Supramolecular assemblies from lysosomal matrix proteins and complex lipids

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1 Eur. J. Biochem. 249, (1997) FEBS 1997 Supramolecular assemblies from lysosomal matrix proteins and complex lipids Michel JADOT, FranL DUBOIS, Simone WATTIAUX-DE CONINCK and Robert WATTIAUX Laboratoire de Chimie Physiologique, Facultts Universitaires Notre-Dame de la Paix, Namur, Belgium (Received 11 Julyll5 September 1997) - EJB /1 Most lysosomal hydrolases are soluble enzymes. Lamp-IT (lysosome-associated membrane protein-11) is a major constituent of the lysosomal membrane. We studied the aggregation of a series of lysosomal molecules. The aggregation-sensitive lysosomal marker enzymes were optimally aggregated at intralysosoma1 ph. A similar ph dependence was recorded for aggregation of Lamp-TI. The ph-dependent loss of solubility of isolated Lamp-TI required components of the lysosome extract. Conditions of mild acid ph promoting aggregation triggered the formation of complexes with lipids of lysosomal origin. We fractionated a membrane-free lysosome extract by gel-filtration chromatography and could reconstitute assemblies in vim from separated fractions. We found some selectivity in the lysosomal proteins binding to compiex lipids, phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine being most effective. We propose that the formation at ph 5. of such supramolecular assemblies between lysosomal proteins and lipids occurs within the intralysosomal environment. Some possible consequences of such an intralysosomal matrix formation on organelle function are discussed. Keywords: lysosome; lysosome-associated membrane protein ; aggregation : protein-lipid interaction. Lysosomes contain a variety of hydrolases active against a wide spectrum of substrates with the common property of working at an acidic ph [l]. Most of the lysosomal hydrolases are devoid of a membrane anchor and are thought to diffuse freely within the organelle. The membrane is essential in the maintenance of the mildly acidic intralysosomal ph. Recently, it was found to be enriched in a specific subset of proteins, which includes lysosome-associated membrane proteins I and TI (Lamp-I and Lamp-TI, respectively), two highly glycosylated type 1 membrane proteins 121. Some lysosomal enzymes were shown previously to undergo aggregation in a ph range corresponding to that recorded within lysosomes [3-51. Although the biological significance of this ph-dependent aggregation is not known, it raises the question of the presence, within lysosomes, of some kind of insoluble matrix or gel. This process is reminiscent of the large insoluble multimolecular complexes that form during the maturation of secretion granules. Likewise, an aggregation triggered by low ph and dependent on calcium appears to be crucial in routing some proteins towards the regulated secretory pathway [6-91. It is tempting to consider that the lysosome matrix might behave in a similar fashion. There could be several advantages in keeping lysosomal components trapped as a matrix of insoluble components. For example, this could assist in intralysosomal retention of soluble proteins or help in the formation of supramolecular assemblies of groups of hydrolases involved sequentially in the same metabolic pathway; it could be a requisite to proper targeting of molecules to and from lysosomes or might explain the observed resistance of lysosomal proteins towards proteolysis by restricting accessibility of acid proteases to this matrix. Correspondence fn M. Jadot, Laboratoire de Chimie Physiologique, Facultts Universitaires Notre-Dame de la Paix, 61, rue de Bruxelles, B-5, Namur, Belgium Fux: E-muil: michel.jadot@fundp.ac.be Ahhreviafion. Lamp, lysosome-associated membrane protein. The present study addresses the problem of intralysosomal aggregation of soluble enzymes. A specificity in the aggregation process is shown for the Lamp-I1 protein and the involvement of complex lipids in the formation of assemblies is demonstrated. Our data indicate the feasibility of reconstituting the organization of the organelle matrix in vitro. EXPERIMENTAL PROCEDURES Aggregation assays. Rat liver lysosomes were prepared