Acid phosphatase 5 is responsible for removing the mannose 6-phosphate recognition marker from lysosomal proteins

Abstract

Most newly synthesized proteins destined for the lysosome reach this location via a specific intracellular pathway. In the Golgi, a phosphotransferase specifically labels lysosomal proteins with mannose 6-phosphate (Man-6-P). This modification is recognized by receptors that target the lysosomal proteins to the lysosome where, in most cell types, the Man-6-P recognition marker is rapidly removed. Despite extensive characterization of this pathway, the enzyme responsible for the removal of the targeting modification has remained elusive. In this study, we have identified this activity. Preliminary investigations using a cell-based bioassay were used to follow a dephosphorylation activity that was associated with the lysosomal fraction. This activity was high in the liver, where endogenous lysosomal proteins are efficiently dephosphorylated, but present at a much lower level in the brain, where the modification persists. This observation, combined with an analysis of the expression of lysosomal proteins in different tissues, led us to identify acid phosphatase 5 (ACP5) as a candidate for the enzyme that removes Man-6-P. Expression of ACP5 in N1E-115 neuroblastoma cells, which do not efficiently dephosphorylate lysosomal proteins, significantly decreased the steady state levels of Man6-P glycoproteins. Analysis of ACP5-deficient mice revealed that levels of Man-6-P glycoproteins were highly elevated in tissues that normally express ACP5, and this resulted from a failure to dephosphorylate lysosomal proteins. These results indicate a central role for ACP5 in removal of the Man-6-P recognition marker and open up new avenues to investigate the importance of this process in cell biology and medicine.

The lysosome is an acidic membrane-delimited organelle that plays a critical role in the cellular digestion of a diverse range of macromolecules including proteins, carbohydrates, nucleic acids, and lipids (1). These catabolic functions are conducted primarily by the concerted actions of over 60 soluble luminal hydrolases and accessory proteins, most of which are targeted to the lysosome by a common pathway (reviewed in ref. 2). Central to this pathway is a posttranslational modification that is largely specific to lysosomal proteins, mannose 6-phosphate (Man-6-P). Like other glycoproteins, soluble lysosomal proteins are synthesized in the endoplasmic reticulum and are cotranslationally glycosylated on select asparagine residues. As these proteins move through the secretory pathway, the lysosomal proteins are selectively recognized by a phosphotransferase that initiates a two-step reaction that results in the generation of the Man-6-P modification on specific N-linked oligosaccharides. The modified proteins are then recognized by two Man-6-P receptors (MPRs), the cation-dependent MPR and the cation-independent (CI-) MPR. These receptors bind the phosphorylated lysosomal proteins in the near neutral pH environment of the transGolgi network and travel to an acidic prelysosomal compartment in which the low pH promotes dissociation of the receptors and ligands. The receptors then recycle back to the Golgi to repeat the process or to the plasma membrane. At the latter location, the CI-MPR can function in the endocytosis and lysosomal targeting of extracellular Man-6-P glycoproteins.

Early studies indicated that Man-6 phosphorylation was a transient modification of lysosomal proteins that was removed with a half-life of 1.4 h in mouse lymphoma cells (3) and also rapidly removed in CHO cells (4). The exact site of dephosphorylation is not clear, and there is evidence suggesting that it may occur in both a prelysosomal compartment (5) and a dense postendosomal compartment that probably represents the lysosome itself (6). The rate and the extent of removal of the Man-6-P recognition marker in cell culture are dependent on cell-type and culture conditions. For example, the Man-6-P modification was found to persist in cells lacking the CI-MPR (4). In addition, mouse L cells remove the Man-6-P modification normally when grown at low density but accumulate Man-6-P glycoproteins when grown at high density or in the absence of serum (7, 8). Man-6-P glycoproteins endocytosed by the l-cells in the absence of serum were restricted to a subset of lysosomes, suggesting that there may be lysosomal populations with distinct functions that differ in their ability to remove the Man-6-P modification (8).

Cell-specific differences in the removal of the Man-6-P recognition marker have also been observed under physiological conditions. Although rat liver has higher levels of most lysosomal enzyme activities than rat brain, the actual proportion of these activities that are represented by the Man-6-P-containing form of each enzyme is dramatically less in the liver than in the brain (9). In the brain, Man-6-P-containing glycoproteins predominantly accumulate within the lysosomes that are present in neuronal cell bodies (10).

