Multiple lysosomal trafficking phenotypes in metastatic mouse mammary tumor cell lines.

Although altered synthesis and trafficking of lysosomal proteins and their receptors are associated with a wide range of human and rodent malignancies, the basis for their involvement remains obscure. Here we describe findings on a set of mouse mammary tumor cell lines that we are using as a model to study the role of these proteins in oncogenesis and tumor progression. Three distinct proteinase-secreting phenotypes were identified among the metastatic cell lines of the set. Two phenotypes displayed a high level of secretion of cathepsin L and the third was characterized by elevated secretion of matrix metalloproteinase 9 (MMP-9). The two cathepsin L-secreting phenotypes were distinct in that they displayed differences in cathepsin trafficking, expression of mannose 6-phosphate/insulin-like growth factor receptor and expression of proliferin, a mannose-phosphorylated angiogenic factor. Although cells representing all three phenotypes are capable of dissemination to distant organs when implanted into mouse mammary glands, only cells with the MMP-9 phenotype were found to be capable of direct intravasation. These findings indicate that multiple proteinase-secreting phenotypes can arise from the same tumor and suggest that cathepsin L and other lysosomal proteins may play a role in dissemination of tumor cells via the lymphatic system.

Lysine-based structure responsible for selective mannose phosphorylation of cathepsin D and cathepsin L defines a common structural motif for lysosomal enzyme targeting.

Previous studies have shown that lysine residues on the surface of cathepsins and other lysosomal proteins are a shared component of the recognition structure involved in mannose phosphorylation. In this study, the involvement of specific lysine residues in mannose phosphorylation of cathepsin D was explored by site-directed mutagenesis. Mutation of two lysine residues in the mature portion of the protein, Lys-203 and Lys-293, cooperated to inhibit mannose phosphorylation by 70%. Other positively charged residues could not substitute for lysine at these positions, and comparison of thermal denaturation curves for the wild type and mutant proteins indicated that the inhibition could not be explained by alterations in protein folding. Structural comparisons of the two lysine residues with those required for phosphorylation of cathepsin L, using models generated from recently acquired crystal structures, revealed several relevant similarities. On both molecules, the lysine residues were positioned approximately 34 A apart (34.06 A for cathepsin D and 33.80 A for cathepsin L). When the lysine pairs were superimposed, N-linked glycosylation sites on the two proteins were found to be oriented so that oligosaccharides extending out from the sites could share a common region of space. Further similarities in the local environments of the critical lysines were also observed. These results provide details for a common lysosomal targeting structure based on a specific arrangement of lysine residues with respect to each other and to glycosylation sites on the surface of lysosomal proteins.

Lysine-based structure in the proregion of procathepsin L is the recognition site for mannose phosphorylation.

The recognition of lysosomal enzymes by UDP-GlcNAc: lysosomal-enzyme GlcNAc-1-phosphotransferase (phosphotransferase) is mediated by a protein structure on lysosomal enzymes. It has been previously demonstrated that lysine residues are required for phosphorylation of procathepsin L and are a common feature of the site on many lysosomal proteins. In this work, the procathepsin L recognition structure was further defined by identification of the region of the protein containing the structure and the critical lysine residues involved. Removal of the cathepsin L propeptide by low pH-induced autocatalytic processing abolished phosphorylation. The addition of either the purified propeptide or a glutathione S-transferase-propeptide fusion protein to the processed protein restored phosphorylation. Mutagenesis of individual lysine residues demonstrated that two propeptide lysine residues (Lys-54 and Lys-99) were required for efficient phosphorylation of procathepsin L. By comparison of the phosphorylation rates of procathepsin L, lysine-modified procathepsin L, and the procathepsin L oligosaccharide, lysine residues were shown to account for most, if not all, of the protein-dependent interaction. On this basis, it is concluded that the proregion lysine residues are the major elements of the procathepsin L recognition site. In addition, lysine residues in cathepsin D were shown to be as important for phosphorylation as those in procathepsin L, supporting a general model of the recognition site as a specific three-dimensional arrangement of lysine residues exposed on the surface of lysosomal proteins.

The proregion of cathepsin L is required for proper folding, stability, and ER exit.