from male Wistar rats weighing 3-35 g as described [lo]. For the aggregation assay, lysosomes were lysed in 5 mm Hepes, 2 mm Mes, ph 8., by sonication at maximum amplitude in an MSE sonicator, using 1 cycles of 3 s with 3 s intervals to avoid overheating. Membranes were removed by pelleting at 1OOOOOXg for 3 min in a Beckman 6Ti rotor. Aliquots from the supernatant were adjusted to decreasing ph values by slowly adding.125 M HCl while vortexing [8]. After incubation for 2 min at room temperature, aggregated material was sedimented by centrifugation at 35 g for 1 min in a Beckman TLA 12.1 rotor. For analysis by gel-filtration chromatography, lysosomes lysates were prepared as described above and applied onto a Superose 6 FPLC column (Pharmacia Biotech) equilibrated and eluted with 5 mm Hepes, 1 mm Mes, ph 8.. Floatation assays. For floatation assays, lysosome extracts (from sonicated lysosomes or from the Superose 6-B chromatography fraction) were adjusted to ph 5. with.125 M HCI and to a density of 1.24 glml with a solution of 1.3 glml sucrose in water at ph 5.. An aliquot of this preparation was poured as a cushion in tubes fitting the Beckman SW65Ti rotor, and overlaid with a continuous, g/ml sucrose density gradient adjusted to ph 5.. The gradient was centrifuged overnight at 25 rpm (u t = 41 1 radz/sx lo ). Immunoblotting. Studies of Lamp-I1 were carried out using mouse monoclonal antibody anti-lamp-i1 1D1. The antibody

2 Jadot et al. (Em J. Biochem. 249) c- N-acetylglucosaminidase p-glucuronidase cathepsin D ---t arylsulfatase ~- I I Lamp-11 ph: 3.3 P PH a-mannosidase S 51 B I I, + Lamu-II 4.,.,.,, 7. i PH Fig. 1. ph-dependent aggregation of lysosomal proteins. (A) Purified lysosome content (membrane free) was prepared in 5 mm Hepes, 1 mm Mes, ph 8., as described in the Experimental Procedures section. The ph was decreased with.125 M HCI as indicated. Pellets and supernatants were recovered after high-speed centrifugation of the samples. The activities of lysosomal marker enzymes measured in pellets are expressed as the percentage of the corresponding total activities (pellet and supernatant). Aliquots of supernatants (S) and pellets resuspended in the same volumes (P) were spotted on a nitrocellulose sheet processed for immunodetection of Lamp-11. (B) The soluble form of Lamp-I1 was purified from a membrane-free, ph 8., lysosome extract by chromatography on an anti-lamp-i1 affinity column. The isolated and iodinated (lactoperoxidase) molecule was submitted to SDSPAGE and autoradiography (inset). 'Z51-Lamp-II was added to a membrane-free lysosome extract titrated and centrifuged as described in A. Radioactivities recovered in pellets are expressed as the percentage of the corresponding total radioactivities (pellet and supernatant). was raised against a detergent extract of membranes obtained from rat lysosomes purified by the method of Wattiaux et al. [lo]. For preparation of radio-iodinated Lamp-11, lysosome lysates (prepared as described above) were passed through an affinity column made from lodlo antibodies coupled to Sepharose 4B according to the manufacturer's protocol (Pharmacia). The binding was performed overnight at 4 C in 5 mm Hepes, 1 mm Mes, ph 8. buffer. Immunopurified Lamp-I1 was eluted at ph 4., dialyzed against 5 mm Hepes, 1 mm Mes, ph 8., and concentrated by ultrafiltration. A 1-pg aliquot was iodinated by the lactoperoxidase method [ll]. Immunoblotting of Lamp-I1 was performed either after SDSPAGE (1 % polyacryl- Lamp-rl Density Wml) Density(dml1 Fig. 2. Floatation of lysosomal proteins. (A) Purified lysosome content (membrane free) was prepared in 5 mm Hepes, 1 mm Mes, ph 8. as described the Experimental Procedures section and titrated to ph 5. with.125 M HCI. This preparation, adjusted to 1.24 g/ml density with a solution of 1.3 g/ml sucrose, ph 5., was poured as a cushion in a SW65 Ti rotor tube and overlaid with a continuous sucrose density gradient ( g/ml) adjusted to ph 5.. The gradient was centrifuged overnight at 25 rpm (w2t = 41 1 rad2/sx1'). Fractions were collected, tested for a-mannosidase activity and used for immunodetection of Lamp-I1 on dot blots. (B) A floatation experiment was performed as described in A but with a