The cellular activity that is responsible for the removal of the Man-6-P recognition marker has not been identified. It could conceivably be a glycosidase or isomerase but is most likely a phosphatases, and several studies have investigated the role of two lysosomal hydrolases, acid phosphatase 2 (ACP2, also called lysosomal acid phosphatase) and acid phosphatase 5 (ACP5, also called tartrate-resistant acid phosphatase or uteroferrin) in the removal of Man-6-P from lysosomal proteins. ACP2 is transported to the lysosome in a membrane-bound form and then is proteolytically cleaved to release a soluble phosphatase (11), whereas ACP5 is a soluble lysosomal protein that is targeted via the Man-6-P pathway (12). Both enzymes hydrolyze a variety of phosphomonoesters at acid pH in vitro, but their physiological substrates are unknown. ACP2 was overexpressed by a factor of 100-fold in mouse L cells, but this failed to increase the slow rate of removal of Man-6-P and purified ACP2 did not dephosphorylate arylsulfatase A in vitro (13). These data suggest that ACP2 is not involved in the removal of Man-6-P under physiological conditions. In contrast, ACP5 can efficiently dephosphorylate arylsulfatase A in vitro and was originally considered a likely candidate for the Man-6-phosphatase (6). However, subsequent studies on fibroblasts from mice that were deficient in both ACP5 and ACP2 concluded that the absence of both of these enzymes had no effect on the dephosphorylation of endocytosed arylsulfatase A (14). Thus, the current view is that ACP2 and ACP5 do not play a significant role in the dephosphorylation of lysosomal proteins under physiological conditions.

In a recent proteomic study of lysosomal proteins in rat tissues, we made the observation that ACP5 protein was present at low levels in the brain but relatively high levels in the liver (15). This finding is consistent with the measurement of a relative abundance of ACP5 transcripts in rodents (16). Given that the levels of ACP5 appeared inversely correlated with levels of glycoproteins containing Man-6-P in rat liver and brain, these observations suggest that this enzyme could play a role in the removal of this carbohydrate modification. In this study, we have examined the potential role of ACP5 in removal of the Man-6-P recognition marker and demonstrate that this enzyme is responsible for this cellular function.

Results

Bioassay for the Activity that Removes Man-6-P.

We initially developed a bioassay for the activity responsible for the removal of the Man-6-P-targeting modification from lysosomal proteins [supporting information (SI) Fig. S1]. This assay entailed monitoring the phosphorylation state of endogenous lysosomal enzymes in N1E-115 cells, a neuroblastoma cell line that does not efficiently dephosphorylate lysosomal proteins. Briefly, N1E-115 cells were cultured in the presence of an analyte [e.g., tissue extract or fractions from a purification procedure for the dephosphorylation activity (Fig. S2)]. The phosphorylation state of lysosomal enzymes was determined by comparing the activities of lysosomal enzymes that were retained on immobilized MPR affinity columns with the total activities, as previously described (9). Approximately 70% of each lysosomal hydrolase retains Man-6-P in N1E-115 cells (data not shown). Culturing the cells in the presence of a mouse liver differential centrifugation fraction enriched in lysosomes resulted in a significant increase in dephosphorylation of cellular lysosomal activities, and this was essentially complete after 7 h (Fig. S3A). There was a lag period of 1 h, which was presumably the time period required for the endocytosis and delivery of the enzyme that removes Man-6-P to the compartment in which dephosphorylation takes place. Dephosphorylation of endogenous lysosomal enzymes depended on the amount of liver extract added (Fig. S3B).

Two lines of investigation indicate that the activity present in the liver extracts is physiologically relevant to lysosome function and does not reflect some other stress condition or signal transduction pathway that indirectly transforms the N1E-115 cells into a dephosphorylation-competent state.

