Comparative kinetics of phosphomannosyl receptor-mediated pinocytosis of fibroblast secretion acid hydrolases and glycopeptides prepared from them

In a previous report we demonstrated that phosphorylated oligosaccharides isolated from acid hydrolases were subject to pinocytosis by phosphomannosyl receptors present on the cell surface of human fibroblasts [9]. However, limiting quantities of oligosaccharides precluded detailed comparison of the kinetics of pinocytosis of these phosphorylated oligosaccharides to those of the acid hydrolases from which they were derived. In this report we present studies comparing the kinetics of pinocytosis of acid hydrolases from NH4Cl-induced fibroblast secretions with those of concanavalin A-binding glycopeptides prepared from them by pronase digestion. The uptake of both secretion acid hydrolases and 125I-labeled glycopeptides was linear for at least 3 hr, saturable, inhibited competitively by mannose 6-phosphate, and destroyed by prior treatment of the ligand with alkaline phosphatase. The inhibition constants of excess unlabeled glycopeptide for the uptake of 125I-labeled glycopeptides (Ki of 1.5 X 10(-6) M) and for the uptake of secretion acid hydrolases (Ki of 2.2 X 10(-6) M) were remarkably similar. Furthermore, the Ki for mannose 6-phosphate inhibition of pinocytosis of glycopeptide uptake (3 X 10(-5) M) compares closely to that previously determined for the pinocytosis of intact "high-uptake" acid hydrolases (3-6 X 10(-5) M). "High-uptake" fractions of both ligands were prepared and quantified by affinity chromatography on immobilized phosphomannosyl receptors purified from bovine liver. Only 10% of the concanavalin A-binding glycopeptides bound to the immobilized phosphomannosyl receptors, while 80% of the acid hydrolases from which they were prepared bound and were eluted with 10 mM mannose 6-phosphate. However, the fraction of each type of ligand that binds to the immobilized phosphomannosyl receptors accounts for all the uptake activity of that ligand.     

Mannose 6-phosphate receptors in an ancient vertebrate, zebrafish

The endosome/lysosome system plays key roles in embryonic development, but difficulties posed by inaccessible mammalian embryos have hampered detailed studies. The accessible, transparent embryos of Danio rerio, together with the genetic and experimental approaches possible with this organism, provide many advantages over rodents. In mammals, mannose 6-phosphate receptors (MPRs) target acid hydrolases to endosomes and lysosomes, but nothing is known of acid hydrolase targeting in zebrafish. Here, we describe the sequence of the zebrafish cation-dependent MPR (CD-MPR) and cation-independent MPR (CI-MPR), and compare them with their mammalian orthologs. We show that all residues critical for mannose 6-phosphate (M6P) recognition are present in the extracellular domains of the zebrafish receptors, and that trafficking signals in the cytoplasmic tails are also conserved. This suggests that the teleost receptors possess M6P binding sites with properties similar to those of mammalian MPRs, and that targeting of lysosomal enzymes by MPRs represents an ancient pathway in vertebrate cell biology. We also determined the expression patterns of the CD-MPR and CI-MPR during embryonic development in zebrafish. Both genes are expressed from the one-cell stage through to the hatching period. In early embryos, expression is ubiquitous, but in later stages, expression of both receptors is restricted to the anterior region of the embryo, covering the forebrain, midbrain and hindbrain. The expression patterns suggest time- and tissue-specific functions for the receptors, with particular evidence for roles in neural development. Our study establishes zebrafish as a novel, genetically tractable model for in vivo studies of MPR function and lysosome biogenesis.     

Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling

Adsorptive pinocytosis of acid hydrolases by fibroblasts depends on phosphomannosyl recognition markers on the enzymes and high-affinity pinocytosis receptors on the cell surface. In this study, beta- glucuronidase binding to the cell surface of attached fibroblasts was found to be saturable and inhibitable by mannose-6-phosphate (Man-6-P). Dissociation of cell-bound beta-glucuronidase occurred very slowly at neutral pH, but was greatly accelerated by lowering the pH below 6.0, or by exposure to Man-6-P. Comparison of the maximal cell surface binding and the observed rate of enzyme pinocytosis suggests that the pinocytosis receptors are replaced or reused about every 5 min. Enzyme pinocytosis was not affected by inhibition of new protein synthesis for several hours, suggesting a large pool of internal receptors and/or reuse of internalized receptors. Chloroquine treatment of normal human fibroblasts had three effects: (a) greatly enhanced secretion of newly synthesized acid hydrolases bearing the recognition marker for uptake, (b) depletion of enzyme-binding sites from the cell surface, and (c) inhibition of pinocytosis of exogenous enzyme. Only the third effect was seen in I-cell disease fibroblasts, which were also less sensitive than control cells to this effect. These observations are consistent with a model for transport of acid hydrolases that proposes that delivery of newly synthesized acid hydrolases to lysosomes requires the phosphomannosyl recognition marker on the enzymes, and intracellular receptors that segregate receptor-bound enzymes into vesicles for transport to lysosomes. This model explains how chloroquine, which raises intralysosomal pH, can disrupt both the intracellular pathway for newly synthesized acid hydrolases, and the one for uptake of exogenous enzyme by cell surface pinocytosis receptors.     

The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors

The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.     

Cell- and ligand-specific dephosphorylation of acid hydrolases: evidence that the mannose 6-phosphatase is controlled by compartmentalization

Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum (Einstein, R., and C. A. Gabel. 1989. J. Cell Biol. 109:1037-1046). To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated. This difference in processing indicates that lysosomes themselves exist in a dephosphorylation-competent and -incompetent state. Man 6-P-bearing acid hydrolases endocytosed by the L+ cells in the absence of serum were not distributed uniformly throughout the lysosomal compartment. The change in the dephosphorylation competence of L cells in response to serum suggests, therefore, that these cells contain multiple populations of lysosomes that differ with respect to their content of a mannose 6-phosphatase, and that serum factors affect the distribution of hydrolases between the different compartments.     

The sorting and trafficking of lysosomal proteins

For a long time lysosomes were considered terminal organelles involved in the degradation of different substrates. However, this view is rapidly changing by evidence demonstrating that these organelles and their content display specialized functions in addition to the degradation of substances. Many lysosomal proteins have been implicated in specialized cellular functions and disorders such as antigen processing, targeting of surfactant proteins, and most lysosomal storage disorders. To date, about fifty lysosomal hydrolases have been identified, and the majority of them are targeted to the lysosomes via the mannose-6-phosphate receptor (M6P-Rc). However, recent studies on the intracellular trafficking of the non-enzymic lysosomal proteins prosaposin and GM2 activator (GM2AP) demonstrated that they use an alternative receptor termed "sortilin". Existing evidence suggests that some hydrolases traffic to the lysosomes in a mannose 6-phophate-indepentend manner. The possibility that sortilin is implicated in the targeting of some soluble hydrolases, as well as the consequences of this process, is addressed in the present review.     

A fascination with sugars

We now recognize that a large number of membrane and soluble proteins contain covalently linked oligosaccharides that exhibit a vast array of structures and participate in a wide variety of biological processes. Nowhere is this better illustrated than the mannose 6-phosphate (Man-6-P) recognition system that mediates the trafficking of newly synthesized acid hydrolases to lysosomes in higher eukaryotes. The Asn-linked high-mannose oligosaccharides of these hydrolases facilitate folding of the nascent proteins in the endoplasmic reticulum via interaction with lectin-type chaperones and after phosphorylation in the Golgi, function as ligands for binding to Man-6-P receptors, a critical step in their transport to lysosomes. Failure to synthesize the Man-6-P recognition marker results in a serious lysosomal storage disease, one of a growing number of genetic conditions, termed congenital disorders of glycosylation, that result from faulty glycan biosynthesis.     

