DEVELOPMENTAL GENETICS 1 1 : 4 5 4 4 6 2 (1990)
Biochemical and Genetic Analysis of the Biosynthesis, Sorting, and Secretion of Dictyostelium Lysosomal Enzymes JAMES A. CARDELLI, JOHN SCHATZLE, JOHN M. BUSH, JAN RICHARDSON, DAVID EBERT, AND HUDSON FREEZE Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport (J.A.C., J.S., J.M.B., J.R.); Baker Medical Research Institute, Prahran, Victoria, Australia (D.E.); La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, California (H.F.) ABSTRACT Dictyostelium discoideum is a useful system to study the biosynthesis of lysosomal enzymes because of the relative ease with which it can be manipulated genetically and biochemically. Previous studies have revealed that lysosomal enzymes are synthesized in vegetatively growing amoebae as glycosylated precursor polypeptides that are phosphorylated and sulfated on their Nlinked oligosaccharide side-chains upon arrival in the Golgi complex. The precursor polypeptides are membrane associated until they are proteolytically processed and deposited as soluble mature enzymes in lysosomes. In this paper we review biochemical experiments designed to determine the roles of post-translational modification, acidic pH compartments, and proteolytic processing in the transport and sorting of lysosomal enzymes. We also describe molecular genetic approaches that are being employed to study the biosynthesis of these enzymes. Mutants altered in the sorting and secretion of lysosomal enzymes are being analyzed biochemically, and we describe recent efforts to clone the genes coding for three lysosomal enzymes in order to better understand the molecular mechanisms involved in the targeting of these enzymes. Key words: Mutants, proteolytic processing,
N-
linked oligosaccharide
INTRODUCTION The biosynthesis and targeting of lysosomal enzymes has been well described in certain mammalian systems such as the human fibroblast [see Kornfeld and Mellman, 1989, for a review]. The enzymes are synthesized hs precursor glycoproteins which are modified upon arrival in the Golgi complex by the addition of phosphate residues to mannose sugars. These phosphomannosyl residues serve as recognition markers for specific membrane bound receptors which mediate transport of the
0 1990 WILEY-LISS, INC.
enzymes to endosomal compartments. The low pH in these compartments facilitates the release of the enzymes from the receptor, and the recycling of these receptors back to the Golgi complex. The enzymes in t u r n are subjected to proteolytic processing which generates mature enzymes that reside in dense secondary lysosomes. A number of different mammalian cell types have been identified which use mannose-6-phosphate receptor independent targeting pathways, but very little is known about the factors which influence targeting of lysosomal enzymes in these systems. Dictyostelium has proven to be a useful system to investigate alternate lysosomal enzyme processing and targeting pathways because it can be manipulated biochemically and genetically [see Cardelli and Dimond, 1988, for a review], and because it lacks detectable mannose-6-phosphate receptors [Cardelli et al., 19871. Dictyostelium synthesizes at least three lysosomal enzymes, a-mannosidase, p-glucosidase, and acid phosphatase, as precursor polypeptides [Pannell et al., 1982; Mierendorf et al., 1985; Cardelli et al., 1986a; Bennett and Dimond, 1986; Bush and Cardelli, 1989al that are N-glycosylated in the lumen of the rough endoplasmic reticulum (RER) with typical mannose rich oligosaccharide side-chains [Freeze, 1986; Cardelli et al., 1986bl. The membrane associated precursors [Mierendorf et aZ.,1985; Cardelli et al., 1986al are transported at nonuniform rates to the Golgi complex [Cardelli et al., 1986a; Bush and Cardelli, 19891 where they are sulfated and phosphorylated on mannose residues [Cladaras and Kaplan, 1984; Gabel et al., 1984; Freeze, 1985; Mierendorf et al., 1985; Freeze and Wolgast, 19861. A small percentage of
Received for publication July 31, 1990. Address reprint requests to James A. Cardelli, Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, LA 71130.
DICTYOSTELIUM LYSOSOMAL ENZYMES the uncleaved precursor polypeptides are exported from cells along a constitutive secretory pathway while the remainder of the enzymes are proteolytically processed through intermediate forms to mature forms which reside in acidic lysosomes [Mierendorf et al., 1985; Cardelli et al., 1986al. These mature, apparently soluble enzymes are secreted a t low rates during growth and at high rates when cells are suspended in a buffered salt solution in the absence of nutrients [Crean and Rossomando, 1979; Burns et al., 1981; Dimond et al., 1981; Wood et al., 1983; Wood and Kaplan, 19851. Although these enzymes are all colocalized in the same lysosomal organelles [Bush and Cardelli, 19891, they are secreted from cells with distinctly different kinetics. This suggests that a n undefined mechanism operates to distinguish these classes of enzymes a t a step after targeting to lysosomes but prior to secretion. In this report we describe recent studies which examine the roles of N-linked oligosaccharide sidechains, low pH compartments, and proteolytic processing in the transport and sorting of lysosomal enzymes. We also summarize our recent approaches to isolate the genes coding for a-mannosidase, p-glucosidase, and acid phosphatase.