lysosome extract kept at ph 8. and a ph 8. sucrose density gradient. (C) A ph 5. floatation experiment was performed as described in A except that.5 % Triton X-1 was added tu the titrated lysosome extract. The ordinate frequency (QEQ. AQ), where Q represents the activity found in the fraction, EQ the total activity recovered in the sum of the fractions, and d~ the density difference between the top and the bottom of the fraction. amide reducing gels) and transfer of proteins to polyvinylidene difluoride (Immobilon-P, Millipore), or after spotting and drying aliquots on nitrocellulose. Membranes were probed with the monoclonal anti-lamp I1 antibody 1D1, and the signal was revealed using a chemiluminescence system (Boehringer-Mannheim). Miscellaneous. Cholesterol was measured by an enzymatic/ colorimetric assay (procedure no. 352 from Sigma). Lysosomal

3 864 Jadot et al. (Eur: J. Biochem. 249).-.* * 8 8o 1 3, Y P % floating at ph 5. El % aggregated at ph 5. % floating at ph 8, % aggregated at PH 8. 1: cathepsin C 2: acid PhosPhatase 3: N-acetylglucosaminidase 4: a-mannosidase 5: 6-glucuronidase Fig. 3. Comparison between the extent of aggregation and floatation for five lysosome marker enzymes. Purified lysosome content (menibrane free) was prepared in 5 mm Hepes, 1 mm Mes, ph 8., as described in the Experimental Procedures section. This preparation was used for floatation assays performed at ph 5. and ph 8. (see Fig. 2A and B). The extent of floatation was monitored for the five lysosomal marker enzymes indicated by measuring the percentage of their activities excluded from the load (bottom gradient fraction no. 13). Values recorded are compared to the extent of aggregation measured in the same conditions as described in Fig. 1 A and the Experimental Procedures section. Both values in the left hand column of each pair refer to floatation; both values in the right hand column of each pair refer to aggregation. Lamp h.3- E. m el Y <.2- m N [ll [21 I I I I I I I I 13 I Fraction LI B 3 B 2 g E. a E h Q h :.3.2 * h 1 : enzymatic activities were assayed according to the following references : P-glucuronidase [ 121; a-mannosidase [ 131; N-acetylglucosaminidase [14]; arylsulfatase [15]; cathepsin C [16]; cathepsin B [ 171;,&galactosidase [ 131; acid phosphatase [ 181; cathepsin D [12]; ~-glucosidase [13]; sphingomyelinase [ 191. RESULTS Lamp-I1 is a lysosomal membrane protein. It is also present as a soluble protein that cannot be pelleted by high speed centrifugation [5]. The data shown in Fig. 1 demonstrate that the soluble form of Lamp-I1 and the aggregation-prone matrix enzymes tested behaved similarly with respect to the ph dependence of their aggregation. A similar ph optimum for aggregation, around was found for several lysosomal proteins, including Lamp-I1 (Fig. 1 A). For the experiment reported in Fig. 1 B, the soluble form of Lamp-I1 was purified from a membrane-free lysosome extract by chromatography on an anti-lamp-i1 affinity column. The purified, radio-iodinated molecule (see inset) was used in aggregation assays performed in the presence of a membrane-free lysosome extract. The data in Fig. 1B clearly show that Lamp-I1 most effectively aggregates at ph Using the same assay, the ph 5. aggregation of '2sI-Lamp-II was shown to require the presence of a lysosome extract (membranefree supernatant from sonicated lysosomes) and could not be induced by comparable protein concentrations of albumin or cytosol (data not shown). These results raise the question of the nature of the lysosome-specific, acid-induced aggregation. To address this question further, the sedimentation properties of the molecular aggregates formed at ph 5. were studied. A membrane-free lysosome extract was prepared at ph 8., adjusted to ph 5. with.125 M HCl, and submitted to the floatation assay as described in the Experimental Procedures section. After centrifugation, the distributions of Lamp-I1 and a-manno- 6 I Fraction Fig. 4. Gel-filtration chromatography of a ph 8. lysosome extract. Purified lysosome content (membrane free) prepared in 5 mm Hepes, 1 mm Mes, ph 8. as described in the Experimental Procedures section was loaded onto a Superose 6 FPLC column (Pharmacia Biotech) equilibrated