First, the liver contains high levels of the dephosphorylation activity compared with the brain. Previous studies from our laboratory (9) indicate that the liver contains higher levels of lysosomal enzyme activities than the brain, but the proportion of these enzymes that contain Man-6-P is much lower. From this observation, it is reasonable to predict that the activity that removes Man-6-P activity is high in the liver and low in the brain. We tested this prediction by culturing N1E-115 cells with equivalent amounts of tissue extracts from the liver and the brain. The liver extract allows the N1E-115 cells to dephosphorylate ≈80% of the Man-6-P containing forms of lysosomal proteins, whereas dephosphorylation in cells cultured with the brain extract was considerably less (≈20%) (data not shown). This finding is congruent with our measurements of steady-state phosphorylation in vivo, indicating physiological relevance.

Second, the dephosphorylation activity is associated with lysosomes. We conducted classical analytical subcellular fractionation to determine what membrane-delimited cellular compartment contains the dephosphorylation activity. From analysis of membrane fractions from postnuclear supernatants, the activity was enriched most highly in the differential centrifugation fraction L, similar to that of lysosomal markers (Fig. S4). Although these results are consistent with lysosomal localization, peroxisomes are also enriched in liver L fractions. We therefore conducted isopycnic centrifugation on liver organelles prepared from control mice and mice injected with Triton WR-1339 4 days before subcellular fractionation. Triton WR-1339 induces a lysosomal lipidosis and results in a selective shift in the buoyant density of lysosomes but not of other organelles. As demonstrated in Fig. S5, the dephosphorylation activity undergoes a shift similar to that of lysosomal markers. These findings are consistent with the conclusion of others (6) that the activity that removes the Man-6-P modification resides in a late endocytic/lysosomal compartment.

Tissue Expression of Lysosomal Proteins.

The observation that the activity that removes Man-6-P resides within the lysosome led us to consider known and potential lysosomal proteins as candidates for this enzyme. Given that the Man-6-P marker is largely retained in the brain and removed in the liver, the protein responsible for this activity would be expected to be expressed at relatively low levels in the former and high levels in the latter. In a recent proteomics study of lysosomal proteins in rat tissues, we made the observation that the distribution of ACP5 fits this profile (15). This finding is also consistent with the measurement of a relative abundance of ACP5 transcripts in mice by Northern blotting (16) and in mice, rats, and humans found by using the Unigene EST expression profiling tool (www.ncbi.nlm.nih.gov/sites/entrez?db=unigene). Given that ACP5 has also been demonstrated to dephosphorylate Man-6-P glycoproteins in vitro (6), we therefore decided to investigate this enzyme further as a candidate for the enzyme that dephosphorylates lysosomal proteins.

Dephosphorylation of Man-6-P Glycoproteins in N1E-115 Cells by ACP5.

If ACP5 were responsible for the removal of the Man-6-P recognition marker, increased levels of this enzyme should result in increased dephosphorylation of lysosomal proteins. We tested this prediction by overexpressing ACP5 in N1E-115 neuroblastoma cells. As a control, we also transfected these cells with another lysosomal phosphatase, ACP2, that is not thought to be involved in the removal of Man-6-P (13). We identified transfected cells by visualization of the cotransfection marker GFP and visualized Man-6-P glycoproteins with biotinylated sCI-MPR (Fig. 1). In cells transfected with GFP alone (data not shown) or cotransfected with GFP and ACP2, Man-6-P glycoproteins were detected at similar levels as observed in untransfected N1E-115 cells (Fig. 1 Upper). However, cells transfected with ACP5 showed significantly reduced levels of Man-6-P glycoproteins compared with untransfected cells (Fig. 1 Lower). These results indicate increased removal of the Man-6-P recognition marker when ACP5 is overexpressed in a cell-based model.

Fig. 1.

Expression of recombinant ACP5 in mouse neuroblastoma cells. N1E-115 cells were cotransfected with plasmids expressing ACP2 or ACP5 and GFP as a marker for transfected cells (also see arrows, Right). Images were obtained at 40× magnification.

Glycoproteins Containing Man-6-P in ACP5 Deficient Mice.

To investigate the potential role of ACP5 in the dephosphorylation of lysosomal proteins under physiological conditions, we examined how the loss of this protein affected levels of Man-6-P glyco-proteins in an ACP5-knockout mouse model. Should ACP5 play a physiological role in the removal of this modification, levels of Man-6-P glycoproteins should be increased in its absence. Soluble extracts were prepared from tissues, and Man-6-P glycoproteins were detected with a radiolabeled Man-6-P receptor affinity probe after fractionation of proteins by SDS/PAGE and transfer to nitrocellulose (17).

