JOURNAL OF CELLULAR PHYSIOLOGY 143:110-117 (1990)

Kinetics of Hydrolysis of Endocytosed Substrates by Mammalian Cultured Cells: Early Introduction of Lysosomal Enzymes Into the Endocytic Pathway ROBERT BOWSER AND ROBERT F. M U R P H Y *

Department of Biological Sciences and Center for Fluorescence Research in Biomedical Sciences, Carnegie-Mellon University, Pittsburgh, Pennsylvania 7 52 13 The kinetics of exposure of endocytosed material to two lysosomal enzymes were determined for a number of cultured cell lines using fluorogenic substrates. Hydrolysis of endocytosed substrates for cathepsin B and acid phosphatase was observed to begin within 3-1 0 min of substrate addition and to proceed linearly for up to 60 min thereafter. Hydrolysis of the cathepsin B substrate was not affected by inhibition of protein synthesis with cycloheximide, indicating that the enzymes present in early endosomes are not exclusively newly synthesized. As had been observed previously for a cathepsin B substrate (Roederer,M., Bowser, R., and Murphy, R.F., 1. Cell. Physiol., 13 1:200-209, 1987), hydrolysis of the acid phosphatase substrate was not blocked at temperatures below 20°C. The results suggest that the endosome is the primary site of initial exposure of endocytosed material to hydrolytic enzymes

The acidic endocytic compartments involved in endocytosis are currently divided into two major classes, endosomes and lysosomes. Two broad lines of evidence support this distinction. First, populations of the two endocytic compartments can be resolved in 27% Percoll density gradients (e.g., Merion and Sly, 1983). Endocytosed material, whether it is internalized by receptormediated or fluid-phase endocytosis, is observed to enter the lighter of these compartments (endosomes) first, and then appears in the denser compartments (lysosomes) a t later times. Second, appearance of endocytosed material in dense compartments, and associated degradation, can be inhibited by incubation at temperatures below 20°C (Dunn e t al., 1980; Marsh et al., 1983). These temperatures have been postulated to block the fusion of late endosomes with lysosomes, although no direct evidence for the occurrence of such a fusion event in vivo is currently available. While most models of endocytosis previously held that endosomes lack lysosomal enzymes, recent evidence indicates that at least some lysosomal enzymes are introduced into the endocytic pathway much earlier than had previously been thought. Diment and Stahl (1985) observed cathepsin D activity in low-density, prelysosomal compartments after as little as 6 min at 37°C. We have previously shown that fluorogenic substrates may be used to determine the kinetics with which endocytosed material is exposed to specific hydrolytic enzymes in living cells (Roederer et al., 1987). Hydrolysis of a substrate for cathepsin B began within 2-5 min after substrate addition and was not blocked at temperatures below 20°C. These results suggested t h a t the inhibition of degradation and transfer to dense compartments previously observed at low temGI 1990 WILEY-LISS,

INC.

peratures may not involve prevention of fusion between endosomes and lysosomes. While exposure of endocytosed material to the full active complement of lysosomal enzymes may be inhibited at reduced temperatures, its exposure to at least some of these enzymes is clearly not. In view of the possibility that only cathepsins or similar peptidases might be present in the endosome, arid the possibility that presence of hydrolytic enzymes in endosomes might be restricted to certain cell types, we have examined the hydrolysis of substrates for cathepsin B and acid phosphatase in a number of cell lines. While quantitative differences were observed between some cell lines, the results indicate that these lysosoma1 enzymes are present and active in early endocytic compartments in many cell types, in a manner that is not blocked by low temperatures. Preliminary accounts of this work have been presented previously (Roederer et al., 1986; Murphy et al., 19891.1 ‘Minisymposium on Secretion and Endocytosis, The American Society for Cell Biology Twenty-Sixth Annual Meeting, Washington, DC, December 9, 1986; American Society for Cell Biology Summer Research Conference, Airlie, VA, June 7, 1987. Received July 12, 1989; accepted December 19, 1989. *To whom reprint requestsicorrespondence should be addressed. Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; cDMEM, DMEM supplemented with 2 mM L-glutamine, 100 unitsiml penicillin, and 100 Fgiml streptomycin; MNA-peptide, N-carbobenzyloxy-ala-arg-arg-4-methoxy-~-naphthylamin~e; MUP, 4-methylumbelliferyl-phosphate;PBS, 137 mM NaCI, 27 mM KC1, 153 m M Na,HPO,, 14.7 mM KH,PO,, 0.6 mM CaCI,, 0.5 mM MgCl,, pH 7.4.