and eluted with 5 mm Hepes, 1 mm Mes, ph 8.. (A) Fractions were collected and used for measurement of the absorbance at 28 nm (), for measurement of the a-mannosidase activity (O), and for immuno-detection of Lamp-I1 on dot blots. (B) A gel-filtration chromatography was performed exactly as described in A to establish the distribution of cholesterol shown by the absorbance at 5 nm (X). () absorbance measured in fractions at 28 nm. sidase were compared as shown in Fig. 2 A. A significant portion of a-mannosidase activity was no longer recovered in the bottom fraction, but was detected in the density region 1.17 g/ml. The distribution of Lamp-11, established by analyzing the gradient fractions by inimunoblotting, was almost identical, with part of its population floating in the gradient. When a similar experiment was carried out with a ph 8. lysosome extract, in a gradient equilibrated to ph 8., both a-mannosidase and Lamp-I1 remained associated with the bottom fraction (Fig. 2B). A possible explanation for the floatation observed at ph 5. could be that aggregated a-mannosidase forms a supramolecular complex with lipids, resulting in an apparent decreased density. This was assessed by repeating the floatation experiment at ph 5. in the presence of.5 % Triton X-1, a concentration that was previously shown to be without effect on the aggregation of Lamp- I1 or a-mannosidase. As shown in Fig. 2C, the presence of the detergent totally abolishes floatation. The unexpected low equilibrium density of the ph 5. Lamp-I1 and a-mannosidase aggregates suggests that this might be a common property of several lysosomal soluble enzymes. Floatation assays at ph 5. and ph 8. were thus carried out to

4 4-3 8 k! & 1 I Jadot et al. (Em J. Biochem. 249) A r k r g r 1 I a-mannosidase m Density (dml) Lamp-I B a-mannosidase L J l O! I 1.5 Lamp-I1 Y i.i Density Wml) Density (g/ml) Fig. 5. ph 5. floatation of a-mannosidase obtained after gel filtration of a lysosome extract. (A) A membrane-free lysosome extract was gel filtrated on Superose 6 at ph 5. (see Fig. 4A). An aliquot of fraction 2, exhibiting maximal a-mannosidase activity was added to 1 vol. 5 mm Hepes, 1 mm Mes, ph 8., titrated to ph 5., and submitted to a floatation assay through sucrose (see Experimental Procedures and legend to Fig. 2A). (B) A ph 5. floatation assay was performed similarly except that fraction 2 was supplemented with 1 vol. fraction 1 (see Fig. 4A) instead of buffer before acidification. After centrifugation overnight at 25 rpm (w2t = 41 1 radl/sx lo7), fractions were collected and tested for a-mannosidase activity. The ordinate indicates frequency (QEQ. A@), where Q represents the activity found in the fraction, CQ the total activity recovered in the sum of the fractions and A@ the density difference between the top and the bottom of the fraction glml and g/ml are the a-mannosidase mean equilibrium densities measured in conditions A and B, respectively. study the behavior of five lysosomal marker enzymes : cathepsin C, acid phosphatase, N-acetyl glucosaminidase, a-mannosidase and P-glucuronidase, with an estimation of the percentage of activities floating in the gradient versus that included in the bottom fraction. In parallel, the propensity for aggregation of the same proteins at two different ph values of 5. and 8. was measured. The results reveal a con-elation between the propensity to form aggregates and the percentage of activity sensitive to floatation (Fig. 3). It is worth noticing cathepsin C, which remains soluble at all ph values and is equally resistant to floatation. These results suggest an acid-induced aggregation of a series of lysosomal proteins into relatively low density supramolecular structures. To investigate the composition of these complexes, membrane-free lysosome extracts prepared at ph 8. were submitted to gel filtration chromatography. As shown in Fig. 4A, the proteins excluded from the gel appeared in fractions 9 and 1 (referred to as [fraction l]), in front of a broad shoulder (fractions 12-24) reflecting the diversity of the lysosomal protein population. a-mannosidase (soluble at ph 8.) showed maximum activity in fraction 14 (referred to as [fraction 21). Soluble Lamp-I1 