Loss of ACP5 had little or no significant effect on the levels or pattern of Man-6-P glycoproteins in brain and heart (Fig. 2). This finding is consistent with the fact that these tissues normally express low levels of ACP5. However, loss of ACP5 had a dramatic effect on levels of Man-6-P glycoproteins in tissues that normally express higher levels of this enzyme. In the knockout mice compared with controls, the patterns of Man-6-P glyco-proteins were similar in such tissues but levels were clearly increased in liver, kidney, spleen, testis, lung, and small intestine (Fig. 2). Total levels of Man-6-P glycoproteins were strikingly high in the spleen and testis in mutant mice.

Fig. 2.

Levels of Man-6-P glycoproteins in tissues from ACP5-deficient mice. Twenty microgram protein equivalents of each tissue extract were fractionated by SDS/PAGE, transferred to nitrocellulose, and probed with 2 nM ¹²⁵I-CI-MPR to specifically detect glycoproteins containing Man-6-P. Tissue extracts were prepared from Acp5^−/− and Acp5^+/+ 129SvEv mice.

Lysosomal Enzyme Activities in ACP5-Deficient Mice.

The finding that levels of Man-6-P glycoproteins were elevated in the absence of ACP5 is consistent with a role for this enzyme in removal of the Man-6-P recognition marker. In this case, the proportion of a given lysosomal protein that contains Man-6-P would be increased. However, an alternative possibility is that the increase in Man-6-P glycoproteins in the absence of ACP5 may simply reflect an elevation in lysosomal protein expression with the proportion of proteins that contain the Man-6-P modification being the same in the mutant and control mice. To distinguish between these possibilities, we measured the activity of 12 representative lysosomal enzymes in tissues (brain, liver, spleen, kidney, heart, and serum) from ACP5-deficient and wild-type control mice (Fig. 3 and Fig. S6). For most of the enzymes measured, there was little or no alteration in activity in the absence of ACP5. In cases where there was an increase in activity, elevations were modest (less than ≈1.5-fold) and did not appear sufficient to account for the increased levels of Man-6-P glycoproteins. These results suggest that the increase in levels of Man-6-P glycoproteins in the absence of ACP5 cannot be attributed to a generalized increase in lysosomal enzyme activities. Interestingly, α-l-fucosidase was significantly decreased in all of the ACP5-deficient tissues tested.

Fig. 3.

Lysosomal enzyme activities in ACP5-deficient mice. Activities of 12 lysosomal enzymes were measured in brain and liver from Acp5^+/+ and Acp5^−/− mice. Results were obtained from 2 animals of each genotype and expressed as activity in Acp5 mutant tissue normalized to wild-type control tissue with the line at y = 1 representing control activity. Mean activities are shown with error bars representing the range.

Dephosphorylation of Lysosomal Proteins in ACP5-Deficient Mice.

Our results suggest that removal of the Man-6-P recognition marker is impaired in the ACP5-deficient mice and in this case, the relative proportion of a given lysosomal protein that retains the targeting modification will be increased. To address this possibility directly, we conducted analytical affinity purification of Man-6-P glycoproteins on immobilized sCI-MPR and measured the proportion of lysosomal activities that contain Man-6-P as the fraction that was specifically eluted from the column compared with the total activity. We measured 4 representative lysosomal enzyme activities, 3 of which contain Man-6-P (β-galactosidase, β-hexosaminidase, and β-glucuronidase) and a control activity (β-glucosidase, also called glucocerebrosidase) that does not contain Man-6-P and is targeted to the lysosome by an independent pathway (18). In the liver, the Man-6-P modification is normally efficiently removed and levels of the Man-6-P containing lysosomal proteins containing this modification were very low in the control mice (Fig. 4). However, in the absence of ACP5, the proportion of these enzymes that contained Man-6-P was elevated greatly. Levels of β-glucosidase that were specifically purified on the immobilized sCI-MPR were unchanged. In the brain, where the Man-6-P modification is retained, the proportion of each activity represented by the Man-6-P-containing form was unchanged in the absence of ACP5. These results indicate that the increased levels of Man-6-P glycoproteins measured in the ACP5-deficient mice do not reflect an increase in lysosomal protein synthesis but result from decreased removal of the targeting modification.