HYDROLYSIS KINETICS OF ENDOCYTOSED SUBSTRATES

MATERIALS AND METHODS Cells and reagents All adherent cell lines were passaged before confluence. Swiss 3T3 cells (originally obtained from American Type Culture Collection, Rockville, MD) were grown in cDMEM containing 10% fetal calf serum. The Chinese hamster ovary (CHO) cell line K1 (obtained from Dr. Rockford Draper) was grown in cDMEM containing 5% fetal calf serum and 40 pgiml proline. A201.11cells (obtained from Dr. John Kappler) were grown in cDMEM containing 10% fetal calf serum and 50 mM P-mercaptoethanol. The human erythroleukemia cell line K562 (obtained from American Type Culture Collection) was grown in RPMI-1640 containing 10% heatinactivated fetal calf serum. Mouse LMTK- cells (obtained from Dr. Rockford Draper) were grown in cDMEM containing 5% fetal calf serum. MNA-peptide was purchased from Research Plus (Bayonne, NJ) and Enzyme Systems Products (Livermore, CAI. Some lots of the substrate contained a n unknown toxic contaminant; lots were screened for toxicity before use. Stock solutions of 20 mM MNA-peptide were prepared in distilled water. MUP was purchased from Sigma (St. Louis, MO) and dissolved in 0.01 M sodium phosphate-citrate buffer, pH 6.3, at a concentration of 20 mM. a-Naphthylphosphate was purchased from Sigma and dissolved in DMEM at a concentration of 400 mM. Dihydrocytochalasin B was purchased from Sigma and dissolved in PBS at a concentration of 1 mg/ml. Fluorometry All measurements were made with a Fluoro IV spectrofluorometer (Gilford Systems, Oberlin, OH). Each sample was mixed with a plastic pipette before each reading to ensure accurate measurement of the fluorescence from all hydrolyzed substrate, including t h a t in the immediate vicinity of the cells (in the case of MNA experiments). For each experiment, the average background fluorescence of samples taken immediately after substrate addition was subtracted from all values. The number of cells per plate was measured for parallel plates for each experiment. All fluorescence values were normalized for cell number and also normalized to constant photomultiplier high voltage (using curves of fluorescence vs. photomultiplier high voltage obtained for calibration samples). The extent to which hydrolysis required a n acidic intracellular compartment was assayed by the addition of 100 mM NH,Cl 20 min prior to substrate addition (the amine was kept in the media during the subsequent incubation). MNA-peptide. Adherent cells were grown on 18 mm x 18 mm coverslips that were split in half and placed in tissue culture dishes before addition of cells. Corresponding halves were washed with PBS and placed back-to-back at a 45" angle in Ultra-Vu cuvettes (Elkay Products Inc., Shrewsbury, MA) containing 2 ml of PBS. Excitation and emission occurred above the top of the coverslips and represented fluorescence due only to soluble MNA. The temperature of the cuvettes was maintained with a circulating water bath. MNApeptide (0.5mM) was added, and the kinetics ofhydrolysis as measured by the generation of fluorescent MNA

111

(excitation 340 nm, emission 420 nm) in the cuvette were determined over a time course of 60 min. Data were recorded on a VAX 111750 as sample emission vs. time since addition of substrate. Each data point was the average emission over a 4 sec interval. The background fluorescence (before substrate addition) was typically 8%of that due to substrate hydrolyzed during 60 min. The specificity of the proteolytic activity was assayed by addition of leupeptin (100 pg/ml) immediately before substrate addition. MUP. Cells on 60 mm culture dishes were washed twice with PBS and 2 ml DMEM was added. The cells were incubated a t the appropriate temperature for 10 min to ensure temperature equilibrium. Substrate was added at the desired concentration (normally 1 mM) and incubation continued at the appropriate temperature. At various times, three samples of 10 p1 of media from the culture dish were removed and each was placed in a cuvette containing 2 ml of PBS, pH 10.5. Thirty microliters of media containing substrate was then placed into the culture dish to maintain a constant volume throughout the experiment. The fluorescence emission at 452 nm resulting from excitation a t 350 nm was measured. To determine whether MUP hydrolysis was sensitive to a competitive inhibitor, anaphthylphosphate (final concentration, 100 mM) was added simultaneously with MUP. To inhibit microfilament-dependent endocytosis (see Results), dihydrocytochalasin B was added (to a final concentration of 2 pg/ml) 10 min before MUP addition. Determination of total cellular activity. For adherent cell lines, flasks were washed with PBS and cells were harvested by scraping into homogenization buffer (250 mM sucrose, 10 mM HEPES, 2 mM EDTA, pH 6.8).The number of cells per flask was determined by trypsinization and counting of a parallel flask. For nonadherent cell lines, the number of cells per flask was determined by counting a n aliquot of the suspension culture, and cells were then pelleted and resuspended in homogenization buffer. In either case, cells were disrupted with a tight-fitting glass-Teflon homogenizer and diluted 1 : l in homogenization buffer containing 0.2% Triton X-100 (pH 6.5 for MNA-peptide and pH 5.0 for MUP). Substrate was added to 0.6 mM (MNA-peptide) or 1 mM (MUP) and hydrolysis measured every 2 min over 15 min a s described above. The hydrolysis rate was determined by linear least-squares fitting and normalized for cell number. RESULTS MNA-peptide hydrolysis To determine the kinetics with which endocytosed materials encounter a proteolytically active compartment, we have used a fluorogenic substrate specific for cathepsin B, MNA-peptide (Roederer et al., 1987). Hydrolysis of internalized MNA-peptide permits free MNA to diffuse across cell membranes and rapidly equilibrate in the external medium. Since MNA-peptide is essentially nonfluorescent a t the wavelengths used to measure liberated MNA, it is possible to estimate the kinetics of hydrolysis of MNA-peptide within intracellular vesicles by measuring the fluorescence of free MNA in the external medium as a function of time after addition of MNA-peptide.

112

BOWSER AND MURPHY

TABLE 1. Comparison of i n vivo and i n vitro hydrolysis of acid phosphatase and cathepsin B substrates

lo

?E a) 0 c 0 Q)

10

(A20

al

0

b 0

20 40

60

0

20

40

60

Time ( m i n ) Fig. 1. Kinetics of hydrolysis of MNA-peptide in different cell types. MNA-peptide (0.5 mM) was added to cells that did not receive pretreatment (0, ). or to cells that were pretreated for 20 min with 100 mM NH,Cl (o,.). The open symbols show the data for A20-1.11 multiplied by a factor of 10. An example of the inhibition of substrate hydrolysis by coincubation with 100 pgiml leupeptin is also shown (XI.