was detected as an intense spot in fraction 1 as well as Lamp-I1 a-mannosidase Density Wml) I, I,,, I I, I I I, N 3-4 " 5 z3z3zzzzz3 2 2 Density (ghnl) 3 N m a r 2 B F 8 1 o " 2 3 Fig. 6. Effect of a proteinase K treatment on the floatation reconstituted from gel filtration chromatography fractions. A membrane-free lysosome extract was gel filtrated on Superose 6 at ph 5. (see Fig. 4A). An aliquot of fraction 2, exhibiting maximal a-mannosidase activity was mixed with 1 vol. fraction 1 with and without proteinase K treatment. The mixture was titrated to ph 5. and submitted to a floatation assay through sucrose (see Experimental Procedures section and legend to Fig. 2A). After centrifugation overnight at 25 rpm (w't = 41 1 rad2/sx lo7), fractions were collected, tested for a-mannosidase activity and spotted on nitrocellulose to perform the immunodetection of Lamp- 11. (A) Control experiment performed with untreated fraction 1. (B) and (C) floatation observed after treatment of fraction 1 for 15 min at room temperature with 1 mg/ml and 5 mg/ml proteinase K, respectively. The ordinate indicates frequency (QEQ AQ), where Q represents the activity found in the fraction, CQ the total activity recovered in the sum of the fractions, and A@ the density difference between the top and the bottom of the fraction. a shoulder extending to fraction 2. The distribution of cholesterol was followed to assess the presence of lipids in the complexes excluded from the gel. Fig. 4B shows that the bulk of cholesterol was found in fraction 1, suggesting that some association between Lamp-IT and lipids occur in these complexes. The absence of Lamp-I1 floatation at ph 8. (Fig. 2B) probably resulted from the relatively small size of the lipid complexes in their non-aggregated state. To get a better idea of the role of lipids in the formation and sedimentation behavior of the assemblies, similar gel-filtration experiments were used to study the floatation of a-mannosidase recovered from fraction 2. As shown in Fig. 5, when the a-man-

5 866 Jadot et al. (Em J. Biochenz. 249) Frequency (wq.6~) Frequency (W9.b) 31 c I Density (g/ml) A: Phosphatidylethanolamine B: Phosphatidylserine C: Phosphatidylinositol D : Galactocer ebroside E: Ceramide F: Lecithin G Sphmgomyelin H: Phosphatidylglycerol I: Phosphatidic Acid Dipalmitoyl Density (g/ml) Fig. 7. Floatation of a-mannosidase can be reconstituted from exogenously added liposome preparations. A membrane-free lysosome extract was gel filtrated on Superose 6 at ph 5. (see Fig. 4A). An aliquot of fraction 2, exhibiting maximal a-mannosidase activity was mixed with 1 vol. liposome prepared as follows: 1 mg/ml lipid was dissolved in chloroform, vacuum dried, resuspended in 5 mm Hepes, 1 mm Mes, ph 8., and sonicated at maximal amplitude for 8x3 s at 4 C. The mixture was titrated to ph 5. before being submitted to a floatation assay through a sucrose gradient adjusted to ph 5.. (see Experimental Procedures section and legend to Fig. 2A and B). After centrifugation overnight at 25 rpm (~*t = 41 1 rad2/sx17), fractions were collected and tested for a-mannosidase activity. The shaded area indicates floatation at ph 5.; the dotted line represents floatation at ph 8.. The ordinate indicates frequency (QEQ A@), where Q represents the activity found in the fraction, ZQ the total activity recovered in the sum of the fractions and A@ the density difference between the top and the bottom of the fraction. nosidase containing fraction 2 is adjusted to ph 5., the bulk of the activity remains associated with the bottom fraction after centrifugation (Fig. 5A), showing that the enzyme did not retain its ability to float in the sucrose gradient (compare with the behavior of a-mannosidase from a crude lysosome extract in Fig. 2A). Despite the loss of floatation, a-mannosidase from fraction 2 is nevertheless prone to aggregation at ph 5., 65 % of the activity being sedimented at ph 5. (data not shown). The floatation behavior of a-mannosidase from fraction 2 can be restored by adding fraction 1 in a 1 : 1 ratio (Fig. 5 B), indicating that fraction 1 can provide the elements, most likely lipids, required to induce floatation of the aggregation complexes. To obtain