Lysosomal enzyme secretory mutants of Dictyostelium discoideum

DAVID L. EBERT1*, KEVIN B. JORDAN1 and RANDALL L. DIMOND1'2

^Department of'Bacteriology; University of Wisconsin, Madison, WI53706, USA2Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA

* Author for correspondence at: Baker Medical Research Institute, PO Box 348, Prahran, Victoria 3181, Australia

Summary

Dictyostelium discoideum secretes a number of lyso-somal enzymes during axenic growth and upon sus-pension in a low ionic strength, non-nutrient buffer(standard secretion conditions). These secretorycharacteristics have allowed us to identify 74 lyso-somal enzyme secretory mutants generated byJV-methyl-iV -nitro-N-nitrosoguanidine mutagenesis.The majority of these mutants fell into one of fourclasses, on the basis of their secretory characteristicsin non-nutrient buffer. The four mutant classes indi-cate that a minimum of three distinct sets of genes arenecessary for proper secretion of lysosomal enzymesfrom D. discoideum cells under standard secretionconditions: one set of genes that is involved in gen-eral lysosomal enzyme secretion, one that is involvedin glycosidase type secretion, and a third that isinvolved in acid phosphatase type secretion. Thesethree classes likely reflect heterogeneity in the intra-

cellular destination of lysosomal enzymes, the se-cretory mechanism, or both. A fourth set of genesmay be necessary for proper secretion duringgrowth, but plays no role under standard secretionconditions. These are likely altered in the regulationof secretion or in lysosomal enzyme targeting. Of the74 secretory mutants, 36 were also modification mu-tants resulting in decreased pi, thermolability, or invivo instability of lysosomal enzyme activities. Thehigh frequency of modification mutants indicates anintegral relationship between lysosomal enzymemodification, and lysosomal enzyme targeting andsecretion in D. discoideum.

Key words: Dictyostelium discoideum, lysosomal enzymes,secretion, secretory mutants.

Introduction

Many cell types secrete lysosomal enzymes. These en-zymes are secreted in either of two forms, as highermolecular weight precursors or as proteolytically pro-cessed mature forms (Jessup et al. 1985). In mammaliancells, excess secretion of the precursor form of lysosomalenzymes may be indicative of absent targeting signals(Reitman et al. 1981), or defective or absent targetingreceptors (Robbins and Myerowitz, 1981; Gabel etal. 1983;Gonzales-Noriega et al. 1980). In these cases the lysosomalenzymes never reach their lysosomal destination andinstead are secreted directly from the Golgi. The secretionof mature lysosomal enzymes directly from lysosomalvesicles has been studied most extensively in phagocyticleukocytes, namely macrophages and neutrophils(Ohsumi and Lee, 1987; Cox et al. 1986; for review seeJessup et al. 1985) and lower eucaryotes such as Dictyo-stelium discoideum (Dimond et al. 1981), Tetrahymenapyriformis (Blum and Rothstein, 1975), T. thermophila(Hunseler et al. 1987), Leishmania donovani (Gottlieb andDweyer, 1981) and Acanthamoeba castellannii (Hohmanand Bowers, 1984). However, the mechanisms and controlsof these secretory processes are poorly understood.

Ashworth and Quance (1972) first noted the secretion oflysosomal enzymes in the cellular slime mold, Dictyo-stelium discoideum, during both axenic growth and devel-opment. They suggested that this secretion may be underdevelopmental control. The developmental process in D.Journal of Cell Science 96, 491-500 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

discoideum is normally triggered by the absence of thebacterial food source, and, on a solid substratum, results inthe differentiation of single cells into a multicellularfruiting structure (Loomis, 1982). However, an early stageof the developmental sequence may be initiated duringaxenic growth in liquid media (Burns et al. 1981). Therelease of lysosomal enzymes during both the early stagesof development and axenic growth is quite rapid (Dimondet al. 1981). Furthermore, Rossomando and coworkers(Crean and Rossomando, 1979; Rossomando et al. 1978)reported that suspension of cells in a non-nutrient, star-vation buffer triggers the release of the lysosomal glycosi-dases, iV-acetylglucosaminidase and a'-mannosidase.

