Fate of plasma membrane during endocytosis. II. Evidence for recycling (shuttle) of plasma membrane constituents

Cultured rat embryo fibroblasts were first allowed to store for 24 h fluorescein-labeled goat immunoglobulins directed against rabbit immunoglobulins (F anti-R IgG), and were subsequently exposed for 24 h to [(3)H]acetylated rabbit immunoglobulins known to bind to the cell membrane either specifically (anti-plasma membrane IgG: A anti-PM IgG) or unspecifically (contol IgG: AC IgG). As a result of immunological interaction between the two antibodies (no effect was found if the cells had been preloaded with control goat FC IgG), a substantial portion of the stored F anti-R IgG was unloaded from its intracellular storage site, appearing in the medium in the form of soluble immune complexes with rabbit A IgG. Part of the unloaded F anti-R IgG also was recovered in association with the plasma membrane, but only when A anti-PM IgG was used. In addition, significant reverse translocation of AC IgG from plasma membrane to lysosomes or some related intracellular storage compartment was also observed. With A anti-PM IgG, this translocation was less marked and affecte at the same time the plasma membrane marker 5’- nucleotidase. Cells that had stored horseradish peroxidase (HRP) simultaneously with F anti-R IgG did not unload HRP when exposed to A anti-PM IgG. These results support strongly, though not unequivocally, the concept that plasma membrane patches interiorized by endocytosis are recycled, or shuttled, back to the cell surface. In the framework of this concept, recycling antibody-coated membrane is taken to serve as vehicle for the selective intracellular capture and extracellular discharge of immunologically bound F anti-R IgG. The alternative explanation of regurgitation triggered off by immune complexes is considered less likely in view of the lack of HRP unloading.     

Fate of plasma membrane during endocytosis. III. Evidence for incomplete breakdown of immunoglobulins in lysosomes of cultured fibroblasts

Rat embryo fibroblasts, when cultured in the presence of control rabbit immunoglobulins (C IgG), doubly labeled by (3)H-acetylation (A) and then conjugated with flourescein (F), take up FAC IgG continuously for at least 72 h. They return the major part of their intake back to the medium in the form of breakdown products of very low molecular weight. Gel filtration and immunological analyses of cells and medium at various times indicate that essentially all the FAC IgG molecules taken up undergo digestion of their Fc part, but that the Fab part of only about three-fourths of the molecules is degraded. The rest remains stored intracellularly in the form of F(ab’)2-type fragments that slowly dissociate into Fab’-type fragments. When FAC IgG was incubated in vitro in the presence of a hepatic lysosomal extract, complete digestion of the Fc part likewise occurred, but the Fab’ part of most if not all the molecules proved resistant to breakdown, and remained as Fab’-type fragments. Cell fractionation experiments have demonstrated that the storage compartment of the FAC IgG and of its digestion residues: (a) shows a density distribution pattern in a sucrose gradient identical to that of the lysosomal marker N-acetyl-β-glucosaminidase and clearly dissociated from that of the Golgi marker galactosyltransferase, and (b) accompanies the lysosomal marker in its density shift induced by exposure of the cells to chloroquine. It is concluded that storage and processing of FAC IgG by rat fibroblasts occur in a single, digestively active compartment of lysosomal nature, and that resistance to digestion of certain Fab’-type fragments accounts largely for the inability of the lysososmal enzymes to completely digest the FAC IgG taken up. This conclusion implies that the intracellular storage compartment through which, in earlier work, plasma membrane patches were found to transit after endocytosis and before recycling to the cell surface consists of authentic lysosomes.     

Fc gamma-receptor-mediated changes in the plasma membrane potential induce prostaglandin release from human fibroblasts

Binding of aggregated human immunoglobulin G (IgG) on diploid human fibroblasts leads to a rapid depolarization of the cells within 1-2 min. We resolved this membrane potential change into its plasma membrane and mitochondrial membrane components by measuring the transmembrane distribution of the lipophilic tritium-labelled cation tetraphenylphosphonium, [3H]Ph4P+. The responsibility of the plasma membrane for the membrane potential change, induced by binding of IgGs, is demonstrated. The IgG-induced membrane depolarization leads to the induction of prostaglandin E2 synthesis. Aggregated immunoglobulins (IgG) are specifically bound via the Fc portion because only binding of Fc fragments, in contrast to (Fab')2 fragments, leads to a stimulation of prostaglandin E2 synthesis comparable to that mediated by IgGs. Depolarization of the plasma membrane by short incubation of the fibroblasts in high-K+ buffer (5 min) results in a stimulation of prostaglandin E2 synthesis comparable to that mediated by either aggregated human IgGs or Fc fragments. Our previous results on Fc gamma-receptor-mediated antigen-IgG-antibody complex internalization showed that a maximum uptake of these complexes could be detected 60-90 min after binding. Therefore, we conclude that not internalisation but binding of aggregated IgGs to the Fc gamma receptors on human fibroblasts is the stimulus for plasma membrane depolarization leading to an enhanced prostaglandin E2 release.     