information on the interactions that occur at ph 5. between n-mannosidase and lipids, experiments involving the mixing of fractions 1 and 2 were repeated, after treatment of fraction 1 by proteinase K. As shown in Fig. 6, proteinase K treatment of fraction 1 decreased the density of the floating structures (compare the distributions of a-mannosidase and Lamp-I1 in Fig. 6A and B). Increasing the proteinase K concentration (Fig. 6C) resulted in an even more prominent floatation of a-mannosidase, while Lamp-I1 was digested by this treatment. Presumably, proteinase K decreased the density of the lipoprotein complexes formed at ph 5. by decreasing the protedlipid ratio in fraction 1. The extensive a-mannosidase floatation observed when fraction 1 has been stripped of its protein content (Fig. 6C) suggests that binding of a-mannosidase to lipids does not require a protein anchor provided by fraction 1. To understand further the role of the lipid component in the complex, the floatation-resistant fraction 2 (Fig. 4) was mixed with various liposomes before being tested in the floatation

6 Jadot et al. (Eu,: J. Biochem. 249) 867 Frequency (QEQ.Ad Density (dd) Lamp-11: I : : i r J Density (g/ml) : Control :+Sphingomyelin A Cathepsin C B Cathepsin B C: f3-galactosidase D Acid Phosphatase E: p-glucuronidase F: N-Acetylglucosaminidase G a-mannosidase H: Cathepsin D I f3-glucosidase J: Sphingomyelinase, I,.,,, I,, I I 1 a ioiiiais Control +Sphingomyelin Fig. 8. Floatation triggered by exogenously added sphingomyelin only concerns enzymes able to bind endogenous lipids. Purified lysosome content (membrane free) was prepared in 5 mm Hepes, 1 mm Mes, ph 8. as described in the Experimental Procedures section and titrated to ph 5. with.125 M HCl. This preparation, untreated or diluted 1/1 with sphingomyelin liposomes prepared as described in legend to Fig. 7, was submitted to the usual floatation assay through a sucrose gradient adjusted to ph 5. (see Experimental Procedures section and legend to Fig. 2A and B). After centrifugation overnight at 25 rpm (oh = 41 1 rad2/sxi ), fractions of the gradients were collected and tested for lysosornal enzymes activities. The ordinate indicates frequency (QEQ. A@), where Q represents the activity found in the fraction, CQ the total activity recovered in the sum of the fractions and A@ the density difference between the top and the bottom of the fraction. assay at ph 5.. The data in Fig. 7 show that some phospholipids such as phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine can promote a-mannosidase floatation at ph 5. and therefore substitute for the lipids of lysosomal origin found in fraction 1. As expected, for all lipids tested, no floatation occurred at ph 8. (data not shown). In another series of experiments, the results of which are summarized in Fig. 8, membrane- free lysosome extracts were used in floatation assays performed at ph 5. in the presence and absence of exogenous sphingomyelin liposomes. The lysosomal enzymes that are known to be aggregation sensitive, such as P-glucuronidase, N-acetylglucosaminidase, a-mannosidase, cathepsin-d, P-glucosidase, and soluble Lamp-11, had their distribution shifted towards the low-density zone of the gradients by sphingomyelin. By contrast, the sedimentation profiles of other enzymes that do not show aggregation, such as cathepsin C, cathepsin B, P-galactosidase, or acid phosphatase, are unaffected by sphingomyelin liposomes (Fig. 8). DISCUSSION Previous studies showed that some lysosomal proteins have a strong tendency to aggregate when the ph becomes slightly acidic [3-51. The present data provide evidence that this phdependent loss of solubility is an intrinsic feature of the intralysosomal milieu. Aggregation is optimal in the ph range known to exist in lysosomes (ph [2]) and the ph dependence of the aggregation process is similar for all these enzymes. Components of lysosome extracts are required to induce aggregation of an isolated lysosomal protein. Although the mechanisms underlying aggregation remain unclear, the observation that it occurs when lysosomal proteins are close to their isoelectric point [21] would suggest the importance of hydrophobic interactions. In the case