In Swiss 3T3 cells, MNA-peptide encounters compartments containing active cathepsin B within 2-3 min after addition to Swiss 3T3 cells (Roederer et al., 1987). After this short lag, the hydrolysis of MNA-peptide was observed to be linear for at least 60 min. Addition of leupeptin, a competitive inhibitor of cathepsins B, H, and L, a s well as other enzymes (Barrett and Kirschke, 1981), reduced the fluorescence more than 90%, demonstrating that the MNA-peptide is being specifically cleaved by a protease of this class. The hydrolysis also required low pH, as addition of a weak base, NH,Cl, greatly reduced the rate. Inhibitors of endocytosis, such as cytochalasin B (Wagner et al., 1971), and metabolic inhibitors, such as 2-deoxyglucose and sodium azide, also inhibited hydrolysis of MNApeptide, demonstrating that endocytosis (rather than diffusion) of the probe is required for liberation of MNA. We concluded that MNA-peptide is internalized within 3T3 cells and encounters active cathepsin B in a n acidic compartment within 3 min. To determine if these results were unique to 3T3 cells, we have performed similar studies on four other cell types (Fig. 1).The kinetics of hydrolysis were qualitatively similar in all, in that hydrolysis began within 5 min and continued linearly for up to 60 min. Addition of NH4Cl partially inhibited hydrolysis in all cell types, as was previously observed for 3T3. We attribute the lack of complete inhibition by NH,C1 to the fact

Cell line Cathepsin B substrate CHO K1 Swiss 3T3 K562 LMTK A20- 1.11 Acid phosphatase substrate CHO K1 Swiss 3T3 K562 LMTK A20-1.11

In vivo'

In vitro'

0.23

2.7 10.0 3.8 0.88 0.47

0.13 ~.

0.15 0.13 0.013 0.018 0.009 0.011 0.009 0.007

1.1 0.82 2.3 0.44 3.3 -

'Rate of substrate hydrolysis per cell over 60 min (fluorescence unitsicellimin) for intact cells. The standard deviation of the rate averaged 24% of the mean 'Rate of substrate hydrolysis per cell equivalent over 15 min (fluorescenceunils/ cellimin) for cell homogenates in the presence of 0.1% Triton-X100.

that cathepsin B activity in vitro is not completely inhibited a t neutral pH (Roederer et al., 1987) and that the high concentration of substrate used can overcome the normal pH dependence by favoring the formation of enzyme-substrate complexes (data not shown). At least some of the decreased hydrolysis may be due to inhibition of endocytosis (Sullivan et al., 1987; Fritsch et al., 1988). In any case, such a n effect would be further evidence that substrate hydrolysis occurs as a result of endocytosis (rather than diffusion). Addition of leupeptin decreased the level of MNA production 85-95% in all cell types, demonstrating that the increase in fluorescence due to MNA-peptide cleavage is due to a cathepsin B-like enzyme (data not shown). The absolute rates of hydrolysis per cell were similar for the different cell lines, with the exception of A20-1.11 (Table 1). This Balbic tumor cell line of presumed B-lymphocyte origin (Kim et al., 1979) showed only 10% of the MNA-peptide hydrolysis rate of Swiss 3T3 cells. To determine whether this difference could be accounted for by a difference in the total amount of cathepsin B per cell, MNA-peptide hydrolysis activities were measured in cell lysates in the presence of detergent (Table 1).These in vitro activities should represent the maximum hydrolysis that can be observed under any conditions, since all enzyme was exposed t o substrate and the assays were carried out a t optimal in vitro reaction conditions (e.g., pH). The observed in vivo hydrolysis rates range from 1 to 15%of the maximum possible. Much of the decreased rate of hydrolysis by A20-1.11 in vivo would appear to be due to a lower amount of active enzyme per cell than in 3T3. This low level of enzyme activity suggests the possibility that A20-1.11 may be unable to adequately degrade its physiological substrates. An indication of the normal substrate load was obtained by measuring rates of fluid-phase endocytosis. The rate of uptake of FITC-dextran (as measured by flow cytometry) in A201.11was only 6.5%that of 3T3. It is possible that one or both of these differences in A20-1.11 may relate to its function a s a n antigen-presenting cell (see Discussion). In order to determine whether the active cathepsin B that is present in early endocytic compartments is newly synthesized enzyme coming from the Golgi complex (as opposed to preexisting enzyme), we determined

113

HYDROLYSIS KINETICS OF ENDOCYTOSED SUBSTRATES

TABLE 2. Lack of inhibition of MNA-peptide hydrolysis by cycloheximide Pretreatment time (h)'

Hvdrolvsis rate'

20 min 0.246 0.269 0.262 0.330 0.230

60 min 0.218 0.213 0.221 0.297 0.207

0.6

1

'Cells were preincuhated with 20 pg/ml cycloheximide for the indicated time. 'Rate of substrate hydrolysis per cell over 20 or 60 min (fluorescence units/ cellimin) for intact cells. The standard deviation of the rate averaged 17% of the mean.

whether MNA-peptide hydrolysis could be inhibited by blocking protein synthesis with cycloheximide. Pretreatment with 20 pg/ml cycloheximide for from 1 to 8 h (which reduced protein synthesis by 95%:) did not significantly alter the rate of MNA-peptide hydrolysis by 3T3 cells (Table 2). There was no effect on either the average rate over 60 min or that over the first 20 min; the latter rate would be expected to be especially sensitive to depletion of enzyme in early compartments. The failure of cycloheximide to affect the rate of substrate hydrolysis indicates that the majority of enzyme present in early endocytic compartments is derived from preexisting hydrolytic compartments (or has taken longer than 8 h, approximately 40% of the generation time, to reach its destination). (The unlikely possibility that cycloheximide treatment perturbs the endocytic pathway so as to result in the same hydrolysis rate a s that observed in untreated cells cannot be ruled out.)

