct delivery to endosomes and lysosomes. Sorting from the Golgi or the plasma membrane into the endosomal system is mediated by signals encoded by the short cytosolic domain of these proteins. This review will discuss the role of lysosomal membrane proteins in the biogenesis of the late endosomal and lysosomal membranes, with particular emphasis on the structural features and molecular mechanisms underlying the intracellular trafficking of these proteins.     

Membrane association, electrostatic sequestration, and cytotoxicity of Gly-Leu-rich peptide orthologs with differing functions

The skins of closely related frog species produce Gly-Leu-rich peptide orthologs that have very similar sequences, hydrophobicities, and amphipathicities but differ markedly in their net charge and membrane-damaging properties. Cationic Gly-Leu-rich peptides are hemolytic and very potent against microorganisms. Peptides with no net charge have only hemolytic activity. We have used ancestral protein reconstruction and peptide analogue design to examine the roles of electrostatic and hydrophobic interactions in the biological activity and mode of action of functionally divergent Gly-Leu-rich peptides. The structure and interaction of the peptides with anionic and zwitterionic model membranes were investigated by circular dichroism with 2-dimyristoyl-sn-glycero-3-phosphatidylcholine or 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol vesicles and surface plasmon resonance with immobilized bilayers. The results, combined with antimicrobial assays, the kinetics of bacterial killing, and membrane permeabilization assays, reveal that Gly, Val, Thr, and Ile can all be accommodated in an amphipathic alpha helix when the helix is in a membrane environment. Binding to anionic and zwitterionic membranes fitted to a 2-stage interaction model (adsorption to the membrane followed by membrane insertion). The first step is governed by hydrophobic interactions between the nonpolar surface of the peptide helix and the membranes. The strong binding of Gly-Leu-rich cationic peptides to anionic membranes is due to the second binding step and involves short-range Coulombic interactions that prolong the residence time of the membrane-inserted peptide. The data demonstrate that evolution has positively selected charge-altering nucleotide substitutions to generate an orthologous cationic variant of neutral hemolytic peptides that bind to and permeate bacterial cell membranes.     

Ultrastructural localization of calcium-activated adenosine triphosphatase (Ca2+-ATPase) in growth-plate cartilage

The electron-microscopic cytochemical localization of calcium-activated adenosine triphosphatase (Ca2+-ATPase) was determined in chick epiphyseal growth-plate cartilage. In the reserve zone, mitochondria and lysosomes contained substantial amounts of reaction product, while the plasma membrane and the Golgi complex showed very weak enzymatic activity, and matrix vesicle membranes did not exhibit the cytochemical reaction. As maturation proceeded, the plasma membrane, Golgi complex, and matrix vesicle membranes also stained and were most intense in the proliferative and early hypertrophic zones. From the hypertrophic to the calcifying zone, cytochemical staining decreased progressively in the plasma membrane, the Golgi complex, and lysosomes, while in some cases mitochondrial reaction product remained intense. Matrix vesicles lost their enzymatic activity at the same time that matrix vesicle calcification commenced. It is proposed that this event allows matrix vesicles to calcify, since efflux of calcium would no longer occur.     

Quantitative aspects of membrane shifts in rat parathyroid cells initiated by decrease in serum calcium

The secretory activity of parathyroid glands in rats was stimulated by decreasing the serum Ca++ concentration through constant intravenous infusion of EGTA. The morphometric analysis of the nuclear and cytoplasmic volume and of the surface area of the rough endoplasmic reticulum, Golgi complex, secretory granules and plasma membrane revealed a membrane shift from secretory granules and Golgi complex to the plasma membrane within 1 hr of calcium depression. Subsequently, between 1 and 3 hr of calcium depression, the membrane shift was from the plasma membrane to the Golgi complex. It is considered likely that these membrane shifts are related to a rise in release of parathyroid hormone by exocytosis and a subsequent increase in retrieval of plasma membrane by endocytosis—probably through the compartment of coated pits and coated and uncoated vesicles.     

Internalization of horseradish peroxidase isozymes by pancreatic acinar cells in vitro

We examined the uptake and fate of four horseradish peroxidase (HRP) isozymes (Type VI, VII, VIII, and IX) in isolated pancreatic acinar cells. The pattern of uptake was similar for all the isozymes examined, with the exception of Type IX. Very little Type IX HRP was internalized by the cells, and what endocytosis did occur was primarily from the apical cell surface in coated vesicles. In contrast, HRP Type VI, VII, and VIII appeared to be endocytosed largely at the basolateral cell surface. Initially, the tracer was found in smooth vesicles and tubules near the plasma membrane. The tubules resembled the basal lysosomes known to be present in these cells. At the early time points, HRP reaction product was also present in multivesicular bodies (MVBs). By 60 min, the HRP was localized in MVBs, vesicles, and tubules adjacent to the Golgi apparatus. By 12 hr after exposure to the isozymes, the tracer was present in small apical vesicles. At no time could reaction product be localized in the rough endoplasmic reticulum, Golgi saccules, or secretory granules. The results of this study suggest that the charge of a soluble-phase marker has little effect on its uptake or intracellular distribution.     

Targeting of lysosomal integral membrane protein LIMP II. The tyrosine-lacking carboxyl cytoplasmic tail of LIMP II is sufficient for direct targeting to lysosomes.

Time course experiments of the localization of rat LIMP II expressed in COS cells show that the protein is transported directly from the Golgi complex to lysosomes. Substitution of the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II for the native cytoplasmic tails of the plasma membrane proteins CD36 and CD8 resulted in straight transport of both proteins to lysosomes. The synthetic tyrosine-containing heptapeptide, RGTGVYG, did not replace the natural carboxyl cytoplasmic tail of LIMP II in its ability to transport both CD36 and CD8 to lysosomes, and the two constructs were transported to and expressed at the plasma membrane. Substitution of the cytoplasmic tails of either CD36 or CD8 for the carboxyl cytoplasmic tail of LIMP II resulted in transport of the mutants to the plasma membrane where they underwent endocytosis before accumulating into lysosomes. The results indicate that a motif contained in the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II is sufficient to target proteins directly from the Golgi complex to lysosomes.     

Prolactin internalisation by the epithelial mammary cell: effects of lysosomotropic agents and transglutaminase inhibitors

Prolactin endocytosis was studied by electron microscopy with 125I-prolactin 125I-hGH (human growth hormone) and prolactin-ferritin. Endocytosis and intracellular transit of the labelled hormone proceeded identically in epithelial cells isolated from the mammary glands of pseudopregnant rabbits and in surviving fragments from mammary glands of lactating rabbits. After binding of the hormone to its receptor, the labelled material was rapidly detectable in vesicles showing an homogeneous aspect; 15 min later part of the labelled material was still localized within the same kind of vesicles, but in addition it appeared to have migrated into microvesicles of the Golgi region and into vesicles of heterogeneous aspect tentatively identified with lysosomes. Endocytosis of bovine serum albumin, labelled with ferritin followed the same intracellular pathway. Native ferritin accumulated in vesicles of various sizes, but seemed excluded from the microvesicles of the Golgi zone. In the presence of lysosomotropic agents labelled prolactin accumulated in cytoplasmic vesicles. In the presence of dansylcadaverine, endocytosis of the labelled material proceeded unimpaired. Conversely, in the presence of bacitracin, the internalisation of labelled prolactin seemed to be reduced. These observations show that the endocytosis of the hormone/receptor complex is linked to membrane movements, which eventually lead to its location within both the Golgi apparatus and the lysosomes.