To investigate the role of the proregion in the biosynthesis and trafficking of mouse cathepsin L, cathepsin L cDNAs encoding proteins with altered proregions were constructed and their expression in COS cells was examined. As in transformed cells, normal mouse cathepsin L was secreted by COS cells. In contrast, two altered proregion cathepsin L proteins, one in which the proregion was deleted and a second in which the proregion was replaced with that of a homologous protein (aleurain), were retained within the cell and degraded over a period of 2-6 h. Immunofluorescence localization and the lack of effect of NH4Cl and brefeldin A on the turnover of the altered cathepsin L proteins indicated that their degradation occurred in the endoplasmic reticulum (ER). By using brefeldin A to induce colocalization of the UDPGlcNAc: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase with the cathepsin L proteins in the ER, it was shown that the altered proteins were not susceptible to mannose phosphorylation as they exist in the ER. Trypsin sensitivity assays indicated that altered proregion proteins synthesized in COS cells or in vitro are misfolded. Taken together, these results indicate that the proregion plays an essential role in proper folding of cathepsin L. ER retention, decreased stability, and lack of mannose phosphorylation of the altered proteins are most likely secondary effects resulting from improper folding.

Lysine is a common determinant for mannose phosphorylation of lysosomal proteins.

The phosphorylation of lysosomal enzymes on high mannose residues is the first step in the targeting of these enzymes to lysosomes in a wide range of mammalian cells. Phosphorylated lysosomal enzymes bind to mannose 6-phosphate receptors, which divert them from the secretory pathway and direct them toward the lysosome. We have been investigating the basis for the specific recognition of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase by using the precursor form of the lysosomal cysteine protease, cathepsin L, as a model lysosomal enzyme in an in vitro assay for mannose phosphorylation. Cathepsin L was found to be phosphorylated in vitro with the same efficiency as other lysosomal enzymes and to contain a conformationally sensitive protein signal that is recognized by phosphotransferase. Biochemical modification of lysine residues on cathepsin L with sulfo-N-hydroxysuccinimide acetate prevented the enzyme from being phosphorylated, indicating that lysine is an important component of the signal. The modification itself did not cause any major conformational changes in cathepsin L. When the same modification was performed on a number of other lysosomal enzymes, phosphorylation was also inhibited. Thus, we conclude that lysine residues are important features of lysosomal enzyme phosphotransferase recognition sites in general, and we discuss the implications of this finding in the ongoing efforts to define the phosphotransferase recognition site.

Comparison of cathepsin L synthesized by normal and transformed cells at the gene, message, protein, and oligosaccharide levels.

The major excreted protein of transformed mouse fibroblasts (MEP) has recently been identified as the lysosomal cysteine protease, cathepsin L. The synthesis and intracellular trafficking of this protein in mouse fibroblasts are regulated by growth factors and malignant transformation. To further define the basis for this regulation, a cDNA encoding MEP/cathepsin L was isolated from a mouse liver cDNA library and used to compare cathepsin L of normal and Kirsten sarcoma virus-transformed NIH 3T3 fibroblasts. Although cathepsin L message levels were elevated 20-fold in the transformed fibroblasts, normal and transformed cells displayed similar cathepsin L genomic DNA digest patterns and gene copy numbers, and cathepsin L mRNA sequences appeared identical by RNase protection analysis. These findings indicate that (i) cathepsin L is synthesized from the same gene in normal and transformed cells and (ii) cathepsin L polypeptides made by these cells are translated with the same primary sequence. Cathepsin L polypeptides synthesized by quiescent, growing, and transformed cells displayed similar isoelectric focusing patterns, suggesting similar post-translational modification. Site-directed mutagenesis of the mouse liver cDNA and expression in COS monkey cells was used to examine the glycosylation of mouse cathepsin L. The results indicated that only one of the two potential N-linked glycosylation sites (the one at Asn221) is glycosylated. Analysis by ion exchange chromatography on QAE-Sephadex, and affinity chromatography on mannose 6-phosphate receptor-Affi-Gel 10, indicated that the cathepsin L oligosaccharide was phosphorylated similarly in normal and transformed cells. Although several phosphorylated oligosaccharide species were observed, the major species contained two phosphomonoester moieties and bound efficiently to the receptor. These findings suggest that cathepsin L made by normal and transformed mouse fibroblasts are identical and substantiate the hypothesis that trafficking of cathepsin L in these cells is regulated by growth-induced changes in the lysosomal protein transport system.

Basis for low affinity binding of a lysosomal cysteine protease to the cation-independent mannose 6-phosphate receptor.