MATERIALS AND METHODS Wild-type Ax3 cells and the mutant cell lines GM1, HL241, HL243, HL244, HMW570, and HMW517 were grown axenically a s described [Cardelli et al., 19871. Details of the protocols for measuring intravesicular pH, radiolabeling of proteins, subcellular fractionation of organelles on sucrose and Percoll gradients, immunoprecipitation of lysosomal enzymes, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can be found in Cardelli et al. [1987,1989] and Freeze et al. [1989aI. RESULTS Factors Affecting the Intracellular Transport and Sorting of Lysosomal Enzymes The role of N-linked oligosaccharide sidechains and post-translational modifications. Mannose 6-phosphate moieties in N-linked oligosaccharide side-chains of mammalian lysosomal enzymes act as sorting signals for transport of these proteins to lysosomes [Kornfeld and Mellman, 19891. Although Dictyostelium lysosomal enzymes are phosphorylated and sulfated on their carbohydrate side-chains [Gabel et al., 1984; Freeze, 1985; Freeze and Wolgast, 19861 little is known about the role these modifications play in the transport and sorting of hydrolases. We have approached this problem by biochemically analyzing mutants [Free et al., 1978; Freeze et al., 1983; Knecht et al., 19841 altered in the biosynthesis and modification of N-linked oligosaccharide side-chains. Details of the
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isolation of the mutants M31, HL241, HL243, and HL244 can be found in Free et al., 119781 and Knecht et al., [1984], and characterization of the structures of the carbohydrate side-chains can be found in Freeze et al. [ 1989a,c]. Radiolabel pulse-chase and subcellular fractionation experiments indicated that the retention of glucose residues (M31), the absence of Man-6-S04 (HL2441, a reduction in phosphorylation (M31, HL241, and HL2431, or size of N-linked oligosaccharide sidechains (HL241 and HL243) did not affect the correct proteolytic processing or targeting of a-mannosidase, p-glucosidase, or acid phosphatase [Ebert et al., 1989a; Bush and Cardelli, 1990; Cardelli et al., 1990; Freeze et al. 1989133. However, except for the sulfation mutant HL244, the lysosomal enzymes in the other mutants were transported at slower rates from the endoplasmic reticulum [ER] to the Golgi complex than in wild-type cells [Ebert et al., 1989a; Freeze et al., 1989a,b; Bush and Cardelli, 19901. The inability of these proteins to fold normally or a change in their three dimensional structure may account for the delay in transport of these enzymes from the ER [Woychik et al., 19861. These studies do however indicate that under normal conditions sulfate does not act as a transport, processing, or sorting signal for lysosomal enzymes in Dictyostelium. Our studies do not eliminate the possibility that phosphate moieties or other uncharacterized modifications act as a sorting signal but as described below information contained in the polypeptide chain may be more critical for the proper localization of lysosomal enzymes. Role of acidic pH compartments. Lysosomes and endosomes are acidic intracellular compartments with pH values ranging from 4.8 to 6.4 [Mellman et al., 19861. It has been demonstrated in mammalian cells that the maintenance of low pH is required for the proper sorting of lysosomal enzymes [Mellman et al.,. 19861. For instance, when cells are exposed to weak bases such as ammonium chloride the intravesicular pH is raised. As a result of this, the phosphomannosyl receptor does not release its lysosomal enzyme cargo, and the receptor protein is unable to cycle back to the Golgi complex to bind newly synthesized lysosomal enzymes resulting in rapid secretion of precursor polypeptides. We have analyzed the effect of increases in intravesicular pH induced by ammonium chloride on the processing and sorting of Dictyostelium lysosomal enzymes. Spectrophotometric techniques have revealed that axenically growing Ax3 cells maintain lysosomal and endosomal compartments a t a n average pH of 5.4 [Cardelli et al., 19891. Exposure of cells to low concentrations of ammonium chloride raised the pH to 6.1 in these compartments and slowed but did not prevent the correct proteolytic processing and localization of amannosidase, p-glucosidase, and acid phosphatase [Cardelli et al., 19891. However, raising the pH to 6.4 resulted in the accumulation of newly synthesized in-
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termediate forms of a-mannosidase and p-glucosidase which were very slowly processed to mature forms. These forms of the enzymes were correctly targeted to lysosomes a s determined by Percoll gradient fractionation [Cardelli et al., 19891. Also the percentage of newly synthesized lysosomal enzymes that escaped targeting and were secreted did not increase significantly under these conditions. The accumulation of intermediate forms of the enzymes suggests that the proteinases which generate mature enzymes from intermediate forms require a low pH environment for their proper functioning. In fact as described below these enzymes most likely are cysteine proteinases. In summary, these results suggest that although acidic endosomal/lysosomal compartments may be important for the complete proteolytic processing of lysosomal enzymes, low pH is not essential for the proper targeting of these enzymes. The intracellular sites and importance of proteolytic processing. All lysosomal enzymes are initially synthesized as precursor polypeptides that are proteolytically processed to mature lysosomally localized enzymes [Von Figura and Hasilik, 19861. Much remains to be determined about this process including the intracellular site(s) where cleavage occurs, the biochemical nature of the proteinases, and the importance of proteolysis in the function and sorting of lysosomal enzymes. To learn more about the role of proteolysis in lysosomal enzyme biosynthesis, we chased 35S-met pulse labeled cells in growth medium containing a variety of chemicals known to inhibit the activity of each of the four currently classified groups of proteinases [Richardson et al., 19881. As indicated in Figure 1,only three inhibitors, leupeptin, antipain, and Z-PheAlaCHN,, prevented the processing of precursor and intermediate forms of a-mannosidase and pglucosidase. The cysteine proteinase inhibitor Z-PheAlaCHN, prevented the processing of intermediate forms to mature forms. The intermediate forms accumulated in lysosomes a s determined by Percoll gradient fractionations [Richardson et al., 19881. This suggests that a lysosomal cysteine proteinase catalyzes the generation of mature forms, and that this process is not required for proper localization of enzymes. This conclusion is consistent with our previous observations concerning the affect of ammonium chloride on the processing and transport of lysosomal enzymes [Cardelli et al., 19891. In contrast, cells treated with the serinel cysteine inhibitors leupeptin and antipain processed precursor forms of a-mannosidase and p-glucosidase very inefficiently, and instead exported the unprocessed polypeptides into the extracellular medium [Richardson et al., 19881. Furthermore, the precursor polypeptides exited cells without ever reaching lysosomes. These results suggest that a t least two classes of proteinases residing in different intracellular compartments (see below) may be involved in processing the precursors to the mature forms of lysosomal enzymes.
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Fig. 1. The effect of 12 proteinase inhibitors on proteolytic processing of a-mannosidase and p-glucosidase. The cotranslational addition of carbohydrate and subsequent processing of a-mannosidase and pglucosidase is depicted in the top of the figure. The numbers correspond to the inhibitors listed below, and depict which inhibitors effectively prevent processing, as well as the step during proteolytic processing which is inhibited. Abbreviations used: TLCK, N-p-tosylL-lysine chloromethyl ketone; 2-Phe-AlaCHN,, benzyloxycarbonylL-phenylalanyl-L-L-alanine-diazomethyl ketone; Z-Gly-PheNH,, carbobenzoxy-L-glycine-phenylalanine-amide;TPCK, L-1-tosylamide 2-phenylethyl chloromethyl ketone; PMSF, phenylmethylsulfonyl fluoride, DAN, diazoacetyl-DL-norleucine methyl ester; EPNP,1,2epoxy-3-(p-nitrophenoxy)propane.
Furthermore, the initial cleavage of precursor polypeptides may be important in determining the correct localization of the enzymes. Because of the potential importance of proteolytic processing in lysosomal enzyme targeting, we initiated studies to determine the compartment in which this process occurs. Fractionation of cell extracts on Percoll gradients indicated that the intermediate forms of radiolabeled a-mannosidase and p-glucosidase first appeared during a chase in cellular compartments intermediate in density between the Golgi complex and dense lysosomes (Wood and Kaplan, 1985; Richardson et al., 19881while mature forms first appeared in dense lysosomes. These early processing compartments may include elements of the Golgi complex or may represent vacuoles that are part of the endosomal pathway. To more accurately determine the nature of the processing compartments, we used a density shift procedure first
DICTYOSTELIUM LYSOSOMAL ENZYMES
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Fig. 2. Subcellular fractionation on Percoll gradients of organelles loaded with HRP and subjected to DAB cytochemistry (density shift). Growing cells were incubated with 35S-met,3H-dextran, and HRP for 4 hours. Crude membranes were prepared and incubated in the absence (panels A, C) or presence of DAB with H,O, (panels B, D).