Fig. 4.

Phosphorylation state of lysosomal proteins in control and ACP5-deficient mouse brain and liver. Tissue homogenates from Acp5^+/+ (open symbols) and Acp5^−/− (filled symbols) mice were fractionated by affinity chromatography on immobilized sCI-MPR (9). Enzyme activities attributable to the Man-6-P glycoforms were calculated as the activity in the Man-6-P eluate/total recovered activity (flow through/wash+glucose-6 phosphate eluate+Man-6-P eluate). Bars represent mean of measurements from 2 animals of each genotype and error bars represent the range.

Localization of Man-6-P Glycoproteins in ACP5-Deficient Mice.

We investigated the location of Man-6-P glycoproteins in the liver and brain from ACP5-deficient mice by using biotinylated sCI-MPR as a specific affinity probe. As a lysosomal marker, we also performed immunodetection for a known lysosomal protein, TPPI. There was no significant difference in the levels of Man-6-P glycoproteins between the ACP5-deficient and wild-type brain sections (Fig. 5), which is consistent with our previous findings (Fig. 2). Man-6-P glycoproteins were not detectable in the livers of wild-type mice. However, in the absence of ACP5, Man-6-P glycoprotein levels were clearly elevated. In both the brain and liver, TPPI staining was unaffected by the loss of ACP5. Superimposed images showed that Man-6-P glycoproteins colocalized with TPPI in both the brains and livers of ACP5-deficient mice. These results indicate that Man-6-P glycoproteins accumulate within the lysosomal compartment in the absence of ACP5.

Fig. 5.

Localization of Man-6-P glycoproteins in ACP5-deficient mice. TPPI and Man-6-P glycoproteins were detected in the brains (A) and livers (B) of wild-type and mutant mice. In the merged images, the signals from GFP and Man-6-P proteins are in green and red, respectively, with yellow indicating colocalization. Images were obtained at 63× magnification.

Discussion

In this study, we have used 2 parallel approaches to demonstrate that ACP5 is essential for removal of the Man-6-P recognition marker from lysosomal proteins. First, in mice that are deficient in ACP5, we show that levels of Man-6-P glycoproteins are dramatically elevated in tissues that normally contain low levels of these proteins. Second, in a murine neuroblastoma cell line that retains the Man-6-P modification on lysosomal proteins, levels of Man-6-P glycoproteins are clearly reduced when ACP5 is overexpressed. Given that ACP5 has been reported to dephosphorylate Man-6-P glycoproteins in vitro (6), the most compelling hypothesis is that ACP5 itself is directly responsible for the removal of Man-6-P targeting modification in many cell types.

An earlier study (14) concluded that neither ACP5 nor ACP2 was essential for dephosphorylating lysosomal proteins. This conclusion was based on the observation that the rate of dephosphorylation of exogenously added Man-6-P-containing arylsulfatase A appeared similar after endocytosis by wild-type or Acp2^−/−/Acp5^−/− embryonic murine fibroblasts. It is possible that there is a compensatory dephosphorylation activity in cultured fibroblasts that is not present in the tissues analyzed here. Alternatively, endocytosed arylsulfatase A may be delivered to a dephosphorylation-incompetent or -deficient lysosomal/endosomal compartment (8). It will therefore be of interest to determine whether the dephosphorylation of lysosomal proteins depends on their route of delivery to the lysosome (e.g., endocytosis or intracellular targeting).

A variety of biological functions has been assigned to ACP5 (reviewed in ref. 19), including roles in iron metabolism and immune function, but it is best known for its participation in skeletal development and collagen metabolism and its importance in these processes are clear from studies of the ACP5-deficient mutant mouse (20, 21). However, the observation that ACP5 is expressed at different levels in mammalian tissues and that its expression appears to be related to the Man-6-phosphorylation status of lysosomal proteins suggests that ACP5 may have more widespread physiological function. It is also worth noting that there is evidence for multiple populations of lysosomes within a cell that differ in their respective abilities to remove the Man-6-P modification (8). Distribution of hydrolases between these compartments appears to be regulated by serum factors (7). The precise significance of the presence of both phosphorylation-competent and -incompetent lysosomes within the cell is not clear but this intracellular compartmentalization may be intricately linked to ACP5 expression, processing, or location.