In cultured mouse fibroblasts, secretion of the lysosomal cysteine protease, MEP (major excreted protein) is regulated by growth factors and viral transformation. The ability of this protein to be regulated has been attributed to its intrinsic low affinity for the cation-independent mannose 6-phosphate (Man-6-P) receptor (Dong, J., Prence, E. M., and Sahagian, G. G. (1989) J. Biol. Chem. 264, 7377-7383). In this study, the basis for this low affinity was examined. Chromatography on a cation-independent Man-6-P receptor affinity matrix was used to assess relative affinities of Man-6-P-containing oligosaccharides and proteins, and the state of phosphorylation of the oligosaccharides was determined by ion exchange chromatography on QAE-Sephadex. MEP proteins synthesized by normal NIH 3T3 cells or NIH cells transformed with Kirsten sarcoma virus displayed a similar low affinity for the receptor and were found to possess oligosaccharide species with two phosphomonoester moieties. The affinity of these oligosaccharides for the receptor was the same as intact MEP protein and as great as phosphorylated oligosaccharides obtained from lysosomal proteins with the usual high affinity for the receptor. These results indicate that the polypeptide portion of MEP has no effect on binding of the protein to the receptor and that the difference in affinity of MEP and lysosomal proteins with high affinity cannot be attributed to differences in oligosaccharide structure. To investigate this further, we examined the binding characteristics of MEP made by CHO cells. In contrast to mouse MEP, CHO MEP bound to the receptor with high affinity. Partial endoglycosidase H treatment indicated that CHO MEP has two phosphorylated oligosaccharides, whereas the mouse protein has only one. Both oligosaccharides of the CHO cell protein contained two phosphomonoester moieties and displayed an affinity for the receptor that was indistinguishable from that of oligosaccharides of the mouse protein. Conversion of CHO MEP to a one-oligosaccharide species by partial endoglycosidase H treatment produced a protein that displayed low affinity binding similar to that of mouse MEP. A substantial portion of the pool of CHO cell lysosomal protein was also converted to a low affinity ligand by this treatment. Taken together, these results suggest that high affinity binding to the cation-independent receptor involves a divalent interaction with lysosomal proteins that contain two or more phosphorylated oligosaccharides, and that the low affinity of MEP results from an inability to form this multivalent interaction.

Modulation of the transport of a lysosomal enzyme by PDGF.

The major excreted protein (MEP) of transformed mouse fibroblasts is the lysosomal protease, cathepsin L. MEP is also secreted by untransformed mouse cells in response to growth factors and tumor promoters, and is thought to play a role in cell growth and transformation. To determine the relationship between MEP synthesis and MEP secretion, we have examined these events in PDGF-treated NIH 3T3 cells. PDGF enhanced MEP synthesis and caused the diversion of MEP from the lysosomal delivery pathway to a secretory pathway. These two effects were found to be regulated independently at various times after growth factor addition. Short PDGF treatments (0.5 or 1 h) resulted in quantitative secretion of MEP although synthesis was near the control level. High levels of both synthesis and secretion occurred between 2 and 14 h of PDGF treatment. Between 18 and 30 h, the amount of secreted MEP returned to the low control level even though synthesis remained elevated. The secretion was specific for MEP; other lysosomal enzymes were not found in the media from PDGF-treated cells. PDGF-induced secretion of MEP was inhibited 84% by cycloheximide, suggesting that protein synthesis is required to elicit this effect. PDGF also caused a time-dependent increase in mannose 6-phosphate (Man-6-P) receptor-mediated endocytosis. These data support a model in which PDGF alters the distribution of Man-6-P receptors such that the Golgi concentration of receptors becomes limiting, thereby causing the selective secretion of the low affinity ligand, MEP.

Bovine testicular beta-galactosidase: purification of enzyme fractions that exhibit high affinity for phosphomannosyl receptors.

An improved method is described for the preparation of bovine testicular beta-galactosidase that allows the isolation of enzyme fractions that bind avidly to phosphomannosyl receptors. The procedure permits removal of a contaminating beta-hexosaminidase and yields nearly homogeneous beta-galactosidase. Enzyme eluted from DEAE-Sephacel was arbitrarily divided into pools that exhibited differing ability to bind phosphomannosyl receptors. A high binding fraction was rapidly assimilated by cultured cells and bound to both low and high molecular weight phosphomannosyl receptors. Carbohydrate analysis of the high binding fraction indicates an average content of one complex and one high mannose oligosaccharide chain per molecule and an average mannose 6-phosphate content of two residues per molecule. However, electrofocusing studies indicated that all the fractions were heterogeneous with respect to sialic acid and phosphate content. The purification procedure also provides highly purified beta-galactosidase suitable for removing beta-galactosidase residues from a variety of complex carbohydrates.