Membranes were separated on 32% Percoll gradients, and fractions were collected and counted for tritium (panels C, D) or immunoprecipitated with antibodies to a-mannosidase and P-glucosidase (panels A, B). Immunoprecipitated proteins were subjected to SDS-PAGE followed by fluorography.
described by Courtoy et al., [1984]. In this procedure, cells are allowed to accumulate horse radish peroxidase (HRP), a fluid phase marker, by endocytosis. Next, cells are fractionated, total membranes are recovered by centrifugation, and incubated with the substrate diaminobenzidine (DAB) in the presence of H202. The enzyme reaction causes the selective retention of dense DAB polymers only in organelles such a s endosomes and lysosomes that have accumulated HRP. Accumulation of dense DAB polymers causes a significant increase in the density of the organelle as revealed by Percoll gradient fractionation. To test this procedure in Dictyostelium, growing cells were labeled with 3H-dextran (to mark endosomal organelles), 35S-met (to label proteins), and HRP (for density shift) for 4 hours. Total membranes were recovered by centrifugation and 112 of this preparation was incubated with DAB and H,O,. As indicated in Figure 2 after centrifugation of untreated membranes on Percoll gradients a major peak of dextran (panel C ) was observed in the same region of the gradient a s the various immunoprecipitated forms of a-mannosidase and P-
glucosidase separated by SDS-PAGE (panel A). Incubation of membranes with DAB resulted in a large increase in density for 112 of the membranes containing labeled dextran (panel D). In addition, approximately 112 of the radiolabeled mature and intermediate forms of a-mannosidase and P-glucosidase also banded near the bottom of the gradient. The reason only 112 of the dextran containing vesicles shifted may be due to the presence of a much smaller amount of cross-linked dense material in the more buoyant vesicles. In any case, we conclude that essentially all of the intermediate and mature forms of the enzymes reside in organelles that are part of the endosomal pathway, and that the final proteolytic events also occur in these compartments. Interestingly, very little of the precursor forms of a-mannosidase and p-glucosidase shifted when membranes were exposed to DAB (compare panels A, B). This suggests that precursor polypeptides are cleaved immediately upon arrival in prelysosomallendosomal compartments, or that transport of intermediate forms of these enzymes occurred concomitantly with cleavage of precursors.
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Fraction Number Fig. 3. Intracellular distribution of 3H-dextran and acid phosphatase in HMW570 and wild-type cells. Wild-type (A,C) and mutant (B,D) cells were labeled with dextran for 4 hours, and intracellular organelles were fractionated on 24% Percoll gradients as described in Richardson et al. [1988].Fractions were analyzed for radioactivity (A,B) and acid phosphatase activity (C,D).
Examination of Mutants Altered in Sorting and Secretion
the mutant appeared to be altered in endocytosis. The fluid phase marker, 3H-dextran, accumulated to 1/2 the extent and at 112 the rate in mutant cells compared Combined genetic and biochemical approaches have to Ax3. Also as indicated in panel A of Figure 3, if Ax3 proven useful in the analysis of the secretory and ly- cells were allowed to endocytose 3H-dextran and were sosomal targeting pathways in yeast [Rothman and then subject to fractionation on Percoll gradients, the Stevens, 19881; we have initiated similar studies in labeled dextran accumulated in high density vesicles Dictyostelium. In this section we will describe recent (fractions 1-6) and low density vesicles (fractions 9experiments characterizing two mutants from over 70 14). The high density vesicles corresponded in position we have isolated that are altered in the secretion of to dense lysosomal vesicles containing acid phoslysosomal enzymes [Cardelli et al., 1987; Cardelli and phatase activity (panel C) while the low density vesiDimond, 19881. cles represent endosomes and other prelysosomal comHMW570 cells oversecrete most lysosomal enzymes partments. In contrast, following endocytosis dextran during growth compared to wild-type cells [Ebert et al., accumulated in mutant cells predominately in com1989bl. Using a radiolabeled pulse-chase protocol, we partments with a low density characteristic of endohave determined that newly synthesized a-mannosi- somes (panel B). Furthermore, the mutant also condase and B-glucosidase are mis-sorted and secreted as tained a lesser percentage of intracellular acid precursor polypeptides from the mutant [Ebert et al., phosphatase activity in dense vesicles compared to Ax3 1989bl. Subcellular fractionation experiments also in- (compare panels C, D). We conclude that the mutation dicated that the precursor polypeptides never reached in HMW570 may be in a gene required for both the lysosomes, and that their hypersecretion was not ac- generation of dense secondary lysosomes and the sortcompanied by major alterations to their N-linked oli- ing of lysosomal enzymes. This further suggests that a gosaccharide side-chains such as charge, size, and functional connection may exist between the process of amount of phosphate and sulfate esters. Interestingly, endocytosis and the intracellular sorting of hydrolases.
DICTYOSTELIUM LYSOSOMAL ENZYMES
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CHLOR Fig. 4. Proteolytic processing and secretion of lysosomal enzymes from wild-type cells and the mutant HMW517. Growing cells were pulse-labeled with 35S-metand chased in medium lacking the isotope. Radiolabeled a-mannosidase and 6-glucosidase were immunoprecipitated from cell extracts and medium, and subjected to SDS-PAGE
followed by fluorography. The last four lanes represent cells (C) and medium (M) collected and immunoprecipitated following incubation of cultures in the presence of the indicated amounts of chloroquine. For additional information see Cardelli et al. [1989a].