Hydrolysis of MUP by Swiss 3T3 cells Cytochemical staining with phosphatase substrates has been used extensively to measure the lysosomal enzyme content of endocytic organelles. Many of these studies have suggested t h a t early endocytic compartments do not contain significant acid phosphatase activity. A complication in interpreting these studies is the presence in quiescent cells of significant numbers of hydrolase-rich residual bodies, defined as compartments of very high density (greater than 1.12 g/ml) which take longer than 60 min to be labeled by endocytosed material (Roederer et al., 1989). Since most previous cytochemical studies have used confluent cells, the possibility exists that endosomal, and even lysosomal, acid phosphatase activity was not detected because i t was small relative to that found in residual bodies. To determine whether acid phosphatase activity is present in early endocytic compartments, we have measured the kinetics of hydrolysis of a fluorogenic substrate for acid phosphatase, 4-methylumbelliferyl-phosphate (MUP). Hydrolysis of MUP results in the production of 4-methylumbelliferone (MU), which fluoresces strongly in ultraviolet light at pH 10.5 (Mead et al., 1955) and is membrane permeant. Thus, i t is possible to estimate the kinetics of hydrolysis of MUP in internal vesicles by measuring the fluorescence of free MU in the extracellular medium a s a function of time. The onlv difference between this method and that used previo&ly for MNA-peptide is that, since fluorescence from MU is low at neutral pH, fluores-

0

20

40

60

Time ( m i n ) Fig. 2. Kinetics of hydrolysis of a fluorogenic substrate for acid phosphatase (MUP)by 3T3 cells. MUP (1 mM) was added to cells that did not receive pretreatment (m), to cells that were pretreated for 20 min with 100 mM NH,C1 ( 0 ) or to cells that also received 100 mM anaphthylphosphate (XI. The hydrolysis due to exocytosed enzymes is also shown (A,see text). Means 2 standard deviations for six experiments are shown.

cence in the media cannot be directly monitored. Instead, aliquots of media must be brought to pH 10.5 and measured separately. When MUP is added to Swiss 3T3 cells, linear hydrolysis is observed after a lag of up to 5 min (Fig. 2). This short lag may be due to the time required for the pinocytic vesicle to fuse with a vesicle containing acid phosphatase, or it may be due to the time required for adequate acidification, or both (see Discussion). Addition of amines such as NH4C1 reduces the rate of hydrolysis by approximately 50% (Fig. 21, demonstrating that a n acidic environment is required for efficient MUP hydrolysis to occur (the failure of NH4C1 to completely block hydrolysis is discussed below). As a control for possible toxic effects of ammonium chloride treatment, cells were treated with 100 mM ammonium chloride, washed, incubated 10 min in fresh media, and then incubated with MUP. The rate of hydrolysis observed was 96 k 2% that of untreated cells. This indicates that the inhibition by NH4C1 is not due to cell lysis, or any other irreversible effect, but most likely to a n increase in intravesicular pH. This provides additional indirect evidence that the hydrolysis is occurring intracellularly . Evidence that endocytosis of the substrate is required is provided by the observation that the acid phosphatase activity in microsomal pellets is 83% latent when assayed with MUP (data not shown); this result demonstrates the inabilitv of MUP to cross membranes and makes unlikely the possibility that the hydrolysis observed in vivo is of MUP that has entered

114

BOWSER AND MURPHY

cells by diffusion. The possibility t h a t MUP hydrolysis is due to exocytosis of enzyme was also ruled out by assaying the medium from cells incubated for 30 min without MUP (Fig. 2). The rate of hydrolysis was less than 10% of that observed with cells. MUP hydrolysis by cells is also inhibited 50% by a-naphthylphosphate, a competitive inhibitor of acid phosphatase (Pondaven and Meijer, 1986). The fact that the rate of MUP hydrolysis was observed to be linear over 60 min (after a n initial lag) suggests that internalized MUP does not encounter a sudden increase in acid phosphatase concentration, a s would be expected if vesicles containing MUP were to fuse with a hydrolase-rich compartment (see Discussion). This is true even though the observed hydrolysis is from millions of vesicles at different temporal and topographic locations. If post-sorting fusion with hydrolase-rich compartments were to occur, the observed hydrolysis curve would be nonlinear with a n initial slope given by the early (endosomal) amount of enzyme and a final slope dominated by the late (lysosomal) amount. However, for this reasoning to be correct, the possibility that endocytosed MUP is completely hydrolyzed early in the endocytic pathway must be ruled out. In order to determine whether this was likely, we measured the concentration dependence of MUP hydrolysis in vivo. Analysis of MUP hydrolysis rates for concentrations from 0.5 to 25 mM yield a K, of 6.2 mM. While the 1 mM concentration used in most of our experiments is below K,, it is sufficiently close that we expect that complete hydrolysis within early compartments is unlikely. Further evidence that substrate is not being exhausted early in the pathway comes from the use of inhibitors. As discussed above, we have previously shown that the hydrolysis of MNA-peptide by 3T3 cells is inhibited by cytochalasin B, which inhibits endocytosis (Wagner et al., 1971) (presumably by disrupting the cytoskeleton). Similarly, the hydrolysis of MUP is inhibited more than 75% by a cytochalasin B analog, dihydrocytochalasin B (Fig. 3). In addition to demonstrating that the majority of substrate hydrolysis observed is due to endocytosis, this result permits measurements of the time required to hydrolyze a n internalized cohort of MUP. The method is based on our previous observation that linear MNA-peptide hydrolysis can be observed for more than 20 min after leupeptin is added t o cells already containing MNApeptide (Roederer et al., 1987). Since MUP hydrolysis is not completely inhibited by coincubation with anaphthylphosphate (as opposed to the nearly complete inhibition of MNA-peptide hydrolysis observed when coincubated with leupeptin), a strictly analogous experiment was not possible. Instead, cells were allowed to internalize MUP for 15 min, and then dihydrocytochalasin B was added to block further endocytosis of substrate. The results in Figure 3 show that hydrolysis of previously internalized MUP continues for a t least 25 min after further endocytosis is blocked. If the amount of internalized MUP had been sufficiently small that it could have been hydrolyzed entirely within early compartments, the addition of dihydrocytochalasin B should have decreased the subsequent hydrolysis rate significantly. We can conclude that sufficient MUP is available throughout the endocytic