Effect of chloroquine and methylamine on endocytosis of fluorescein-labelled controlled IgG and of anti-(plasma membrane) IgG by cultured fibroblasts

We report here the effect of chloroquine and methylamine two lysosomotropic drugs, on the binding, uptake and subcellular localization of fluorescein-labelled control immunoglobulin G (control IgG) a marker for non-specific adsorptive endocytosis and of anti-(plasma membrane) IgG (specific IgG), a specific ligand of cell-surface antigens. At 4 degrees C, methylamine and chloroquine inhibit the binding of control IgG to the cell surface, probably by a reversible competition. These two drugs, methylamine more than chloroquine, considerably slow down the rate at which control IgG is transferred from its binding sites on the phagosomal membrane to the lysosomal compartment; both drugs block almost completely the intralysosomal digestion of this IgG as well as the release of degradation products into the culture medium. They do not affect the binding and uptake of the specific IgG. In addition, methylamine seems to inhibit partially the return of the cell surface of membrane antigens and of membrane fragments bearing 5'-nucleotidase or binding sites for control IgG. We conclude that important steps (binding to cell surface, delivery to lysosomes, digestion and recycling of plasma membrane) involved in the uptake and the processing of IgG by fibroblasts are inhibited by these two substances. The effects of lysosomotropic agents on the regulation and function of the endocytic pathway and of lysosomes could have many pharmacological and therapeutic implications.     

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.     

Relations between plasma membrane and lysosomal membrane. 1. Fate of covalently labelled plasma membrane protein

To quantify the kinetics of the plasma membrane flow into lysosomes, we covalently labelled at 4 degrees C the pericellular membrane of rat fibroblasts and followed label redistribution to the lysosomal membrane using purified lysosomal preparations. The polypeptides were, either labelled with 125I by the lactoperoxidase procedure, or conjugated to [3H]peroxidase using bisdiazobenzidine as a bifunctional reagent. Both labels were initially bound to plasma membrane, as indicated by their equilibrium density in sucrose or Percoll gradients and their displacement by digitonin, as well as by electron microscopy. Upon cell incubation at 37 degrees C, both covalent labels were lost from cells with diphasic kinetics: a minor component (35% of cell-associated labels) was rapidly released (half-life less than 1 h), and most label (65%) was released slowly (half-life was 20 h for incorporated 125I and 27 h for 3H). Immediately after labelling up to 30 h after incubation at 37 degrees C, the patterns of 125I-polypeptides quantified by autoradiography after SDS-PAGE were indistinguishable, indicating no preferential turnover for the major plasma membrane polypeptides. The redistribution of both labels to lysosomes was next quantified by cell fractionation. At equilibrium (between 6 and 25 h of cell incubation) 2-4% of cell-associated 125I label was recovered with the purified lysosomal membranes. By contrast, when 3H-labelled cells were incubated for 16 h, most of the label codistributed with lysosomes. However, only 6% of cell-associated 3H was bound to lysosomal membrane. These results indicate that in cultured rat fibroblasts, a minor fraction of plasma membrane polypeptides becomes associated with the lysosomal membrane and is constantly equilibrated by membrane traffic.     

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.     

Divergent fates of P- and E-selectins after their expression on the plasma membrane.

P-selectin and E-selectin are related adhesion receptors for monocytes and neutrophils that are expressed by stimulated endothelial cells. P-selectin is stored in Weibel-Palade bodies, and it reaches the plasma membrane after exocytosis of these granules. E-selectin is not stored, and its synthesis is induced by cytokines. We studied the fate of the two proteins after their surface expression by following the intracellular routing of internalized antibodies to the selectins. By immunofluorescent staining, P-selectin antibody was first seen in endosomes, then in the Golgi region, and finally in Weibel-Palade bodies. In contrast, the E-selectin antibody was detected only in endosomes and lysosomes. Subcellular fractionation of cells after 4 h chase confirmed the localization of P-selectin antibody in storage granules and of the E-selectin antibody in lysosomes. In AtT-20 cells, a mouse pituitary cell line, transfected with P- or E-selectin, only P-selectin was delivered to the endogenous adrenocorticotrophic hormone storage granules after endocytosis. Deletion of the cytoplasmic domain abolished internalization. In summary, after a brief surface exposure, internalized E-selectin is degraded in the lysosomes, whereas P-selectin returns to the storage granules from where it can be reused.     