This connection has also been suggested to exist in cosidase were immunoprecipitated and subjected to yeast cells [Rothman and Stevens, 19881. SDS-PAGE followed by fluorography. Figure 4 indiWe have also biochemically characterized a mutant cates that the precursor forms of these enzymes were which is defective in the secretion of lysosomal en- processed to mature forms at approximately the same zymes during growth and in response to starvation. rate in the mutant and wild-type cells. Moreover, subStarvation of Ax3 cells induces a five fold increase in cellular fractionation studies have indicated that the the secretion of glycosidase enzymes and of acid phos- processed forms of a-mannosidase and P-glucosidase in phatase. Although the two classes of enzymes are re- HMW517 resided in organelles that have densities leased with dissimilar kinetics, they are colocalized in equal to those of secondary lysosomes in wild-type cells the same population of lysosomes [Bush and Cardelli, (unpublished results). Mature forms of the enzymes be1989al. This suggests that differences in secretion may gan to be secreted from Ax3 cells by 2 hours of chase be dependent on preferential retention of certain lyso- and by 8 hours of chase >90% of the polypeptides had soma1 enzymes in lysosomes. The mutant HMW517 re- been released. In contrast, M
Fig. 6. Transport, targeting, and secretion pathways for Dzctyostelium lysosomal enzymes. The figure represents the various transport, processing, and secretory pathways followed by precursor forms (p), intermediate forms (i), and mature forms (m) of acid phosphatase (AP), a-mannosidase (M), and pglucosidase (G).
cells, and was first detected in developing cells by 30 minutes following starvation. The mRNA accumulated to its highest level by 4-8 hours in cells developing on filters and in suspension under slow shaking conditions. The concentration of the mRNA decreases dramatically late in development (results not shown). These results are very similar to our previous studies which measured changes in the concentration of functional mRNA [Livi et al., 19851.
DISCUSSION Figure 6 summarizes our current model describing the synthesis, transport, processing, sorting, and secretion of Dictyostelium lysosomal enzymes. In many respects the biosynthesis of Dictyostelium enzymes is similar to the synthesis of mammalian enzymes. For instance, the enzymes are proteolytically cleaved, they are phosphorylated and sulfated, and they are transported to dense secondary lysosomes. However, it is clear that the mechanism of lysosomal enzyme localization in the slime mold differs in many aspects from the localization process in many animal cells. For in-
stance, Dictyostelium lacks phosphomannosyl receptors which are critical in the localization of lysosomal enzymes in fibroblasts. Because other animal cells have been identified that target lysosomal enzymes independently of these receptors, the slime mold will be a useful system to investigate sorting of hydrolases by alternate pathways. Furthermore, the maintenance of acidic pH compartments is not required for targeting of the enzymes in Dictyostelium, and inhibition of cleavage of the precursor forms of the enzymes leads to missorting and hypersecretion of uncleaved polypeptides. This suggests that processing and sorting may be coupled. Also the precursor forms of the enzymes are tightly associated with membranes prior to cleavage and release of mature enzymes into the lumen of lysosomes. Finally, the secretion of mature forms of the enzymes is a regulated process and although all the enzymes reside in the same population of lysosomes they are secreted with distinctly different kinetics. Future studies will involve determining the molecular nature of the sorting signal on lysosomal enzymes. and the importance of proteolysis in the localization event. We will also investigate the molecular mecha-
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nisms regulating secretion of enzymes from lysosomes. Finally, we will continue to analyze the developmental regulation of the expression of genes coding for lysosoma1 enzymes.
ACKNOWLEDGMENTS This work was supported by a grant to J.A.C. from the National Institutes of Health, DK 39232. One of the oligonucleotide probes used to screen for acid phosphatase clones was a kind gift of Drs. J i m Lenhardt and Steve Free. REFERENCES Bennett VD, Dimond RL (1986): Biosynthesis of two developmentally distinct acid phosphatase isozymes in Dictyostelium discoideum. J Biol Chem 2615355-5362. Burns RA, Livi GP, Dimond RL (1981): Regulation and secretion of early developmentally controlled enzymes during axenic growth in Dictyostelium discoideum. Dev Biol 84:407-416. Bush J, Cardelli, J A (1989a): Processing, transport, and secretion of the lysosomal enzyme acid phosphatase in Dictyostelium discoideum. J Biol Chem 264:7630-7636. Bush J, Cardelli J A (1990): Alterations to N-linked oligosaccharide structures which affect intracellular transport rates and regulated secretion but not sorting of acid phosphatase in Dictyostelium discoideum. Arch. Bioch. Biophy. (in press). Cardelli JA, Richardson J, Miears D (1989): Role of acidic intracellular compartments in the biosynthesis of Dictyostelium discoideum lysosomal enzymes: The weak bases ammonium chloride and chloroquine differentially affect proteolytic processing and sorting. J Biol Chem 264:3454-3463. Cardelli JA, Bush J, Ebert D, Freeze HH (1990): Sulfated N-linked oligosaccharides effect secretion but are not essential for the transport, proteolytic processing, and sorting of lysosomal enzymes in Dictyostelium discoideum. J Biol Chem 26523847-8853. Cardelli JA, Dimond Rl(1988): Transport and targeting of lysosomal enzymes in Dictyostelium discoideum, Das, Robbins (eds): in “Protein Transfer and Organelle Biogenesis.” San Diego: Academic Press, pp 363-399. Cardelli JA. Golumbeski GS. Wovchik NA, Ebert DL, Mierendorf RC, Dimond RL (1987): Defining the intracellular localization pathways followed by lysosomal enzymes in Dictyostelium discoideum. Methods Cell Biol 28:139-155. Cardelli JA, Golumbeski GS, Dimond RL (1986a): Lysosomal enzymes in Dictyostelium discoideum are transported to lysosomes a t distinctly different rates. J Cell Biol 102:1264-1270. Cardelli JA, Mierendorf RC, Dimond RL (198613): Initial events involved in the synthesis of the lysosomal enzyme a-mannosidase in Dictyostelium discoideum. Arch Biochem Biophys 244:338-345. Cladaras MH, Kaplan A (1984): Maturation of a-mannosidase in Dictyostelium discoideum. J Biol Chem 259:14165-14619. Courtoy PJ, Quintart J, Bandhuin P (1984):Shift of equilibrium density induced by 3,3’-diaminobenzidinecytochemistry: a new procedure for the analysis and purification of peroxidase containing organelles. J Cell Biol 98870-876. Crean EV, Rossomando EF (1979): Effects of sugars on glycosidase secretion in Dictyostelium discoideum. J Gen Microbiol 110:315322. Dimond RL, Burns RA, Jordan KB (1981): Secretion of lysosomal enzymes in the cellular slime mold, Dictyostelium discoideum. J Biol Chem 256:6565-6572. Dimond RL, Knecht DA, Jordan KB, Burns RA, Livi G P (1983):Secretory mutants in the cellular slime mold, Dictyostelium discoideum. Methods Enzymol 96:815-828. Ebert DL, Bush JM, Dimond RL, Cardelli J A (1989a): Biogenesis of lysosomal enzymes in the a-glucosidase 11-deficient modA mutant of Dictyostelium discoideum: Retention of a-1,3-linked glucose on N-linked oligosaccharides delays intracellular transport but does not alter sorting of a-mannosidase and P-glucosidase. Arch Biochem Biophys 273:479-490.