0

10

20

30

40

Time ( m i n ) Fig. 3. Hydrolysis of previously endocytosed MUP continues after further endocytosis is inhibited. MUP (1 mM) was added to 3T3 cells for 15 min. Dihydrocytochalasin B (2 kg/ml) was then added to one sample (A),while no addition was made to the other (w). The incubation was continued for an additional 25 min. In parallel, MUP was added to cells pretreated for 10 min with the same concentration of dihydrocytochalasin B ( 0 ) .

pathway so that it will be sensitive to any significant increases in enzyme concentration, such as might occur through fusion with hydrolase-rich compartments (at least during the first 25-60 rnin). Hydrolysis of MUP by other cell t y p e s MUP hydrolysis kinetics were also measured for the other four cell lines described above. All four lines exhibit NH,Cl-sensitive MUP hydrolysis, which begins within 5 min of substrate addition. The rates of hydrolysis for each cell line are shown in Table 1. The results indicate that early exposure to acid phosphatase activity is a widespread phenomenon. Similar rates of hydrolysis (within 2.5-fold) were observed for all cell types, even though the total acid phosphatase activity per cell varied by up to eightfold. In contrast to the case for cathepsin B, the in vivo rates were only 0.2-2% of the maximum possible. One explanation for these low rates is the possibility that at least some of the acid phosphatase activity is contained in compartments that are not part of the active endocytic cycle (at least as defined by that which can be reached within 60 min of endocytosis).

Temperature dependence of MUP hydrolysis A number of previous reports have indicated that degradation of endocytosed material and delivery to lysosomes is inhibited at temperatures at or below 20°C (reviewed by Helenius et al., 1983). In contrast, we have previously shown that the hydrolysis of MNi4peptide (by cathepsin B-like activity) is not blocked a t

115

HYDROLYSIS KINETICS OF ENDOCYTOSED SUBSTRATES

.

I

.

.

6 -

0.8

Temperature 37°C 24°C 19°C 17°C

W

0.6 W 0

20 min 0.018 0.012 0.008 0.000

Hvdrolvsis rate' 60 min 60 min 0.017 0.013 0.010 0.0051

NH, sensitive2 0.010 0.0086 0.0067 0.0038

'Rate of substrate hydrolysis per cell over 20 or 60 min (fluorescence units1 cellimin) for intact cells from the data in Figure 4A. 'Rate of amine-sensitive substrate hydrolysis per cell over 60 min (fluorescence unitslcellimin), calculated from the data in Figure 4C.

c

$

TABLE 3. Effect of temperature on hydrolysis of acid phosphatase substrate

0.4

m W L

0 3

-

0.2

LL

0 0 20 40 60 0 20 40 6 0 0 20 40 60

Time ( m i n ) Fig. 4. Temperature dependence of MUP hydrolysis in 3T3 cells. Cells were preincubated for 20 min a t 37°C (W, 24°C (a), 19°C (A), or 17°C (*) and incubated for a n additional 20 min in the absence (A) or presence (B) of 100 mM NH,CI. MUP (1 mM) was then added and incubation continued a t the same temperature. The difference between the hydrolysis in the absence and presence of NH,Cl is also shown (0. This difference is a n estimate of the minimum amount of hydrolysis that must be occurring in acidic compartments. Each point represents the mean of three measurements; the average standard error of the mean was less than 10%.

low temperatures, but rather that i t proceeds a t between 31 and 45% of the normal rate a t 37°C at temperatures between 13 and 21°C (Roederer et al., 1987). In order to determine whether exposure of endocytosed material to acid phosphatase activity was blocked a t reduced temperatures, the temperature dependence of MUP hydrolysis was measured in the presence and absence of NH4C1 (Fig. 4). Significant hydrolysis was observed a t all temperatures, although some reduction in rate was seen, especially at 17°C (Table 3). For MUP hydrolysis a t 17"C, the rate was 31% ofthat a t 37°C. At 19°Cthe rate of hydrolysis was 63% of that a t 37°C. The hydrolysis could be inhibited by NH4C1 a t all temperatures (Fig. 4B); the NH,Cl-sensitive hydrolysis (Fig. 4C) showed a similar temperature dependence to that of the total hydrolysis (the 17°C and 19°C amine-sensitive rates were 38% and 65% of the 37°C rate, respectively). Clearly, reduced temperatures do not cause a complete inhibition of hydrolysis of MUP, extending the previous results for MNA-peptide. It is likely that the well-documented absence of degradation of endocytosed material a t low temperatures (e.g., Dunn et al., 1980; Marsh et al., 1983) is due to the high molecular weight of the physiological ligands used, which require extensive degradation before acid-soluble material is produced. It is interesting to note that a 20-30 min lag in amine-sensitive hydrolysis was observed a t 17°C; it is reflected in the average rate over the first 20 min (Table 3). This lag may be due to the significant inhibition of acidification observed a t this temperature (Roederer et al., 1987). While inhibition of MNA-peptide hydrolysis was not observed to correlate com-

pletely with inhibition of acidification, a better correlation was observed with MUP. The lower pH requirement of acid phosphatase activity may account for this difference. We conclude that low-temperature inhibition of substrate hydrolysis may be accounted for by a combination of the temperature dependence of the enzymes and the reduced acidification rate at low temperatures.