Rod/cone dysplasia in Irish setters. Presence of an altered rhodopsin.

The subcellular distribution of beta-glucuronidase acquired by deficient human fibroblasts during co-culture with peritoneal macrophages was compared with that taken up by receptor-mediated endocytosis. Labelled enzyme taken up via receptors was located initially in a low-density endosomal fraction and was transferred to lysosomes within a few minutes. The beta-glucuronidase acquired during 24 h of co-culture was present almost entirely within lysosomes and had a distribution profile identical with that of endogenous beta-hexosaminidase. Monensin prevented transfer of radiolabelled enzyme from endosomes to lysosomes and had a similar effect on the distribution of enzyme acquired by direct transfer, causing beta-glucuronidase to accumulate within endosomes. When the temperature was lowered from 37 degrees C to 19 degrees C, the rate of transfer of enzyme from endosomes to lysosomes was decreased during both direct transfer and indirect receptor-mediated endocytosis. These results show that a lysosomal enzyme acquired by direct transfer during cell-to-cell contact follows a similar intracellular route and has a similar distribution to that of enzymes taken up via cell-surface receptors.     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages. Chloroquine and monensin inhibit transfer of the ligand from endocytic vesicles to lysosomes

Earlier studies have shown that immunoglobulin G (IgG)-coated colloidal gold particles bind to specific receptors on the macrophage surface and accumulate in coated pits. They are then internalized via endocytic vesicles and transferred to lysosomes. During this process the plasma membrane is depleted of binding sites for IgG, suggesting that both the receptor and the ligand end up in lysosomes. Here, we have examined the effects of the weak base chloroquine and the Na+-H+ ionophore monensin on endocytosis and intracellular transport of IgG-coated colloidal gold particles in cultured macrophages. The results indicate that chloroquine and monensin do not arrest uptake of IgG-coated particles bound to the cell surface. On the other hand, the drugs strongly inhibit transfer of the particles from endocytic vesicles to lysosomes, the latter marked by prior pulse-chase labeling of the cells with horseradish peroxidase. Since the main effect shared by chloroquine and monensin is to raise pH in acid compartments such as endocytic vesicles and lysosomes, the findings suggest that the transfer of IgG-coated particles into the lysosomes is a pH-dependent process. It remains to be shown whether it is the membrane fusion as such that is controlled by pH or, more specifically, the transfer of receptor-bound ligands into the lysosomes.     

Protein kinase C regulates MARCKS cycling between the plasma membrane and lysosomes in fibroblasts.

MARCKS is a protein kinase C (PKC) substrate that is phosphorylated during neurosecretion, phagocyte activation and growth factor-dependent mitogenesis. MARCKS binds calcium/calmodulin and crosslinks F-actin, and both these activities are regulated by PKC-dependent phosphorylation. We present evidence here that PKC-dependent phosphorylation also regulates the cycling of MARCKS between the plasma membrane and Lamp-1-positive lysosomes. Immuno-fluorescence and immunoelectron microscopy, and subcellular fractionation, demonstrated that MARCKS was predominantly associated with the plasma membrane of resting fibroblasts. Activation of PKC resulted in MARCKS phosphorylation and its displacement from the plasma membrane to Lamp-1-positive lysosomes. MARCKS phosphorylation is required for its translocation to lysosomes since mutating either the serine residues phosphorylated by PKC (phos-) or the PKC inhibitor staurosporine, prevented MARCKS phosphorylation, its release from the plasma membrane, and its subsequent association with lysosomes. In the presence of lysosomotropic agents or nocodazole, MARCKS accumulated on lysosomes and returned to the plasma membrane upon drug removal, further suggesting that the protein cycles between the plasma membrane and lysosomes. In contrast to wild-type MARCKS, the phos- mutant did not accumulate on lysosomes in cells treated with NH4Cl, suggesting that basal phosphorylation of MARCKS promotes its constitutive cycling between these two compartments.     

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.     