Mechanism for selective secretion of a lysosomal protease by transformed mouse fibroblasts.

Studies in recent years have indicated that secretion of certain lysosomal hydrolases can be enhanced under various conditions. One such protein, the major excreted protein (MEP) of Kirsten virus-transformed NIH 3T3 (KNIH) fibroblasts, is a lysosomal cysteine protease whose synthesis and secretion are affected by viral transformation and growth factors. We have been studying the synthesis and transport of MEP in order to understand better the mechanisms responsible for regulation of lysosomal enzyme secretion. Synthesis of MEP in KNIH cells was found to be 25-fold greater than that in untransformed NIH cells, and 94% of the MEP made was secreted. This was in contrast to NIH cells which secreted only 11% of the newly synthesized MEP. The high level of secretion by the transformed cells was relatively specific in that most other lysosomal enzymes were retained. MEP isolated from both NIH and KNIH cells exhibited a low intrinsic affinity for the mannose-6-phosphate receptor which was at least 10-fold lower than that of other lysosomal enzymes. On the basis of these results, we suggest that both the high level of MEP synthesis and the intrinsic low affinity of MEP for the receptor are responsible for the specific increase in MEP secretion by transformed cells.

Transport of surface mannose 6-phosphate receptor to the Golgi complex in cultured human cells.

The cation-independent mannose 6-phosphate receptor (MPRCI) functions in the packaging of both newly made and extracellular lysosomal enzymes into lysosomes. The subcellular location of MPRCI reflects these two functions; receptor is found in the Golgi complex, in endosomes, and on the cell surface. To learn about the intracellular pathway followed by surface receptor and to study the relationship between the receptor pools, we examined the entry of the surface MPRCI into Golgi compartments that contain sialyltransferase. Sialic acid was removed from surface-labeled K562 cultured human erythroleukemia cells by neuraminidase treatment. When the cells were returned to culture at 37 degrees C, surface MPRCI was resialylated by the cells with a half-time of 1-2 h. Resialylation was inhibited by reduced temperature, a treatment that allows surface molecules to reach endosomes but blocks further transport. These results indicate that surface MPRCI is transported to the sialyltransferase compartment in the Golgi complex. After culture at 37 degrees C, a small fraction (10-20%) of the resialylated receptor was found on the cell surface. Because a similar fraction of the total receptor pool is found on the cell surface, it is likely that cell surface MPRCI mixes with the cellular pool after resialylation. These data also support the idea that extracellular and newly made lysosomal enzymes are transported to lysosomes through a common compartment.

Insulin-like growth factor-II (IGF-II) inhibits both the cellular uptake of beta-galactosidase and the binding of beta-galactosidase to purified IGF-II/mannose 6-phosphate receptor.

The insulin-like growth factor-II/mannose 6-phosphate receptor which targets acid hydrolases to lysosomes, has two different binding sites, one for the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal enzymes and the other for insulin-like growth factor-II (IGF-II). We have asked whether IGF-II can regulate the cellular uptake of the lysosomal enzyme 125I-beta-galactosidase by modulating the binding of 125I-beta-galactosidase to the IGF-II/Man-6-P receptor. We first isolated high affinity 125I-beta-galactosidase by affinity chromatography on an IGF-II/Man-6-P receptor-Sepharose column. Specific uptake (mannose 6-phosphate-inhibitable) of 125I-beta-galactosidase in BRL 3A2 rat liver cells and in rat C6 glial cells was 3.7-4.8 and 4.0-8.0% of added tracer, respectively. The cell-associated 125I-beta-galactosidase in the uptake experiments largely represented internalized radioligand as measured by acid or mannose 6-phosphate washing. The uptake of 125I-beta-galactosidase was inhibited by an antiserum (No. 3637) specific for the IGF-II/Man-6-P receptor. Low concentrations of IGF-II also inhibited the uptake of 125I-beta-galactosidase. Maximal concentrations of IGF-II inhibited uptake by 73 +/- 8% (mean +/- S.D.) in C6 cells and by 77 +/- 6% in BRL 3A2 cells compared to the level of inhibition by mannose 6-phosphate. The relative potency of IGF-II, IGF-I, and insulin (IGF-II much greater than IGF-I; insulin, inactive) were characteristic of the relative affinities of the ligands for the IGF-II/Man-6-P receptor. IGF-II also partially inhibited the binding of 125I-beta-galactosidase to C6 and BRL 3A2 cells at 4 degrees C and inhibited the binding to highly purified IGF-II/Man-6-P receptor by 58 +/- 14%. We conclude that IGF-II inhibits the cellular uptake of 125I-beta-galactosidase and that this inhibition is partly explained by the ability of IGF-II to inhibit binding of 125I-beta-galactosidase to the IGF-II/Man-6-P receptor.