Ebert DL, Freeze HH, Richardson J , Dimond RL, Cardelli J A (1989b): A Dictyostelium discoideum mutant that missorts and oversecretes lysosomal enzymes precursors is defective in endocytosis. J Cell Biol 109:1445-1456. Free SJ, Schimke RT, Freeze HH, Loomis WF (1978): Characterization and genetic mapping of modA. A mutation in the posttranslational modification of the glycosidases of Dictyostelium discoideum J Biol Chem 253:4102-4106. Freeze HH (1985): Mannose 6-sulfate is present in the N-linked oligosaccharides of lysosomal enzymes of Dictyostelium discoideum. Arch Biochem Biophys 243:690-693. Freeze HH (1986): Modifications of lysosomal enzymes in Dictyostelium discoideum. Mol Cell Biochem 72:47-65. Freeze HH, Bush JM, Cardelli J A (1989a): Biochemical and genetic analysis of a n antigenic determinant found on N-linked oligosaccharides in Dictyostelium. Dev Genet 11 (this issue). Freeze HH, Koza-Taylor P, Saunders A, Cardelli J A (1989b): The effects of altered N-linked oligosaccharide structures on maturation and targeting of lysosomal enzymes in Dictyostelium discoideum. J Biol Chem 264:19278-19286. Freeze HH, Willies L, Hamilton S, Koza-Taylor P (1989~):Two mutants of Dictyostelium discoideum that lack a sulfated carbohydrate antigenic determinant synthesize a truncated lipid-linked precursor of N-linked oligosaccharides. J Biol Chem 2645653-5659. Freeze HH, Wolgast D (1986): Biosynthesis of methyl-phosphomannosy1 residues in the oligosaccharides of Dictyostelium discoideum glycoproteins. J Biol Chem 261:135-141. Freeze HH, Yeh R, Miller AL, Kornfeld S (1983): The m o d mutant of Dictyostelium discoideum is missing the a-l,3-glucosidase involved in asparagine-linked oligosaccharide processing. J Biol Chem 258:14880-14884. Gabel CA, Costello CE, Reinhold VN Kurz L, Kornfeld S (1984): Identification of methylphosphomannosyl residues as conipouents of the high mannose oligosaccharides of Dictyostelium discoideum glycoproteins. J Biol Chem 259:13762-13769. Golumbeski GS, Dimond RL (1986): The use of tolerization in the production of monoclonal antibodies against minor antigenic determinants. Anal Biochem 154:373-381. Knecht DA, Dimond RL, Wheeler S, Loomis WF (1984): Antigenic determinants shared by lysosomal proteins of Dictyostelium discoideum. J Biol Chem 259:10633-10640. Kornfeld S, Mellman I (1989): The biogenesis of lysosomes. Ann Rev Cell Biol 5483-526. Livi GP, Cardelli JA, Mierendorf RC, Dimond RL (1985): Regulation of lysosomal a-mannosidase-l synthesis during development of Dictyostelium discoideum. Dev Biol 110:514-520. Mellman I, Fuchs R, Helenius A (1986): Acidification of the endocytic and exocytic pathways. Ann Rev Biochem 55663-700. Mierendorf RC, Cardelli JA, Dimond RL (1985): Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium discoideum. J Cell Biol 110:1777-1787. Pannell R, Wood L, Kaplan A (1982): Processing and secretion of a-mannosidase forms by Dictyostelium discoideum. J Biol Chem 257:9861-9865. Richardson JR, Woychik NA, Ebert DL, Dimond RL, Cardelli J A (1988):Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum. J Cell Biol 107: 2097-2107. Rothman JH, Stevens TH (1988):Protein sorting and biogenesis of the lysosome-like vacuole in yeast. In Das, Robbins (eds): “Protein Transfer and Organelle Biogenesis.” San Diego: Academic Press, pp 318-362. Von Figura K, Hasilik A (1986): Lysosomal enzymes and their receptors. Ann Rev Biochem 55:167-194. Wood L, Kaplan A (1985): Transit of a-mannosidase during its maturation in Dictyostelium discoideum. J Cell Biol 101:2063-2069. Wood L, Pannell RN, Kaplan A (1983): Linked pools of processed a-mannosidase in Dictyostelium discoideum. J Biol Chem 258: 9426-9430. Woychik NA, Cardelli JA, Dimond RL (1986): A conformationally altered precursor to the lysosomal enzyme a-mannosidase accumulates in the endoplasmic reticulum in a mutant strain of Dictyostelium discoideum. J Biol Chem 261:9595-9602.
Biochemical and Genetic Analysis of the Biosynthesis, Sorting, and Secretion of Dictyostelium Lysosomal Enzymes JAMES A. CARDELLI, JOHN SCHATZLE, JOHN M. BUSH, JAN RICHARDSON, DAVID EBERT, AND HUDSON FREEZE Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport (J.A.C., J.S., J.M.B., J.R.); Baker Medical Research Institute, Prahran, Victoria, Australia (D.E.); La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, California (H.F.) ABSTRACT Dictyostelium discoideum is a useful system to study the biosynthesis of lysosomal enzymes because of the relative ease with which it can be manipulated genetically and biochemically. Previous studies have revealed that lysosomal enzymes are synthesized in vegetatively growing amoebae as glycosylated precursor polypeptides that are phosphorylated and sulfated on their Nlinked oligosaccharide side-chains upon arrival in the Golgi complex. The precursor polypeptides are membrane associated until they are proteolytically processed and deposited as soluble mature enzymes in lysosomes. In this paper we review biochemical experiments designed to determine the roles of post-translational modification, acidic pH compartments, and proteolytic processing in the transport and sorting of lysosomal enzymes. We also describe molecular genetic approaches that are being employed to study the biosynthesis of these enzymes. Mutants altered in the sorting and secretion of lysosomal enzymes are being analyzed biochemically, and we describe recent efforts to clone the genes coding for three lysosomal enzymes in order to better understand the molecular mechanisms involved in the targeting of these enzymes. Key words: Mutants, proteolytic processing,
N-
linked oligosaccharide
INTRODUCTION The biosynthesis and targeting of lysosomal enzymes has been well described in certain mammalian systems such as the human fibroblast [see Kornfeld and Mellman, 1989, for a review]. The enzymes are synthesized hs precursor glycoproteins which are modified upon arrival in the Golgi complex by the addition of phosphate residues to mannose sugars. These phosphomannosyl residues serve as recognition markers for specific membrane bound receptors which mediate transport of the
0 1990 WILEY-LISS, INC.