A yeast homolog of the mammalian mannose 6-phosphate receptors contributes to the sorting of vacuolar hydrolases

The soluble hydrolases of the mammalian lysosome are marked for delivery to this organelle by the addition of mannose 6-phosphate to their N-glycans. Two related mannose 6-phosphate receptors (MPRs) recognize this feature in the trans Golgi network (TGN) and deliver the hydrolases to the late endosome. In contrast, the vacuolar hydrolases of the yeast Saccharomyces cerevisiae do not contain 6-phosphate monoesters on their N-glycans, and the only sorting receptor so far identified in this organism is the product of the VPS10 gene. This protein also cycles between the Golgi and the late endosome, but is unrelated to the vertebrate MPRs, and recognizes a specific amino acid sequence of carboxypeptidase Y (CPY). This has led to the notion that although yeast and mammals share many components in Golgi to endosome traffic, they use unrelated receptor systems to sort their abundant soluble hydrolases. In this paper, we report that the yeast genome does in fact contain an uncharacterized ORF (YPR079w) that encodes a membrane protein that is distantly related to mammalian MPRs. The protein encoded by this gene (which we term MRL1) cycles through the late endosome. Moreover, there is a strong synergistic effect on the maturation of proteinases A and B when both MRL1 and VPS10 are deleted, which suggests that Mrl1p may serve as a sorting receptor in the delivery of vacuolar hydrolases.     

Synthesis of novel bivalent mimetic ligands for mannose-6-phosphate receptors

Mannose-6-phosphate (M6P)-containing N-linked glycans are essential signaling molecules for sorting hydrolases in eukaryotic cells. Their receptors, especially the cation-independent M6P receptors (CI-MPRs), have emerged as promising protein targets for targeted drug delivery for the treatment of lysosomal storage disease and liver fibrosis. In this Letter, we describe the design and synthesis of novel bivalent mimetic ligands for CI-MPRs. We report that for the first time, a newly-discovered binding motif, GlcNAc-M6P, has been incorporated in mimetic ligands. M6P- and GlcNAc-M6P-containing building blocks, equipped with NH₂ and CO₂H handles, have been prepared and assembled with an ornithine linker through amide coupling reactions. Efficient global deprotection protocols have also been developed which have been showcased in the synthesis of our novel bivalent mimetic ligands.     

Proteomic analysis of lysosomal acid hydrolases secreted by osteoclasts: implications for lytic enzyme transport and bone metabolism

Osteoclasts, the bone-digesting cells, are polarized cells that secrete acid hydrolases into a resorption lacuna where bone degradation takes place. The molecular mechanisms underlying this process are poorly understood. To analyze the nature of acid hydrolases secreted by osteoclasts, we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into mature osteoclasts in the presence of the receptor activator of NF-kappaB ligand. Upon differentiation, we observed a strong increase in the secretion of mannose 6-phosphate-containing acid hydrolases. A proteomic analysis of the secreted proteins captured on a mannose 6-phosphate receptor affinity column revealed 58 different proteins belonging to several families of acid hydrolases of which 16 are clearly involved in bone homeostasis. Moreover these acid hydrolases were secreted as proproteins. The expression of most of the identified acid hydrolases is unchanged during osteoclastogenesis. Thus, our data strongly support the notion that the polarized secretion of acid hydrolases by osteoclasts results from a reorganization of key steps of membrane traffic along the lysosomal pathway rather than from a fusion of lysosomes with the membrane facing the resorption lacuna.     