Biochemical evidence that the type II insulin-like growth factor receptor is identical to the cation-independent mannose 6-phosphate receptor.

Cloning and sequencing of the human type II insulin-like growth factor (IGF) receptor cDNA revealed an 80% deduced amino acid sequence homology with the bovine cation-independent mannose 6-phosphate (Man-6-P) receptor, suggesting identity of the two receptors (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). We have performed biochemical experiments that support this proposal. Rat liver type II IGF receptor, purified by the conventional method of IGF-II affinity chromatography, bound quantitatively to a beta-galactosidase affinity column and was eluted with Man-6-P. Bovine liver Man-6-P receptor, prepared by the conventional method of affinity chromatography on phosphomannan-Sepharose, bound IGF-II with high affinity (Kd = 1 nM). Affinity cross-linking of 125I-IGF-II to the Man-6-P receptor and analysis by sodium dodecyl sulfate-gel electrophoresis showed that beta-galactosidase, but not Man-6-P, inhibited the formation of the 250-kDa 125I-IGF-II-receptor complex. The inhibition by beta-galactosidase was prevented by coincubation with Man-6-P. 125I-IGF-II did not bind to the 46-kDa cation-dependent Man-6-P receptor. For immunologic studies we purified type II IGF receptors and Man-6-P receptors in parallel from rat placental membranes using either IGF-II- or beta-galactosidase affinity chromatography. A panel of five antisera that previously had been raised against either type II IGF receptor or Man-6-P receptor behaved identically toward type II IGF receptor versus Man-6-P receptor in ligand blocking and immunoprecipitation assays. Our data support the conclusion that the type II IGF receptor and the cation-independent Man-6-P receptor are the same protein and that the IGF-II and Man-6-P-binding sites are distinct.

Transmembrane orientation of the mannose 6-phosphate receptor in isolated clathrin-coated vesicles.

The mannose 6-phosphate (Man-6-P) receptor is an integral membrane glycoprotein which mediates intracellular transport and receptor-mediated endocytosis of lysosomal proteins. Clathrin-coated vesicles, which have been shown to be significantly involved in these processes, have also been shown to be a major subcellular site of the receptor. In order to define the orientation of the Man-6-P receptor within the coated vesicle membrane, highly purified preparations of coated vesicles were prepared from bovine brain employing D2O/sucrose gradient centrifugation and Sephacryl S-1000 column chromatography. Using [35S]methionine-labeled lysosomal enzymes secreted by Chinese hamster ovary cells as receptor ligand, significant binding activity was detected only upon permeabilization of the coated vesicle membranes with detergent. Prior treatment of intact vesicles with proteinase K resulted in similar binding activity upon permeabilization. However, examination of the receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with rabbit anti-receptor serum revealed that proteinase K treatment of intact vesicles reduced the size of the receptor by 12,000 daltons. A similar decrease in size was obtained when the vesicles were treated with carboxypeptidase Y. These results suggest that the Man-6-P receptor is a transmembrane protein with its lysosomal enzyme binding site oriented toward the lumen of the coated vesicle and its C-terminal end exposed to the exterior or cytoplasmic portion of the vesicle membrane.

The mannose 6-phosphate receptor: function, biosynthesis and translocation.

This report summarizes studies concerning the role of the lysosomal protein: Man-6-P receptor and describes some recent data on its biosynthesis and cellular translocation. The receptor functions both in the Golgi apparatus (or GERL) and on the cell surface where it binds lysosomal proteins and mediates their transport to lysosomes. Consistent with its dual role, the receptor in several cell types has been localized to the plasma membrane and Golgi cisternae, to clathrin-coated structures at both locations, and to vesicles characteristic of endosomes or CURL. Biosynthetic studies have shown that the receptor undergoes several post-translational modifications including the processing of its asparagine-linked oligosaccharides, phosphorylation of serine residues, and unknown modifications required for acquisition of immunoreactivity and functional activity. Cellular pools of mature receptor readily mix as evidenced by rapid labeling of intracellular receptor by exogenously added receptor antibodies. Degradation of the receptor occurs non-lysosomally and is perhaps mediated by extracellular Man-6-P-containing hydrolases. A working hypothesis for the mechanism of Man-6-P receptor function that is consistent with these observations is presented.