enzymes to endosomal compartments. The low pH in these compartments facilitates the release of the enzymes from the receptor, and the recycling of these receptors back to the Golgi complex. The enzymes in t u r n are subjected to proteolytic processing which generates mature enzymes that reside in dense secondary lysosomes. A number of different mammalian cell types have been identified which use mannose-6-phosphate receptor independent targeting pathways, but very little is known about the factors which influence targeting of lysosomal enzymes in these systems. Dictyostelium has proven to be a useful system to investigate alternate lysosomal enzyme processing and targeting pathways because it can be manipulated biochemically and genetically [see Cardelli and Dimond, 1988, for a review], and because it lacks detectable mannose-6-phosphate receptors [Cardelli et al., 19871. Dictyostelium synthesizes at least three lysosomal enzymes, a-mannosidase, p-glucosidase, and acid phosphatase, as precursor polypeptides [Pannell et al., 1982; Mierendorf et al., 1985; Cardelli et al., 1986a; Bennett and Dimond, 1986; Bush and Cardelli, 1989al that are N-glycosylated in the lumen of the rough endoplasmic reticulum (RER) with typical mannose rich oligosaccharide side-chains [Freeze, 1986; Cardelli et al., 1986bl. The membrane associated precursors [Mierendorf et aZ.,1985; Cardelli et al., 1986al are transported at nonuniform rates to the Golgi complex [Cardelli et al., 1986a; Bush and Cardelli, 19891 where they are sulfated and phosphorylated on mannose residues [Cladaras and Kaplan, 1984; Gabel et al., 1984; Freeze, 1985; Mierendorf et al., 1985; Freeze and Wolgast, 19861. A small percentage of
Received for publication July 31, 1990. Address reprint requests to James A. Cardelli, Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, LA 71130.
DICTYOSTELIUM LYSOSOMAL ENZYMES the uncleaved precursor polypeptides are exported from cells along a constitutive secretory pathway while the remainder of the enzymes are proteolytically processed through intermediate forms to mature forms which reside in acidic lysosomes [Mierendorf et al., 1985; Cardelli et al., 1986al. These mature, apparently soluble enzymes are secreted a t low rates during growth and at high rates when cells are suspended in a buffered salt solution in the absence of nutrients [Crean and Rossomando, 1979; Burns et al., 1981; Dimond et al., 1981; Wood et al., 1983; Wood and Kaplan, 19851. Although these enzymes are all colocalized in the same lysosomal organelles [Bush and Cardelli, 19891, they are secreted from cells with distinctly different kinetics. This suggests that a n undefined mechanism operates to distinguish these classes of enzymes a t a step after targeting to lysosomes but prior to secretion. In this report we describe recent studies which examine the roles of N-linked oligosaccharide sidechains, low pH compartments, and proteolytic processing in the transport and sorting of lysosomal enzymes. We also summarize our recent approaches to isolate the genes coding for a-mannosidase, p-glucosidase, and acid phosphatase.
MATERIALS AND METHODS Wild-type Ax3 cells and the mutant cell lines GM1, HL241, HL243, HL244, HMW570, and HMW517 were grown axenically a s described [Cardelli et al., 19871. Details of the protocols for measuring intravesicular pH, radiolabeling of proteins, subcellular fractionation of organelles on sucrose and Percoll gradients, immunoprecipitation of lysosomal enzymes, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can be found in Cardelli et al. [1987,1989] and Freeze et al. [1989aI. RESULTS Factors Affecting the Intracellular Transport and Sorting of Lysosomal Enzymes The role of N-linked oligosaccharide sidechains and post-translational modifications. Mannose 6-phosphate moieties in N-linked oligosaccharide side-chains of mammalian lysosomal enzymes act as sorting signals for transport of these proteins to lysosomes [Kornfeld and Mellman, 19891. Although Dictyostelium lysosomal enzymes are phosphorylated and sulfated on their carbohydrate side-chains [Gabel et al., 1984; Freeze, 1985; Freeze and Wolgast, 19861 little is known about the role these modifications play in the transport and sorting of hydrolases. We have approached this problem by biochemically analyzing mutants [Free et al., 1978; Freeze et al., 1983; Knecht et al., 19841 altered in the biosynthesis and modification of N-linked oligosaccharide side-chains. Details of the
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isolation of the mutants M31, HL241, HL243, and HL244 can be found in Free et al., 119781 and Knecht et al., [1984], and characterization of the structures of the carbohydrate side-chains can be found in Freeze et al. [ 1989a,c]. Radiolabel pulse-chase and subcellular fractionation experiments indicated that the retention of glucose residues (M31), the absence of Man-6-S04 (HL2441, a reduction in phosphorylation (M31, HL241, and HL2431, or size of N-linked oligosaccharide sidechains (HL241 and HL243) did not affect the correct proteolytic processing or targeting of a-mannosidase, p-glucosidase, or acid phosphatase [Ebert et al., 1989a; Bush and Cardelli, 1990; Cardelli et al., 1990; Freeze et al. 1989133. However, except for the sulfation mutant HL244, the lysosomal enzymes in the other mutants were transported at slower rates from the endoplasmic reticulum [ER] to the Golgi complex than in wild-type cells [Ebert et al., 1989a; Freeze et al., 1989a,b; Bush and Cardelli, 19901. The inability of these proteins to fold normally or a change in their three dimensional structure may account for the delay in transport of these enzymes from the ER [Woychik et al., 19861. These studies do however indicate that under normal conditions sulfate does not act as a transport, processing, or sorting signal for lysosomal enzymes in Dictyostelium. Our studies do not eliminate the possibility that phosphate moieties or other uncharacterized modifications act as a sorting signal but as described below information contained in the polypeptide chain may be more critical for the proper localization of lysosomal enzymes. Role of acidic pH compartments. Lysosomes and endosomes are acidic intracellular compartments with pH values ranging from 4.8 to 6.4 [Mellman et al., 19861. It has been demonstrated in mammalian cells that the maintenance of low pH is required for the proper sorting of lysosomal enzymes [Mellman et al.,. 19861. For instance, when cells are exposed to weak bases such as ammonium chloride the intravesicular pH is raised. As a result of this, the phosphomannosyl receptor does not release its lysosomal enzyme cargo, and the receptor protein is unable to cycle back to the Golgi complex to bind newly synthesized lysosomal enzymes resulting in rapid secretion of precursor polypeptides. We have analyzed the effect of increases in intravesicular pH induced by ammonium chloride on the processing and sorting of Dictyostelium lysosomal enzymes. Spectrophotometric techniques have revealed that axenically growing Ax3 cells maintain lysosomal and endosomal compartments a t a n average pH of 5.4 [Cardelli et al., 19891. Exposure of cells to low concentrations of ammonium chloride raised the pH to 6.1 in these compartments and slowed but did not prevent the correct proteolytic processing and localization of amannosidase, p-glucosidase, and acid phosphatase [Cardelli et al., 19891. However, raising the pH to 6.4 resulted in the accumulation of newly synthesized in-