Role of cation independent mannose 6-phosphate receptor protein in sorting and intracellular trafficking of lysosomal enzymes in chicken embryonic fibroblast (CEF) cells

Delivery of soluble lysosomal proteins to the lysosomes is dependent primarily on the mannose 6-phosphate receptors (MPRs) in mammals. However, in non-mammalian cells the role of MPR300 in sorting and trafficking of acid hydrolases to lysosomes is not fully understood till now. In this paper, we tested the role of MPR300 in sorting and trafficking of lysosomal enzymes in CEF cells using a small interfering RNA (siRNA) technology. Inactivation of MPR300 resulted in the secretion of large amounts of newly synthesized hydrolases into the medium and also inhibited the endocytosis of mannose 6-phospharylated ligands. Knockdown of MPR300 in CEF cells results in missorting of fucosidase and arylsulfatse A enzymes into the medium. The results indicated that the MPR300 in CEF cells plays a key role in sorting and trafficking of these soluble hydrolases.     

Structural basis for recognition of phosphorylated high mannose oligosaccharides by the cation-dependent mannose 6-phosphate receptor.

Mannose 6-phosphate receptors (MPRs) play an important role in the targeting of newly synthesized soluble acid hydrolases to the lysosome in higher eukaryotic cells. These acid hydrolases carry mannose 6-phosphate recognition markers on their N-linked oligosaccharides that are recognized by two distinct MPRs: the cation-dependent mannose 6-phosphate receptor and the insulin-like growth factor II/cation-independent mannose 6-phosphate receptor. Although much has been learned about the MPRs, it is unclear how these receptors interact with the highly diverse population of lysosomal enzymes. It is known that the terminal mannose 6-phosphate is essential for receptor binding. However, the results from several studies using synthetic oligosaccharides indicate that the binding site encompasses at least two sugars of the oligosaccharide. We now report the structure of the soluble extracytoplasmic domain of a glycosylation-deficient form of the bovine cation-dependent mannose 6-phosphate receptor complexed to pentamannosyl phosphate. This construct consists of the amino-terminal 154 amino acids (excluding the signal sequence) with glutamine substituted for asparagine at positions 31, 57, 68, and 87. The binding site of the receptor encompasses the phosphate group plus three of the five mannose rings of pentamannosyl phosphate. Receptor specificity for mannose arises from protein contacts with the 2-hydroxyl on the terminal mannose ring adjacent to the phosphate group. Glycosidic linkage preference originates from the minimization of unfavorable interactions between the ligand and receptor.     

Serum factors alter the extent of dephosphorylation of ligands endocytosed via the mannose 6-phosphate/insulin-like growth factor II receptor

Mouse L-cells that contain the cation-independent (CI) mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor endocytose acid hydrolases and deliver these enzymes to lysosomes. The postendocytic loss of the Man 6-P recognition marker from the cell-associated acid hydrolases was assessed by CI-Man 6-P receptor affinity chromatography. 125I-labeled acid hydrolases internalized by L-cells grown at high density were delivered to lysosomes but were not dephosphorylated. In contrast, the same 125I-labeled hydrolases internalized by L-cells maintained at low density were delivered to lysosomes and were extensively dephosphorylated. The dephosphorylation at low density required 5 h for completion suggesting that the phosphatase responsible for the dephosphorylation is located within the lysosomal compartment. Transition from the high to low density state was rapid and was not inhibited by cycloheximide. Medium substitution experiments indicated that serum factors were necessary to maintain the L-cells in the dephosphorylation-competent (low density) state, and that serum-free conditions led to a dephosphorylation-incompetent (high density) state. Addition of IGF II to cells in serum-free medium allowed acid hydrolases subsequently introduced by endocytosis to be dephosphorylated. The results indicate that the removal of the Man 6-P recognition marker from endocytosed acid hydrolases is regulated by serum factors in the growth medium, including IGF II.     