DISCUSSION The data we have presented indicate that cathepsin B and acid phosphatase are present and active a t a n early stage of the endocytic pathway and that this phenomenon is common to a number of cell types. The results make unlikely the possibility that the presence of lysosomal enzymes in early compartments is limited to specific enzymes and specific cell types. In addition to our results for cathepsin B and acid phosphatase, Storrie et al. (1984) have observed colocalization with acid phosphatase within 13 min after addition of horseradish peroxidase; Diment and Stahl (1985) have presented evidence that early endosomes in macrophages contain cathepsin D activity; Geuze et al. (1985) have observed cathepsin D in CURL structures in Hep G2 cells; Korc and Magun (1985) have demonstrated that partial processing of EGF in light endocytic compartments occurs within 15 rnin after internalization in human pancreatic carcinoma cells; and Gabel and Foster (1987) have shown that lysosomal enzymes can be processed in endosomes. The experiments reported here used soluble enzyme substrates internalized by fluid-phase endocytosis, rather than a ligand taken up by receptor-mediated endocytosis. As discussed above, the possibility that hydrolysis results from diffusion rather than endocytosis may be ruled out by 1) the high degree of latency of the enzyme activity measured with the substrates (indicating that the substrate does not cross membranes) and 2) the inhibition of hydrolysis by cytochalasin derivatives. While hydrolysis must therefore be the result of endocytosis, the possibility that it occurs in a pathway different from that described previously for receptor-mediated ligands must be considered. At least three lines of evidence suggest that the fluid-phase and receptor-mediated pathways share a common step. First, the kinetics of initial acidification of FITC-dextran are very similar to those of EGF and transferrin (Murphy, 1988). Second, low-temperature incubations with FITC-dextran result in measured pH values close to those of endosomes labeled by receptor-mediated endocytosis (Roederer and Murphy, 1986; Roederer et al.,

116

BOWSER AND MURPHY

1987; Sipe and Murphy, 1987). Third, the in vitro acidification characteristics of endosomes labeled with FITC-dextran and FITC-transferrin are indistinguishable (Fuchs et al., 1989). Regardless of whether the pathways followed by receptor-mediated and fluidphase probes a r e identical, the results reported here reflect the characteristics of compartments which are part of the normal endocytic apparatus. As noted above, a slight difference was observed between the times of onset of MUP and MNA-peptide hydrolysis (5 min and 2-3 min, respectively). Cathepsin B has a pH optimum of 6.6 (Knight, 1980), whereas acid phosphatase has a pH optimum near 5.0 (Georgatsos, 1965; Verjee, 1969). Studies on the kinetics of acidification in 3T3 cells (Murphy et al., 1984; Roederer et al., 1987; Sipe and Murphy, 1987) have shown that early endosomes rapidly acidify to a pH near 6 within 5 min, followed by a slow acidification to a pH as low as 5.2 within 50 min. Therefore, cathepsin B present in early endosomes would reach optimal activity within 5 min, explaining the observed results for the hydrolysis of MNA-peptide. However acid phosphatase, if also present in early endosomes, would not exhibit substantial activity until the pH of the vesicle decreased below 6. Since this should occur after 5 min, the observed kinetics of acid phosphatase activity in 3T3 cells agree with previously reported acidification kinetics for these cells. Further experimentation is needed to confirm whether the difference in initial kinetics of MUP and MNA-peptide hydrolysis is significant. The distinction between the results described above for acid phosphatase and those of Storrie et al. (1984) should be noted. In the latter study, colocalization between acid phosphatase and endocytosed horseradish peroxidase was not observed a t 6 min after addition of label, but was observed by 13min. The buoyant density and morphology of the compartment labeled by this time were suggestive of late (post-sorting) endosomes; in contrast, the time of onset of MUP hydrolysis reported here ( 5 min) is consistent with a n early endosoma1 location. It is possible that the cytochemical assay used by Storrie et al. may not have been sensitive enough to detect acid phosphatase in early endosomes. Some differences in the rates of substrate hydrolysis were observed in the cell lines tested. A decreased rate of hydrolysis of MNA-peptide by A20-1.11 was particularly evident. The main explanation would appear to be a lower steady-state level of enzyme activity per cell. The possibility that the low amount of enzyme results in incomplete digestion of endocytosed proteins, as would be required for antigen presentation (Unanue, 1984), suggests itself. Whether the reduced level of fluid-phase endocytosis in A20-1.11 is also involved remains unclear. The possibility that surface proteases, which have been detected on antigen-presenting cells (Buus and Werdelin, 1986), may contribute to the hydrolysis observed in A20-1.11 must also be considered. Since inhibition of protein synthesis by cycloheximide does not affect hydrolysis of MNA-peptide, the presence of lysosomal enzymes in the endosome is not restricted to newly synthesized enzymes or enzyme precursors. The linearity of MNA-peptide and MUP hydrolysis curves indicates that a fusion event between late endosomes and a hydrolase-rich compartment is unlikely (see above). While alternative models may not