Why the Man-6-P modification is removed in some cell types (e.g., hepatocytes) but retained in others (e.g., neurons) remains an intriguing question. Studies using cultured fibroblasts suggest that retention of the Man-6-P modification may allow lysosomal enzymes to be anchored at the plasma membrane by interaction with the CI-MPR, allowing for their extracellular function in a locally concentrated and spatially controlled manner (22, 23). In the brain, it is conceivable that lysosomal enzymes tethered at the cell surface may play an important role in axon growth and neuronal migration. Conversely, retention of the Man-6-P modification may have a protective role in some cell types. In most cell types, lysosomal proteins are largely retained within this compartment after intracellular targeting, which limits potential deleterious effects associated with inappropriate release of hydrolases. However, in some cell types that are engaged in extensive vesicular trafficking (e.g., neurons), one unintended consequence may be inappropriate release of lysosomal hydrolases into the extracellular milieu. In such cases, retention of the Man-6-P modification may facilitate the rapid reuptake of secreted lysosomal proteins and their delivery to the lysosome via CI-MPR-mediated endocytosis.

Alterations in the lysosomal system accompany and may play a mechanistic role in widespread disorders, including cancer. Studies on human breast cancer specimens revealed a marked increase in the levels of Man-6-P containing glycoproteins in approximately 1/3 of tumors, and this was associated with the more aggressive cases (24). This finding does not appear to simply reflect increased lysosomal enzyme synthesis but probably reflects decreased dephosphorylation. Retention of the Man-6-P modification in malignant cells could allow presentation of these hydrolases on the cell surface via interaction with the CI-MPR, and this could play a role in pathogenic processes such as metastasis (22, 23, 25). It may be relevant in this respect that ACP5 has been reported to be down-regulated in some forms of hepatocarcinoma (26).

In summary, we have identified a key enzyme responsible for removal of the Man-6-P targeting modification from lysosomal proteins. This identification should allow in-depth investigation regarding the biomedical importance for removal and retention of this posttranslational modification.

Materials and Methods

Materials.

N1E-115 mouse neuroblastoma cells (27) and eukaryotic pCMV-SPORT6 expression plasmids encoding murine ACP5 (MGC:7956) and ACP2 (MGC:31030) were obtained from the American Type Culture Collection. The eukaryotic expression plasmid pCS2-GFP was constructed and kindly provided by P. Matteson (University of Medicine and Dentistry of New Jersey, Piscataway, NJ). Rabbit polyclonal antisera Rb72–5 raised against tripeptidylpeptidase 1 (TPPI) and biotinylated and ¹²⁵I-labeled derivatives of the soluble cation-independent mannose 6-phosphate receptor (sCI-MPR) were described previously (17, 24, 28). Rabbit anti-GFP was from Molecular Probes. Goat anti-rabbit IgG conjugated with Alexa Fluor 488 and streptavidin conjugated with Alexa Fluor 555 were purchased from Invitrogen. ACP5-deficient-knockout mice in a 129SvEv genetic background were described previously (20).

Detection of Man-6-P Glycoproteins in Mouse Tissue Extracts.

Twelve-week-old male wild-type and ACP5-knockout mice were killed, and tissues were rapidly frozen in liquid nitrogen and stored at −80°C before use. Frozen tissue powders were prepared by using a Bessmann tissue pulverizer (Thermo Fisher Scientific), and 100–150 mg of each were transferred to a 1.5-ml microcentrifuge tube. Ten volumes (wt/vol) homogenization buffer [PBS (PBS) containing 0.1% Triton X-100, 5 mM β-glycerophosphate, and 1 mM EDTA] were added, and samples were homogenized on ice by using a Polytron homogenizer (Brinkmann Instruments). The resulting homogenate was cleared by centrifugation at 14,000 rpm at 4 °C for one hour in a BHG Hermle Z229 microcentrifuge and supernatants transferred to fresh tubes. Protein concentration was measured by using Bio-Rad protein assay (Bio-Rad LaboratoriesInvitrogen), transferred to nitrocellulose membrane, and probed with ¹²⁵I labeled sCI-MPR receptor as described previously (17). Radioactive signal was detected by using a PhosphorImager (GE Healthcare) and quantified by using ImageQuant 5.2 software (Molecular Dynamics).