4-sulfomuconolactone hydrolases from Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2

The 4-carboxymethylen-4-sulfo-but-2-en-olide (4-sulfomuconolactone) hydrolases from Hydrogenophaga intermedia strain S1 and Agrobacterium radiobacter strain S2 are part of a modified protocatechuate pathway responsible for the degradation of 4-sulfocatechol. In both strains, the hydrolase-encoding genes occur downstream of those encoding the enzymes that catalyze the lactonization of 3-sulfomuconate. The deduced amino acid sequences of the 4-sulfomuconolactone hydrolases demonstrated the highest degree of sequence identity to 2-pyrone-4,6-dicarboxylate hydrolases, which take part in the meta cleavage pathway of protocatechuate. The 4-sulfomuconolactone hydrolases did not convert 2-pyrone-4,6-dicarboxylate, and the 2-pyrone-4,6-dicarboxylate hydrolase from Sphingomonas paucimobilis SYK-6 did not convert 4-sulfomuconolactone. Nevertheless, the presence of highly conserved histidine residues in the 4-sulfomuconolactone and the 2-pyrone-4,6-dicarboxylate hydrolases and some further sequence similarities suggested that both enzymes belong to the metallo-dependent hydrolases (the "amidohydrolase superfamily"). The 4-sulfomuconolactone hydrolases were heterologously expressed as His-tagged enzyme variants. Gel filtration experiments suggested that the enzymes are present as monomers in solution, with molecular weights of approximately 33,000 to 35,000. 4-Sulfomuconolactone was converted by sulfomuconolactone hydrolases to stoichiometric amounts of maleylacetate and sulfite. The 4-sulfomuconolactone hydrolases from both strains showed pH optima at pH 7 to 7.5 and rather similar catalytic constant (k(cat)/K(M))values. The suggested 4-sulfocatechol pathway from 4-sulfocatechol to maleylacetate was confirmed by in situ nuclear magnetic resonance analysis using the recombinantly expressed enzymes.     

Remarkable conservation of distinct nonclassical MHC class I lineages in divergent amphibian species

Pulmonary surfactant contains homeostatic and antimicrobial hydrolases. When Mycobacterium tuberculosis is initially deposited in the terminal bronchioles and alveoli, as well as following release from lysed macrophages, bacilli are in intimate contact with these lung surfactant hydrolases. We identified and measured several hydrolases in human alveolar lining fluid and lung tissue that, at their physiological concentrations, dramatically modified the M. tuberculosis cell envelope. Independent of their action time (15 min to 12 h), the effects of the hydrolases on the M. tuberculosis cell envelope resulted in a significant decrease (60-80%) in M. tuberculosis association with, and intracellular growth of the bacteria within, human macrophages. The cell envelope-modifying effects of the hydrolases also led to altered M. tuberculosis intracellular trafficking and induced a protective proinflammatory response to infection. These findings add a new concept to our understanding of M. tuberculosis-macrophage interactions (i.e., the impact of lung surfactant hydrolases on M. tuberculosis infection).     

Murine UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase lacking the gamma-subunit retains substantial activity toward acid hydrolases

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on acid hydrolases. The transferase exists as an alpha(2)beta(2)gamma(2) hexameric complex with the alpha- and beta-subunits derived from a single precursor molecule. The catalytic function of the transferase is attributed to the alpha- and beta-subunits, whereas the gamma-subunit is believed to be involved in the recognition of a conformation-dependent protein determinant common to acid hydrolases. Using knock-out mice with mutations in either the alpha/beta gene or the gamma gene, we show that disruption of the alpha/beta gene completely abolishes phosphorylation of high mannose oligosaccharides on acid hydrolases whereas knock-out of the gamma gene results in only a partial loss of phosphorylation. These findings demonstrate that the alpha/beta-subunits, in addition to their catalytic function, have some ability to recognize acid hydrolases as specific substrates. This process is enhanced by the gamma-subunit.     

Role of protein phosphatases in insulin-like growth factor II (IGF II)-stimulated mannose 6-phosphate/IGF II receptor redistribution.