be ruled out, all of these results, taken together, suggest, but do not prove, that the primary site of exposure of endocytosed material to lysosomal enzymes is the endosome. Whether the enzymes that are present in endosomes a r e resident or transient remains to be determined. Some enzymes (e.g., acid phosphatase) are present in both light (endosomal) and dense (lysosomal) structures (Khan et al., 1982; Merion and Sly, 1983; Roederer et al., 1989), suggesting t h a t these enzymes are transferred between light and dense compartments. Endocytosed and newly synthesized lysosomal enzymes are transferred from light to dense compartments (Brown and Swank, 1983; Sahagian and Neufeld, 19831, supporting this suggestion (although not all enzymes, or forms of enzymes, may behave this way). Additional support for this theory may be found in the observation that cathepsin B substrate, which is initially exposed to enzyme in early endosomes, can be transferred to late cathepsin B-containing compartments which are inaccessible to a chase with leupeptin (Roederer et al., 1987). The failure of low temperatures to block the hydrolysis of MNA-peptide (Roederer e t al., 1987) or MUP (Fig. 4) is further evidence that initial exposure to lysosomal enzymes does not occur through a late fusion event. We have previously shown (Murphy et al., 1984; Roederer and Murphy, 1986; Roederer et al., 1987) that acidification of ligands destined for lysosomes is biphasic, with a rapid acidification to pH 6 within 5 min after ligand addition followed by a slower acidification to pH 5. These results have been confirmed using a mutant of Semliki Forest virus (Kielian et al., 1986). For transferrin, which is recycled along with its receptor to the cell surface, the second phase of acidification does not occur (Sipe and Murphy, 1987). The elevated endosoma1 pH (relative to lysosomes) has been shown to result from a n inhibition of proton pumping by the N a + / K + ATPase (Cain et al., 1989; Fuchs et al., 1989).The second phase of acidification is inhibited a t temperatures below 20°C. On the basis of these results, we have suggested that the primary effect of low temperatures on appearance of ligands in dense lysosomes is due to inhibition of the sorting of receptors and ligands in the early endosome (Roederer et al., 1987). Whether the initial site of exposure of endocytosed material to cathepsin B and acid phosphatase is the early endosome, as would be suggested by the kinetics of substrate hydrolysis, or a later, post-recycling compai-tment, as suggested by morphological studies (Storrie et al., 1984; Griffiths et al., 1988) remains unclear. However, since substrate hydrolysis can occur at temperatures below 20"C, it is likely that addition of enzymes occurs before ligand-receptor sorting. If this is the case, mechanisms must exist to limit the degradation of material to be recycled (Murphy, 1988).Whether lysosoma1 enzymes may be delivered by more than one pathway also remains to be determined; however, current evidence suggests that regulation of hydrolysis of endocytosed material occurs through mechanisms other than simple control of enzyme delivery.

ACKNOWLEDGMENTS We thank Mario Roederer, Russell Wilson, and Robert Preston for helpful discussions and critical reading

117

HYDROLYSIS KINETlCS OF ENDOCYTOSED SUBSTRATES

of the manuscript, Mario Roederer for providing FITCdextran uptake data, and Greg LaRocca and Elizabeth Wickert for technical assistance. This work was supported by National Institutes of Health grant GM32508 and National Science Foundation Presidential Young Investigator Award DCB-8351364, with matching funds from Becton Dickinson Monoclonal Center, Inc.

NOTE ADDED IN PROOF Braun, Waheed and von Figura (1989) have recently demonstrated that lysosomal acid phosphatase is transported to lysosomes via the cell surface in BHK cells. If a similar phenomenon occurs in the cell types studied here, initial hydrolysis of MUP may be due to internalized enzyme. If so, the 5 min lag before the onset of MUP hydrolysis may be seen as further evidence of the role of endosomal pH in regulating lysosoma1 enzyme activity. LITERATURE CITED Barrett, A.J., and Kirschke, H. (1981) Cathepsin B, cathepsin H, and cathepsin L. Methods Enzymol. 80535-561. Braun, M., Waheed, A., and von Figura, K. (1989) Lysosomal acid phosphatase is transported to lysosomes via the cell surface. EMBO J. 8:3633-3640. Brown, J.A., and Swank, R.T. (1983) Subcellular redistribution of newly synthesized macrophage lysosomal enzymes: Correlation between delivery to the lysosomes and maturation. J. Biol. Chem., 258.15323-15328. Buus, S., and Werdelin, 0. (1986) Oligopeptide antigens of the angiotensin lineage compete for presentation by paraformaldehydetreated accessory cells to T cells. J. Immunol., 136:459-465. Cain, C.C., Sipe, D.M., and Murphy, R.F. (1989) Regulation of endocytic pH by the Na ' ,K ' -ATPase in living cells. Proc. Natl. Acad. Sci. U.S.A., 86.544-548. Diment, S., and Stahl, P. (1985) Macrophage endosomes contain proteases which degrade endocytosed protein ligands. J. Biol. Chem., 260t15311-15317, Dunn, W.A., Hubbard, A.L., and Aronson, N.N., J r . (1980) Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of '251-asialofetuin by the perfused rat liver. J. Biol. Chem., 2555971-5978. Fritsch, J.E., Buckmaster, M.J., and Storrie, B. (1988) Fibroblasts maintain a complete endocytic pathway in the presence of lysosomotropic amines. Exp. Cell Res., 175277-285. Fuchs, R., Schmid, S., and Mellman, I. (1989) A possible role for Na +;K -ATPase in regulating ATP-dependent endosome acidification. Proc. Natl. Acad. Sci. U.S.A., 86539-543. Gabel, C.A., and Foster, S.A. (1987)Postendocytic maturation of acid hydrolases: Evidence of prelysosomal processing. J. Cell Biol., 105: 1561-1570. Georgatsos, J.G. (1965) Acid phosphatases of human erythrocytes. Arch. Biochem. Biophys., llOt354-356. Geuze. H.J.. Slot. J.W.. Strous. G.J.A.M.. Hasilik. A.. and von Fieura. K. (1985)'Possiblepathways for lysosomal enzyme delivery. J:Cell Biol., 1012253-2262. Griffiths, G., Hoflack, B., Simons, K., Mellman, I., and Kornfeld, S. (1988) The mannose 6-~hosphatereceptor and the biogenesis of lysosomes. Cell, 52r329-341.Helenius, A,, Mellman, I., Wall, D., and Hubbard, A. (1983) Endosomes. Trends Biochem. Sci., 8245-249. +