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termediate forms of a-mannosidase and p-glucosidase which were very slowly processed to mature forms. These forms of the enzymes were correctly targeted to lysosomes a s determined by Percoll gradient fractionation [Cardelli et al., 19891. Also the percentage of newly synthesized lysosomal enzymes that escaped targeting and were secreted did not increase significantly under these conditions. The accumulation of intermediate forms of the enzymes suggests that the proteinases which generate mature enzymes from intermediate forms require a low pH environment for their proper functioning. In fact as described below these enzymes most likely are cysteine proteinases. In summary, these results suggest that although acidic endosomal/lysosomal compartments may be important for the complete proteolytic processing of lysosomal enzymes, low pH is not essential for the proper targeting of these enzymes. The intracellular sites and importance of proteolytic processing. All lysosomal enzymes are initially synthesized as precursor polypeptides that are proteolytically processed to mature lysosomally localized enzymes [Von Figura and Hasilik, 19861. Much remains to be determined about this process including the intracellular site(s) where cleavage occurs, the biochemical nature of the proteinases, and the importance of proteolysis in the function and sorting of lysosomal enzymes. To learn more about the role of proteolysis in lysosomal enzyme biosynthesis, we chased 35S-met pulse labeled cells in growth medium containing a variety of chemicals known to inhibit the activity of each of the four currently classified groups of proteinases [Richardson et al., 19881. As indicated in Figure 1,only three inhibitors, leupeptin, antipain, and Z-PheAlaCHN,, prevented the processing of precursor and intermediate forms of a-mannosidase and pglucosidase. The cysteine proteinase inhibitor Z-PheAlaCHN, prevented the processing of intermediate forms to mature forms. The intermediate forms accumulated in lysosomes a s determined by Percoll gradient fractionations [Richardson et al., 19881. This suggests that a lysosomal cysteine proteinase catalyzes the generation of mature forms, and that this process is not required for proper localization of enzymes. This conclusion is consistent with our previous observations concerning the affect of ammonium chloride on the processing and transport of lysosomal enzymes [Cardelli et al., 19891. In contrast, cells treated with the serinel cysteine inhibitors leupeptin and antipain processed precursor forms of a-mannosidase and p-glucosidase very inefficiently, and instead exported the unprocessed polypeptides into the extracellular medium [Richardson et al., 19881. Furthermore, the precursor polypeptides exited cells without ever reaching lysosomes. These results suggest that a t least two classes of proteinases residing in different intracellular compartments (see below) may be involved in processing the precursors to the mature forms of lysosomal enzymes.
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Fig. 1. The effect of 12 proteinase inhibitors on proteolytic processing of a-mannosidase and p-glucosidase. The cotranslational addition of carbohydrate and subsequent processing of a-mannosidase and pglucosidase is depicted in the top of the figure. The numbers correspond to the inhibitors listed below, and depict which inhibitors effectively prevent processing, as well as the step during proteolytic processing which is inhibited. Abbreviations used: TLCK, N-p-tosylL-lysine chloromethyl ketone; 2-Phe-AlaCHN,, benzyloxycarbonylL-phenylalanyl-L-L-alanine-diazomethyl ketone; Z-Gly-PheNH,, carbobenzoxy-L-glycine-phenylalanine-amide;TPCK, L-1-tosylamide 2-phenylethyl chloromethyl ketone; PMSF, phenylmethylsulfonyl fluoride, DAN, diazoacetyl-DL-norleucine methyl ester; EPNP,1,2epoxy-3-(p-nitrophenoxy)propane.
Furthermore, the initial cleavage of precursor polypeptides may be important in determining the correct localization of the enzymes. Because of the potential importance of proteolytic processing in lysosomal enzyme targeting, we initiated studies to determine the compartment in which this process occurs. Fractionation of cell extracts on Percoll gradients indicated that the intermediate forms of radiolabeled a-mannosidase and p-glucosidase first appeared during a chase in cellular compartments intermediate in density between the Golgi complex and dense lysosomes (Wood and Kaplan, 1985; Richardson et al., 19881while mature forms first appeared in dense lysosomes. These early processing compartments may include elements of the Golgi complex or may represent vacuoles that are part of the endosomal pathway. To more accurately determine the nature of the processing compartments, we used a density shift procedure first
DICTYOSTELIUM LYSOSOMAL ENZYMES
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Membranes were separated on 32% Percoll gradients, and fractions were collected and counted for tritium (panels C, D) or immunoprecipitated with antibodies to a-mannosidase and P-glucosidase (panels A, B). Immunoprecipitated proteins were subjected to SDS-PAGE followed by fluorography.
described by Courtoy et al., [1984]. In this procedure, cells are allowed to accumulate horse radish peroxidase (HRP), a fluid phase marker, by endocytosis. Next, cells are fractionated, total membranes are recovered by centrifugation, and incubated with the substrate diaminobenzidine (DAB) in the presence of H202. The enzyme reaction causes the selective retention of dense DAB polymers only in organelles such a s endosomes and lysosomes that have accumulated HRP. Accumulation of dense DAB polymers causes a significant increase in the density of the organelle as revealed by Percoll gradient fractionation. To test this procedure in Dictyostelium, growing cells were labeled with 3H-dextran (to mark endosomal organelles), 35S-met (to label proteins), and HRP (for density shift) for 4 hours. Total membranes were recovered by centrifugation and 112 of this preparation was incubated with DAB and H,O,. As indicated in Figure 2 after centrifugation of untreated membranes on Percoll gradients a major peak of dextran (panel C ) was observed in the same region of the gradient a s the various immunoprecipitated forms of a-mannosidase and P-
glucosidase separated by SDS-PAGE (panel A). Incubation of membranes with DAB resulted in a large increase in density for 112 of the membranes containing labeled dextran (panel D). In addition, approximately 112 of the radiolabeled mature and intermediate forms of a-mannosidase and P-glucosidase also banded near the bottom of the gradient. The reason only 112 of the dextran containing vesicles shifted may be due to the presence of a much smaller amount of cross-linked dense material in the more buoyant vesicles. In any case, we conclude that essentially all of the intermediate and mature forms of the enzymes reside in organelles that are part of the endosomal pathway, and that the final proteolytic events also occur in these compartments. Interestingly, very little of the precursor forms of a-mannosidase and p-glucosidase shifted when membranes were exposed to DAB (compare panels A, B). This suggests that precursor polypeptides are cleaved immediately upon arrival in prelysosomallendosomal compartments, or that transport of intermediate forms of these enzymes occurred concomitantly with cleavage of precursors.