The regulated expression of mannose 6-phosphate/insulin-like growth factor II (M6P/IGF II) receptors in plasma membranes has previously been shown to be accompanied by marked changes in the phosphorylation state of the receptors (Corvera, S., Folander, K., Clairmont, K. B., and Czech, M. P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7567-7571). In the present study we show that protein phosphatase 2A dephosphorylates the human M6P/IGF II receptor in vitro. Incubation of human fibroblasts with okadaic acid, a specific inhibitor of this phosphatase, resulted in a depletion of M6P/IGF II receptors at the cell surface without affecting their internalization kinetics. The phosphorylation state of the remaining cell surface receptors was 3-fold increased. Thus, the endocytosis rate of M6P/IGF II receptors appears to be unaltered by increased phosphorylation. While the decreased cell surface expression of receptors was reversible upon removal of okadaic acid the IGF II-induced redistribution of M6P/IGF II receptors to the plasma membrane (Braulke, T., Tippmer, S., Neher, E., and von Figura, K. (1989) EMBO J. 8, 681-686) was irreversibly inhibited by the phosphatase inhibitor. Receptor redistribution in response to protein kinase C activation was not affected by okadaic acid. These results suggest that the cell surface expression of M6P/IGF II receptor can be regulated by phosphatase-dependent and -independent pathways. In addition, the phosphorylation state and the steady-state cell surface number of transferrin receptors were not affected by okadaic acid, whereas it impaired the IGF II-stimulated receptor redistribution similarly as for M6P/IGF II receptors. The data indicate that okadaic acid-sensitive protein phosphatases may play a general role in terms of IGF II-modulated receptor recycling.     

Lysosomes in Tetrahymena pyriformis. I. Some properties and lysosomal localization of acid hydrolases

In homogenates of Tetrahymena pyriformis, five hydrolases — phosphatase, ribonuclease, deoxyribonuclease, proteinase, amylase — with acid pH optima were found. Over 75% of their activity is sedimentable with a centrifugal force of 250,000 g. min. Only 17% of the acid phosphatase and ribonuclease is active when assayed in the presence of 0.25 M sucrose at 0°. Exposure to a lowered osmotic pressure, freezing and thawing, and incubation at temperatures over 0° result in activation of the latent phosphatase and ribonuclease. After isopycnic centrifugation in a sucrose density gradient the hydrolases show a broad distribution which differs greatly from those of enzymes associated with mitochondria (succinate dehydrogenase) or with peroxisomes (catalase). The results are interpreted as evidence that the five acid hydrolases studied are localized in lysosomes which represent a distinct population of subcellular particles in Tetrahymena.     

Identification of novel lysosomal matrix proteins by proteome analysis

The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins.     

Sortilin and prosaposin localize to detergent-resistant membrane microdomains

Most soluble lysosomal hydrolases are sorted in the trans-Golgi network (TGN) and delivered to the lysosomes by the mannose 6-phosphate receptor (M6PR). However, the non-enzymic sphingolipid activator protein (SAP), prosaposin, as well as certain soluble lysosomal hydrolases, is sorted and trafficked to the lysosomes by sortilin. Based on previous results demonstrating that prosaposin requires sphingomyelin to be targeted to the lysosomes, we hypothesized that sortilin and its ligands are found in detergent-resistant membranes (DRMs). To test this hypothesis we have analyzed DRM fractions and demonstrated the presence of sortilin and its ligand, prosaposin. Our results showed that both the M6PR and its cargo, cathepsin B, were also present in DRMs. Cathepsin H has previously been demonstrated to interact with sortilin, while cathepsin D interacts with both sortilin and the M6PR. Both of these soluble lysosomal proteins were also found in DRM fractions. Using sortilin shRNA we have showed that prosaposin is localized to DRM fractions only in the presence of sortilin. These observations suggest that in addition to interacting with the same adaptor proteins, such as GGAs, AP-1 and retromer, both sortilin and the M6PR localize to similar membrane platforms, and that prosaposin must interact with sortilin to be recruited to DRMs.