Intra-endosomal membrane traffic

Following endocytosis, ubiquitinated signaling receptors are incorporated within intraluminal vesicles of forming multivesicular endosomes. These vesicles then follow the pathway from early to late endosomes, remaining within the endosomal lumen, and are eventually delivered to lysosomes, where they are degraded together with their protein cargo. However, intraluminal vesicles do not always end up in lysosomes for degradation; they can also fuse back with the limiting membrane of late endosomes. This route, which might be regulated by lyso-bisphosphatidic acid and its putative effector Alix, can be hijacked by the anthrax toxin and vesicular stomatitis virus and is presumably exploited by proteins and lipids that transit through intraluminal vesicles. Alternatively, these vesicles can be released extracellularly, like HIV in macrophages, upon fusion of endosomes or lysosomes with the plasma membrane.     

Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair

Rapid plasma membrane resealing is essential for cellular survival. Earlier studies showed that plasma membrane repair requires Ca²⁺-dependent exocytosis of lysosomes and a rapid form of endocytosis that removes membrane lesions. However, the functional relationship between lysosomal exocytosis and the rapid endocytosis that follows membrane injury is unknown. In this study, we show that the lysosomal enzyme acid sphingomyelinase (ASM) is released extracellularly when cells are wounded in the presence of Ca²⁺. ASM-deficient cells, including human cells from Niemann-Pick type A (NPA) patients, undergo lysosomal exocytosis after wounding but are defective in injury-dependent endocytosis and plasma membrane repair. Exogenously added recombinant human ASM restores endocytosis and resealing in ASM-depleted cells, suggesting that conversion of plasma membrane sphingomyelin to ceramide by this lysosomal enzyme promotes lesion internalization. These findings reveal a molecular mechanism for restoration of plasma membrane integrity through exocytosis of lysosomes and identify defective plasma membrane repair as a possible component of the severe pathology observed in NPA patients.     

Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D1, was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane on self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label in secondary lysosomes increased by first order kinetics (k = [56 min]-1) from less than 0.1% (background level) to a steady-state level of approximately 2.5% of the total label. As analyzed by NaDodSO4 PAGE, labeled molecules of Mr 160-190 kD were depleted and of Mr 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all Mr classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constituents to secondary lysosomes is a limited and selective process, and that only approximately 1% of internalized membrane is recycled via a membrane pool of secondary lysosomes.     

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.     

Contact formation and transfer of mannose BSA gold from macrophages to cocultured fibroblasts

When macrophages were cocultured with fibroblasts many of the cells formed firm contacts. In some of these contacts both cell types were closely apposed and in others they were more clearly separated with numerous pseudopodia extending from macrophages toward the fibroblasts. Many small vesicles similar in structure to caveoli were observed immediately beneath the plasma membrane of some fibroblasts in regions immediately adjacent to areas of contact with macrophages. The membrane integrity of both cell types was always maintained and no connecting cytoplasmic strands were observed between contacting cells. Junctions were freely permeable to ruthenium red and less permeable to the larger cationized ferritin. Gold conjugated to mannose BSA was taken up readily by macrophages but not by fibroblasts. When fibroblasts were cocultured with macrophages that had been labeled with endocytosed gold, increasing amounts were transferred to them. Gold was observed within gaps formed between cocultured cells and within recipient fibroblasts in vesicles anatomically similar to lysosomes. These points of contact thus appear to provide a series of specialized protected clefts into which directed exocytosis of ligands from donor cells can take place and from which endocytosis into recipient cells is facilitated.     

Relations between plasma membrane and lysosomal membrane. 2. Quantitative evaluation of plasma membrane marker enzymes in the lysosomes

We have quantified, in cultured rat fibroblasts, the association to the lysosomal membrane of two classical plasma membrane markers, 5'-nucleotidase and alkaline phosphodiesterase I. To isolate highly purified lysosomal preparations, lysosomes were loaded with horseradish peroxidase (2-h cell uptake, 16-h chase) and isolated by isopycnic centrifugation in linear Percoll gradients, followed by a 3,3'-diaminobenzidine-induced density shift in sucrose gradients. Purified lysosomal preparations contained up to 50% of N-acetyl-beta-glucosaminidase of the homogenate. This lysosomal enzyme was enriched 33-fold in the most purified preparations. In the electron microscope, these preparations appeared to be highly purified and only contained organelles filled with diaminobenzidine reaction products. Analysis of purified preparations indicates that 0.5-0.8% of 5'-nucleotidase, but as much as 10.9-14.3% of alkaline phosphodiesterase I activities of the homogenate, are associated with lysosomes. After freezing-thawing, these activities remained essentially membrane-associated. The larger value obtained for alkaline phosphodiesterase I could not be ascribed to other lysosomal enzymes, as no such activity was detected at acidic pH. These two plasma membrane markers are thus unevenly distributed in the lysosomal compartment.     

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent ("caveolar-like") mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a "vital marker" for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.     

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.