Biosynthesis and turnover of the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells.

The natural history of the mannose 6-phosphate receptor was examined by radiolabeling cells in monolayers or in suspension; the receptor was isolated by immuno- or affinity precipitation followed by polyacrylamide gel electrophoresis. The receptor was found to contain asparagine-linked oligosaccharide chains and phosphorylated serine residues. Newly made receptor was sensitive to endo-beta-N-acetylglucosaminidase H (endo-H) and was slowly converted to a mature endo-H resistant form; phosphate was found on the mature receptor only. The receptor had an apparent molecular weight of 215,000 at all times, as determined under reducing and denaturing conditions; unreduced receptor had a greater electrophoretic mobility, suggesting the presence of intrachain disulfide linkages. The synthesis of immunoreactive receptor occurred with a lag of 50 min and of functional receptor with a lag of 70 min, indicating a requirement for some post-translational event(s) for acquisition of immunoreactivity and binding activity. Maturation of asparagine-linked oligosaccharides was not the requisite modification, since endo-H sensitive or deglycosylated receptor bound to both antibody and to insoluble phosphomannan; however, much less immunoreactive and functional receptor was detected in the presence of tunicamycin. Immunoprecipitable [3H]leucine-labeled receptor was degraded with a t1/2 of 16 h and 6 h for cells in monolayers and suspension, respectively, whereas 32P was lost with a corresponding t1/2 of 2.3 and 4 h. A pool of cell surface mannose 6-phosphate receptor was identified by separation on Percoll gradients as well as by iodination of cells with 125I; receptor in this pool was resistant to endo-H and had a t1/2 similar to that of the total [3H]leucine-labeled receptor, even in the presence of a saturating concentration of ligand. During endocytosis, ligand (beta-galactosidase) and 125I-receptor separated, the ligand accumulating within lysosomes. These results are consistent with current concepts of recycling of the mannose 6-phosphate receptor.

Ultrastructural immunocytochemical localization of the phosphomannosyl receptor in Chinese hamster ovary (CHO) cells.

Using ultrastructural immunocytochemistry and antibodies directed against bovine liver phosphomannosyl (PM) receptor, we have localized the receptor in Chinese hamster ovary (CHO) cells. The majority of the receptor was found within the cell. Only a small fraction of the receptor was found on the surface and most of it was clustered in coated pits. Because these cells contain endogenous ligands for the receptor, it was not possible to determine if this clustered state was dependent on occupancy of the receptor. The bulk of the cell's receptor was found in the endoplasmic reticulum, nuclear envelope, and in the Golgi system. Most of the Golgi localization was associated with peripheral Golgi elements, suggesting a possible concentration of receptor in GERL. Very little receptor was found associated with mature lysosomes. PM receptor was also localized in structures that were identified as receptosomes by the presence of alpha 2-macroglobulin (alpha 2M)-gold, a ligand previously shown to enter CHO cells by the coated pit-receptosome pathway. This finding is consistent with the notion that during receptor-mediated endocytosis, receptors accompany ligand from the coated pit into the receptosome. The observation that the majority of the receptor was found in the endoplasmic reticulum and structures similar to GERL raises the possibility that the PM receptor plays an important role in compartmentalization of lysosomal enzymes in the GERL system.

The predominant secreted protein of transformed murine fibroblasts carries the lysosomal mannose 6-phosphate recognition marker.

We have found that the major excreted protein (MEP) of transformed mouse fibroblasts, a phosphoglycoprotein of Mr = 35,000, carries the mannose 6-phosphate recognition marker. MEP secreted by Kirsten virus-transformed NIH 3T3 cells binds to a purified preparation of lysosomal enzyme phosphomannosyl receptor, and this binding is specifically inhibited by mannose 6-phosphate. 32Pi introduced into MEP by metabolic labeling of intact cells is exclusively associated with asparagine-linked oligosaccharides as indicated by sensitivity to endohexosaminidase H. Labeling studies utilizing [2-3H]mannose indicate that approximately one-fifth of the mannose residues of MEP are phosphorylated. Comparative studies of the synthesis, secretion, and uptake of MEP and the lysosomal enzyme beta-galactosidase indicate that MEP made by Kirsten virus-transformed NIH 3T3 cells is not handled in the same manner as are other lysosomal enzymes. MEP may be an unusual lysosomal protein, or both.