Enzyme Activity Measurements.

Fluorescence was measured with a CytoFluor 4000 multiwell plate reader (PerSeptive Biosystems) with excitation at 360 nm and emission at 460 nm. Measurements from different dilutions were averaged after correction for dilution factor. Enzyme activity measurements were conducted by using fluorescent substrates either as described (9) or with the following protocols. Individual reactions conditions for each enzyme were as follows (enzyme, substrate/buffer/incubation time): β-glucosidase, 5 mM 4-MU-β-glucoside/acetate, pH 5.0, 0.25% sodium taurocholate/60min; α-mannosidase, 1 mM 4-MU-α-mannoside/acetate, pH 4.0/60 min; β-glucuronidase, 1 mM 4-MU-β-glucuronide/acetate, pH 4.0/30 min; α-fucosidase, 0.2 mM 4-MU-α-fucoside/citrate, pH 5.0/120 min; cathepsin B, 0.2 mM Z-Arg-Arg-AMC/acetate with 5 mM DTT and 2.5 mM EDTA, pH 6.0/30 min; dipeptidylpeptidase I, 0.5 mM Gly-Arg-AMC/acetate with 5 mM DTT and 2.5 mM EDTA, pH 5.5/60 min; dipeptidylpeptidase II, 0.5 mM Lys-Ala-AMC/acetate, pH 5.5/120 min; tripeptidylpeptidase I, 0.25 mM Ala-Ala-Phe-AMC/acetate pH 4.5/60 min; and legumain, 0.2 mM Z-Ala-Ala-Asn-AMC/citrate-phosphate with 1 mM DTT, pH 5.8/120 min. Buffers consisted of 0.1 M citric acid or 0.1 M sodium acetate, adjusted to the indicated pH by using sodium hydroxide or acetic acid, respectively, and contained 0.1% Triton X-100. For all reactions, substrates were prepared as stocks in dimethyl sulfoxide that were stored at −20 °C and diluted shortly before use.

Immunohistochemistry.

Tissues from 8-week-old mice were collected and fixed overnight in Bouin's reagent (Electron Microscopy Sciences), then treated with 15%, followed by 30% sucrose. Tissues were then embedded in Tissue-Tek OCT medium (Sakura) and frozen in liquid nitrogen. Serial sections (10 μm) were cut and mounted on glass slides. Sections were incubated with the rabbit polyclonal antisera Rb72–5 (to detect the lysosomal protein TPPI) and then probed with Alexa Fluor 488 goat anti IgG at a dilution of 1:400. Man-6-P proteins were probed with biotinylated sCI-MPR as described (10) by using Alexa Fluor 555 strepavidin at a dilution of 1:1,000 as a secondary detection reagent. Slides were mounted by using Aqua Poly/Mount (Polysciences). Fluorescence was detected by using a Zeiss LSM 410 confocal microscope.

Transfection of N1E-115 Cells.

N1E-115 cells were grown to 50% confluence in 4-well chamber slides and were cotransfected with 0.05 μg of pCS2-GFP and 0.5 μg of either pCMV-SPORT6-ACP5 or pCMV-SPORT6-ACP2 using lipofectamine (Invitrogen). After 24 h, cells were fixed with Bouin's reagent for 15 min and then permeabilized with PBS containing 0.5% Triton X-100. Probing was as described above except that anti-GFP antibody (1:1,000 dilution) was used instead of anti-TPPI antibody. Images were captured by using a Nikon Eclipse E600 fluorescence microscope (Nikon Instruments) at magnification 40×. Images were taken by using a SPOT software version 4.5.9.9 (Diagnostic Instruments).

Other.

Determination of the phosphorylated proportion of lysosomal proteins was conducted by analytical purification of Man-6-P glycoproteins as described previously (9). The cell-based dephosphorylation bioassay is described in Fig. S1. Subcellular fractionation is described in Fig. S2.