Previously, we developed conditions, referred to asstandard secretion conditions, under which optimal se-cretion of several lysosomal glycosidases and acid phos-phatase occurs (Dimond et al. 1981). Suspension of D.discoideum cells in a low ionic strength, non-nutrientbuffer stimulates lysosomal enzyme secretion and inhibitslysosomal enzyme synthesis with no loss of existingenzyme activity. Therefore, under these conditions thesecreted enzyme activities all come from preformed lyso-somal vesicles and, consequently, act as markers for thevesicles from which they were released. In addition, thesecretion process requires energy, is not due to cell lysis,and is distinct from phagosomal egestion (Dimond et al.1981).

Our data suggested that the lysosomal system in thewild-type strain, Ax3, of D. discoideum is functionally

491

heterogeneous (Dimond et al. 1981). Ax3 secretes threelysosomal enzymes, Af-acetylglucosaminidase, or-manno-sidase and /3-glucosidase more rapidly and more exten-sively than it secretes acid phosphatase, and it secretes/3-galactosidase-2 to only a minor extent, if at all. Inaddition, the presence of either the protein synthesisinhibitor cycloheximide, or the lysosomotropic aminechloroquine diphosphate, inhibits the secretion of acidphosphatase, but enhances the secretion of the threeefficiently secreted glycosidases. From this information,we suggested that there are at least three functionallydistinct classes of lysosomes (Dimond et al. 1981). Thiscould be due to differential sorting of lysosomal enzymes ordifferential release from lysosomal vesicles. Such hetero-geneous secretory characteristics have also been describedfor T. pyriformis (Banno et al. 1987).

The synthesis, processing and localization ofa-mannosidase in D. discoideum are similar to those oflysosomal enzymes in mammalian systems (Cardelli et al.1987). It is synthesized as a high molecular weight precur-sor on membrane-bound polysomes and cotranslationallyinserted into the lumen of the rough endoplasmic reticu-lum where it is glycosylated (Cardelli et al. 19866).Approximately 90 % of the precursor form is proteolyti-cally processed to mature forms just prior to reaching thelysosomes (Mierendorf et al. 1985; Wood and Kaplan,1985). The remainder of the o--mannosidase precursor issecreted constitutively and accounts for up to 20 % of thesecreted enzyme activity (Mierendorf et al. 1983, 1985).These events are quite similar for /3-glucosidase (Cardelliet al. 1986a) and acid phosphatase (Bush and Cardelli,1989). The oligosaccharide modifications to lysosomalenzymes are also similar to modifications in the mam-malian system, in that they are of the high mannose typeand are phosphorylated on the carbon-6 of mannose(Freeze et al. 1983a). They differ in that the phosphates arecapped with a methyl group (Gabel et al. 1984), the carbon-6 of mannose may be sulfated (Freeze and Wolgast, 1986),and the mannose residue a{l-6)-linked to the core man-nose may be modified with an AT-acetylglucosamine resi-due (Cuoso et al. 1987).

We have taken advantage of the secretory ability of thisorganism to identify 74 secretory mutants. These strainsdiffer from the wild-type strain in the extent and rate ofsecretion of two or more lysosomal enzymes. Our datasuggest that there is a minimum of three sets of genesinvolved in secretion under starvation conditions, andanother set that is necessary for secretion only duringgrowth. Many of these mutant strains were also altered inpost-translational modification, resulting in less nega-tively charged enzymes and in enzyme instability.

Materials and methods

MaterialsAll chemicals were purchased from Sigma Chemical Co., St Louis,MO, unless otherwise noted.

OrganismDictyostelium discoideum was grown axenically in TM medium(Free and Loomis, 1974). Ax3 was the parent strain of all variantsdescribed in this paper unless otherwise noted. The HMW500series strains were generated in our laboratory, except forHMW516, HMW517, HMW545, HMW571 and HMW579, whichwere gifts from Dr Stephen Free (State University of New York atBuffalo). HMW516 was derived from G4, an Ax3 derivative thatlacks /3-glucosidase activity. Ul and UM1 were isolated as UDP-

glucose pyrophosphorylase-deficient mutants (Dimond et al.1976). UM1 was derived from M2, a derivative of Ax3 that lackso--mannosidase activity (Free and Loomis, 1974). M31 is ana--glucosidase-2-deficient strain (Freeze et al. 19836) isolated byFree and Loomis (1974). Strains HL240-HL244 do not have thesulfate-containing common antigenic determinant (Knecht et al.1984), referred to as common antigen 1 (Judelson et al. 1987).

Further information regarding strains can be obtained from DrJames Cardelli, LSU Medical Center, PO Box 33932, Shreveport,LA, 71130-3932.