of the so-called soluble Lamp-11, our data indicate that the protein is associated with lipids. Conditions promoting aggregation of lysosomal enzymes also trigger their association to these lipid complexes. The binding observed between the ph 5. aggregates and artificial liposomes indicates that this association cannot be triggered by every lipid. Interestingly, two of the lipids promoting floatation (sphingomyelin and phosphatidylcholine) are concentrated on the exoplasmic leaflet of the plasma membrane and therefore on the luminal aspect layer of the lysosomal membrane. At least two, not mutually exclusive, hypotheses may account for our data. Indeed, the observed binding of lysosomal proteins to lipid containing Lamp-I1 positive structures could reflect the binding taking place in situ between soluble enzymes and the lysosomal membrane if for example the lipoprotein structures excluded from the size-exclusion chromatography arise directly from the membrane. One cannot exclude, however, that this binding mimics the formation of large aggregates from lipid-containing material pre-existing within the organelle matrix. This would explain some very early observations such as the ones from Baudhuin et al. [22] or de Duve [23] reporting that small coacervates of non-homogeneous material resembling lysosomes stripped of their limiting membrane can still be sedimented from lysosome-rich fractions resuspended in water and would provide support for the presence of some organizational principle within lysosomes, as postulated by Baccino et al. [24]. We would like to suggest that the aggregation into supramolecular complexes that we show in vitro also occurs within the lysosomal environment, at ph , and in the presence of a full set of lysosome components. Our previous observation that the ph 5. aggregation of a lysosomal enzyme is reversible [5] and our measurements of enzymatic activities performed at ph 5. indicate that complex aggregate formation does not lead to denaturation of the proteins, suggesting that the lipid binding concerns native proteins. The binding of soluble lysosomal enzymes to an insoluble, gel-like substrate could be important for the regulation of protein traffic to and from secondary lysosomes, as it would prevent

7 868 Jadot et al. (Eul: J. Biochem. 249) leaking of lysosomal proteins from lysosomes involved in vesicular transport, an idea proposed before 131. This transport incompetence would result from the budding of transport vesicles from a specialized area of the lysosomal membrane not involved in binding of the enzymes; alternatively, one could conceive that soluble enzymes trapped within a naturally occurring stroma could not be included in any type of shuttle vesicles budding from secondary lysosomes. Involvement of mature lysosomes as donors of membranes has been demonstrated recently with their CaZ+ triggered fusion with the plasma membrane in normal fibroblasts and epithelial cells [25]. The observation by Traub et al. [26] that clathrin coats assemble on mature lysosomes provides support for a retrograde traffic out of the lysosomal compartment. Involvement of aggregation to promote retention within an organelle has been established in the case of the Golgi apparatus. For example, oligomerization of the M-protein of coronavirus was shown to correlate with its localization to the cis-golgi is retained in the Golgi apparatus because of its ability to form large oligomers in vivo [28]. The selectivity of the associations between Golgi proteins lead Warren and coworkers to propose a model in which Golgi enzymes tend to associate provided they belong to the same Golgi subcompartment [29-3 I], while an alternative hypothesis exists in which the length of the transmembrane domain is the key determinant in Golgi retention [ The protein p63 was similarly shown to be retained because of its ability to form oligomers [ 351. This oligomerization is reversible and occurs at slightly acidic ph values. The above mentioned retention processes concern intrinsic membrane proteins. Our observation that a proportion of Lamp-I1 behaves similarly to soluble lysosomal enzymes with respect to acidic aggregation raises the possibility that lysosomal