Khan, M.N., Posner, B.I., Khan, R.J., and Bergeron, J.J.M. 11982) Internalization of insulin into rat liver Golgi elements: Evidence for vesicle heterogeneity and the path of intracellular processing. J. Biol. Chem., 257t5969-5976. Kielian, M.C., Marsh, M., and Helenius, A. (1986) Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus. EMBO J., 5t3103-3109. Kim, K.J., Kanellopoulos-Langevin, C., Merwin, R.M., Sachs, D.H., and Asofsky, R. (19791 Establishment and characterization of Balbic lymphoma lines with B cell properties. J. Immunol., 122: 549-554. Knight, C.G. (1980) Human cathepsin B. Biochem. J., 189r447-453. Korc, M., and Magun, B.E. (1985) Recycling of epidermal growth factor in a human pancreatic carcinoma cell line. Proc. Natl. Acad. Sci. U.S.A., 82:6172-6175. Marsh, M., Bolzau, E., and Helenius, A. (1983)Penetration of Semliki Forest virus from acidic prelysosomal vacuoles. Cell, 32931-940. Mead, J.A.R., Smith, J.N., and Williams, R.T. (1955)Studies in detoxication. Biochem. J., 61569-574. Merion, M., and Sly, W.S. (1983) The role of intermediate vesicles in the adsorptive endocytosis and transport of ligand to lysosomes by human fibroblasts. J. Cell Biol., 96:644-650. Murphy, R.F. (1988) Processing of endocytosed material. Adv. Cell Biol., 2:159-180. Murphy, R.F., Powers, S., and Cantor, C.R. (1984) Endosomal pH measured in single cells by dual fluorescence flow cytometry, rapid acidification of insulin to pH 6. J. Cell Biol., 98:1757-1762. Murphy, R.F., Roederer, M., Sipe, D.M., Cain, C.C., and Bowser, R. (1989) Determination of the biochemical characteristics of endocytic compartments by flow cytometric and fluorometric analysis of cells and organelles. In Flow Cytometry: Advanced Research and Clinical Applications. A. Yen, ed. CRC Press, Boca Raton, FL. Vol. 11, pp. 221-254. Pondaven, P., and Meijer, L. (1986) Protein phosphorylation and oocyte maturation. Exp. Cell Res., 163.477-488. Roederer, M., Bowser, R., and Murphy, R.F. (1986) Temperature deoendence of acidification and exDosure of endocvtosed material to iysosomal hydrolases: Evidence tor a maturatioi model of endocytosis J . Cell Biol , I03.148a Roederer, M , Bowser, R , and Murphy, R F (1987) Kinetics and temperature dependence of exposureofendocytosed material to proteolytic enzymes and low pH, evidence for a maturation model for the formation of lysosomes. J. Cell. Physiol., 131t200-209. Roederer, M., Mays, R.W., and Murphy, R.F. (1989) Effect of confluence on endocytosis by 3T3 fibroblasts: Increased rate of pinocytosis and accumulation of residual bodies. Eur. J. Cell Biol., 48:37-44. Roederer, M., and Murphy, R.F. (1986) Cell-by-cell autofluorescence correction for low signal-to-noise systems, application to EGF endocytosis by 3T3 fibroblasts. Cytometry, 7t558-565. Sahagian, G.G., and Neufeld, E.F. (1983) Biosynthesis and turnover of the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells. J . Biol. Chem., 258:7121-7128. Sipe, D.M., and Murphy, R.F. (1987)High resolution kinetics of transferrin acidification in BALB/c 3T3 cells. Exposure to pH 6 followed by temperature-sensitive alkalization during recycling. Proc. Natl. Acad. Sci. U.S.A., 84:7119-7123. Storrie, B., Pool, R.R., Sachdeva, M., Maurey, K.M., and Oliver, C. (1984)Evidence for both prelysosomal and lysosomal intermediates in endocytic pathways. J. Cell Biol., 98:108-115. Sullivan, P.C., Ferris, A.L., and Storrie, B. (1987) Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles. J . Cell. Physiol.. 131:58-63. Unanue, E.R. (1984) Antigen-presenting function of the macrophage. Annu. Rev. Immunol., 2t395-428. Verjee, Z.H.M. (1969)Isolation of three acid phosphatases from wheat germ. Eur. J. Biochem., 9t439-444. Wagner, R., Rosenberg, M., and Estensen, R. (1971) Endocytosis in Chang liver cells. J. Cell Biol., 50:804-817. ~~