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TOP
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Fraction Number Fig. 3. Intracellular distribution of 3H-dextran and acid phosphatase in HMW570 and wild-type cells. Wild-type (A,C) and mutant (B,D) cells were labeled with dextran for 4 hours, and intracellular organelles were fractionated on 24% Percoll gradients as described in Richardson et al. [1988].Fractions were analyzed for radioactivity (A,B) and acid phosphatase activity (C,D).
Examination of Mutants Altered in Sorting and Secretion
the mutant appeared to be altered in endocytosis. The fluid phase marker, 3H-dextran, accumulated to 1/2 the extent and at 112 the rate in mutant cells compared Combined genetic and biochemical approaches have to Ax3. Also as indicated in panel A of Figure 3, if Ax3 proven useful in the analysis of the secretory and ly- cells were allowed to endocytose 3H-dextran and were sosomal targeting pathways in yeast [Rothman and then subject to fractionation on Percoll gradients, the Stevens, 19881; we have initiated similar studies in labeled dextran accumulated in high density vesicles Dictyostelium. In this section we will describe recent (fractions 1-6) and low density vesicles (fractions 9experiments characterizing two mutants from over 70 14). The high density vesicles corresponded in position we have isolated that are altered in the secretion of to dense lysosomal vesicles containing acid phoslysosomal enzymes [Cardelli et al., 1987; Cardelli and phatase activity (panel C) while the low density vesiDimond, 19881. cles represent endosomes and other prelysosomal comHMW570 cells oversecrete most lysosomal enzymes partments. In contrast, following endocytosis dextran during growth compared to wild-type cells [Ebert et al., accumulated in mutant cells predominately in com1989bl. Using a radiolabeled pulse-chase protocol, we partments with a low density characteristic of endohave determined that newly synthesized a-mannosi- somes (panel B). Furthermore, the mutant also condase and B-glucosidase are mis-sorted and secreted as tained a lesser percentage of intracellular acid precursor polypeptides from the mutant [Ebert et al., phosphatase activity in dense vesicles compared to Ax3 1989bl. Subcellular fractionation experiments also in- (compare panels C, D). We conclude that the mutation dicated that the precursor polypeptides never reached in HMW570 may be in a gene required for both the lysosomes, and that their hypersecretion was not ac- generation of dense secondary lysosomes and the sortcompanied by major alterations to their N-linked oli- ing of lysosomal enzymes. This further suggests that a gosaccharide side-chains such as charge, size, and functional connection may exist between the process of amount of phosphate and sulfate esters. Interestingly, endocytosis and the intracellular sorting of hydrolases.
DICTYOSTELIUM LYSOSOMAL ENZYMES
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CHLOR Fig. 4. Proteolytic processing and secretion of lysosomal enzymes from wild-type cells and the mutant HMW517. Growing cells were pulse-labeled with 35S-metand chased in medium lacking the isotope. Radiolabeled a-mannosidase and 6-glucosidase were immunoprecipitated from cell extracts and medium, and subjected to SDS-PAGE
followed by fluorography. The last four lanes represent cells (C) and medium (M) collected and immunoprecipitated following incubation of cultures in the presence of the indicated amounts of chloroquine. For additional information see Cardelli et al. [1989a].
This connection has also been suggested to exist in cosidase were immunoprecipitated and subjected to yeast cells [Rothman and Stevens, 19881. SDS-PAGE followed by fluorography. Figure 4 indiWe have also biochemically characterized a mutant cates that the precursor forms of these enzymes were which is defective in the secretion of lysosomal en- processed to mature forms at approximately the same zymes during growth and in response to starvation. rate in the mutant and wild-type cells. Moreover, subStarvation of Ax3 cells induces a five fold increase in cellular fractionation studies have indicated that the the secretion of glycosidase enzymes and of acid phos- processed forms of a-mannosidase and P-glucosidase in phatase. Although the two classes of enzymes are re- HMW517 resided in organelles that have densities leased with dissimilar kinetics, they are colocalized in equal to those of secondary lysosomes in wild-type cells the same population of lysosomes [Bush and Cardelli, (unpublished results). Mature forms of the enzymes be1989al. This suggests that differences in secretion may gan to be secreted from Ax3 cells by 2 hours of chase be dependent on preferential retention of certain lyso- and by 8 hours of chase >90% of the polypeptides had soma1 enzymes in lysosomes. The mutant HMW517 re- been released. In contrast, M
Fig. 6. Transport, targeting, and secretion pathways for Dzctyostelium lysosomal enzymes. The figure represents the various transport, processing, and secretory pathways followed by precursor forms (p), intermediate forms (i), and mature forms (m) of acid phosphatase (AP), a-mannosidase (M), and pglucosidase (G).
cells, and was first detected in developing cells by 30 minutes following starvation. The mRNA accumulated to its highest level by 4-8 hours in cells developing on filters and in suspension under slow shaking conditions. The concentration of the mRNA decreases dramatically late in development (results not shown). These results are very similar to our previous studies which measured changes in the concentration of functional mRNA [Livi et al., 19851.
DISCUSSION Figure 6 summarizes our current model describing the synthesis, transport, processing, sorting, and secretion of Dictyostelium lysosomal enzymes. In many respects the biosynthesis of Dictyostelium enzymes is similar to the synthesis of mammalian enzymes. For instance, the enzymes are proteolytically cleaved, they are phosphorylated and sulfated, and they are transported to dense secondary lysosomes. However, it is clear that the mechanism of lysosomal enzyme localization in the slime mold differs in many aspects from the localization process in many animal cells. For in-
stance, Dictyostelium lacks phosphomannosyl receptors which are critical in the localization of lysosomal enzymes in fibroblasts. Because other animal cells have been identified that target lysosomal enzymes independently of these receptors, the slime mold will be a useful system to investigate sorting of hydrolases by alternate pathways. Furthermore, the maintenance of acidic pH compartments is not required for targeting of the enzymes in Dictyostelium, and inhibition of cleavage of the precursor forms of the enzymes leads to missorting and hypersecretion of uncleaved polypeptides. This suggests that processing and sorting may be coupled. Also the precursor forms of the enzymes are tightly associated with membranes prior to cleavage and release of mature enzymes into the lumen of lysosomes. Finally, the secretion of mature forms of the enzymes is a regulated process and although all the enzymes reside in the same population of lysosomes they are secreted with distinctly different kinetics. Future studies will involve determining the molecular nature of the sorting signal on lysosomal enzymes. and the importance of proteolysis in the localization event. We will also investigate the molecular mecha-
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nisms regulating secretion of enzymes from lysosomes. Finally, we will continue to analyze the developmental regulation of the expression of genes coding for lysosoma1 enzymes.
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