membrane proteins, including Lamps, intervene in the organization of the lysosomal stroma. For example, intermolecular bridges could form at lysosomal ph between lysosomal content proteins and membrane proteins such as Lamp. It is to be noted that similar association between secretory granule content proteins and the luminal domains of the organelle membrane proteins has been shown to take place in vitro at mildly acidic ph [8]. Differential binding of lysosomal enzymes to membranes has been proposed to explain the differential secretion of lysosomal enzymes in Acanthamoeba [36]. In this study, a correlation between the rate of secretion and the membrane association of a series of lysosomal marker enzymes was shown. Membrane binding was estimated from the proportion of various hydrolases that sediment with membranes at ph 4.5 during high speed centrifugation. One should nevertheless be uming that this assay measures authentic membrane binding. Our data indeed suggest that for some enzymes, their aggregation at acidic ph is sufficient to account for their sedimentability. Intralysosomal degradation of the organelle s membrane could be an important means to compensate for transport vesicles fusion and resulting membrane surface increase, providing a partial explanation for the fate of the lysosomal membrane. In the conventional model, degradation of membrane molecules derived from the plasma membrane during endocytosis for example, occurs selectively within the lysosomal membrane. Sandhoff and his group [37, 381 propose an alternative model for the topology of endocytosis in which plasma membrane molecules become incorporated into the membrane of intra-endosomal vesicles. These vesicles are then transferred into lysosomes, which accounts for their selective degradation. According to our observations, such intralysosomal vesicles could be actively involved in the formation of the intralysosomal matrix. If binding of lysosoma1 enzymes involved in the degradation of these internal ves- icles occurs in acidic endosomes, this could interfere with the trafficking of hydrolases to lysosomes. In fibroblasts obtained from patients with I-cell disease (unable to properly target their hydrolases because of a deficiency of the Man6-P signalling pathway [39]), residual activities of some enzymes can be found [4]. It is interesting to note that enzymes severely reduced in these cells ul-galactosidase, a-fucosidase) are resistant to aggregation and floatation in our experiments, while hydrolases showing a partial reduction only in I-cells fibroblasts (,!-glucuronidase, a-iduronidase, N-acetylglucosaminidase, a-mannosidase, arylsulfatase A, sphingomyelinase) are all sensitive to ph 5. aggregation and lipid binding. In conclusion, the present data show that the lysosomal content tends to form a matrix of supramolecular assemblies of soluble enzymes complexed with lipids. However, more data is needed to understand the significance of this process, particularly its role in vivo, which may be of key importance for lysosome function, and experiments are presently in progress to pursue this idea further. We thank Dr Andr6 M. Goffinet for critical comments on the manuscript and Michel Savels for technical assistance. This work was supported by grants from the Fonds de la Recherche Scientifigue Midicale (contracts no and no ). REFERENCES I. de Duve, C. & Wattiaux, R. (1966) Functions of lysosomes, Annu. Rev. Physiol. 28, Fukuda, M. (11994) Biogenesis of the lysosomal membrane, in Subcelluhr biochemistry (Maddy, A. H. & Harris, J. R., eds) vol. 22, pp , Plenum Press, New York. 3. Buckmaster, M. J., Ferris, A. L. & Storrie, B. (1988) Effects of ph, detergent and salt on aggregation of Chinese-hamster-ovary-cell lysosomal enzymes, Biochem. J. 249, Koenig, H. (1969) Lysosomes in the nervous system, in Lysosomes in biology and pathology (Dingle, J. T. & Fell, H. B., eds) vol. 2, pp , North-Holland Publishing Co., Amsterdam. 5. Jadot, M., Wattiaux, R., Mainferme, F., Dubois, F., Claessens, A. & Wattiaux-De Coninck, S. (1996) Soluble form of Lamp-I1 in purified rat liver lysosomes, Biochem. Biophys. Res. Commun. 223, Chanat, E. & Huttner, W. 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