Enzyme assaysAll enzyme assays were performed at 35 °C with the appropriatep-nitrophenyl-derivatized monosaccharide substrates as de-scribed (iV-acetylglucosaminidase (Loomis, 1969), <r-mannosidase(Loomis, 1970), /3-glucosidase (Dimond and Loomis, 1976), acidphosphatase (Dimond et al. 1981), a-galactosidase-2 (Dimond etal. 1976)). The enzymatic reactions were terminated by theaddition of an equal volume of 1 M Na2CO3. The amount ofp-nitrophenol released during the reaction was measured spectro-photometrically at 420 nm, with units of activity defined asnanomoles of p-nitrophenol released per minute. Protein concen-trations were determined by the method of Lowry et al. (1951).

Isolation of mutantsDimond et al. (1983) previously described the procedure for theisolation of secretory mutants. Briefly, cells treated with themutagen iV-methyl-AT-nitro-iV-nitrosoguanidine (NTG) wereinoculated into 96-well microtiter trays using a multiple-channelpipette (Dimond et al. 1983). Each well received, on the average,one cell. After incubation at 21 °C for 3-4 weeks, plates werereplica plated. After the replicas grew to an easily visible colony,enzyme substrates were added to the wells of one of the replicasusing the multiple pipette. Each well of a replica was assayed forthe presence of a single extracellular enzyme activity, eithero'-mannosidase or AT-acetylglucosaminidase, using the appropri-ate p-nitrophenyl-derivatized substrate. A total of 55000 colonieswere screened of which 11781 were clonal, as calculated, usingthe Poisson distribution, from the number of empty wells per tray.A total of 2976 wells were identified as putative secretory mutantsby visually comparing the intensity of yellow color in eachmicrotiter well with the intensity of color in a control well.

Using the second replica plate, putative secretory mutants wereinoculated into test tubes containing 2 ml of fresh TM andincubated at 21 °C on a reciprocating shaker. A total of 1512 ofthese grew and were harvested in the late exponential phase ofgrowth (2xlO6 to 5xlO6 cells ml"1) and separated from the mediaby centrifugation (1000g, 3min). Cellular and supernatantsamples were assayed for enzymatic activity. A total of 925 ofthese strains had a mean pellet over supernatant activity thatdiffered from the wild-type ratio by greater than fivefold. The 96most extreme variants were chosen for final characterization oftheir secretory abilities.

Of these 96, a total of 66 were actually characterized accordingto the criteria described below. Sixty of these had secretorycharacteristics significantly different from the wild-type strain.This gives a minimum mutation frequency of 0.5 % (60 mutants/11781 clones). This is probably an underestimate, since someclones were lost after both the primary and secondary screenings.

Standard secretionThe secretion of four lysosomal enzymes, AT-acetyl-glucosaminidase, cr-mannosidase, /3-glucosidase and acid phos-phatase, was monitored upon suspension of amoebae from lateexponential phase cultures in a low ionic strength, non-nutrientbuffer (standard secretion conditions) as described by Dimond etal. (1981). The cells were incubated at 27 °C on a reciprocatingshaker. Two-ml samples were removed at 0, 10, 30, 60, 120, 240and 360 min after inoculation. The cells were separated frombuffer by centrifugation and lysed in 0.1% Triton X-100. Theamount of extracellular activity is given as a percentage of thetotal activity at that time point. The kinetics of secretion weremonitored by comparing the amount of extracellular activity

492 D. L. Ebert et al.

present at a particular time point with the final amount ofactivity present at the 360 min time point. To control for non-specific release of enzymes due to cell lysis, both total protein and/3-galactosidase-2 release were measured (Dimond et al. 1981). Iftotal protein secretion was higher than 10% after 360 min, thedata were considered invalid. For each variant, the extracellularactivity for each time point, normalized to total intracellular plusextracellular activity, was compared statistically with wild-type(Ax3) averages.

Secretion during growthThe secretion of the same four lysosomal enzymes was monitoredduring growth in TM as described by Burns et al. (1981). Two-mlsamples were removed at various cell concentrations duringexponential growth (between 5xlO5 and 5xl06cellsml"1). Thesamples were centrifuged to separate cells from media, then lysedin 0.1% Triton X-100. The amount of extracellular activity wasdetermined as a percentage of the total activity present. Theaverage percentage extracellular activity was determined foreach enzyme and the averages were compared statistically withthe Ax3 averages.

Statistical analysisAll variant standard secretion assay averages were statisticallycompared with Ax3 averages using Student's t-test at a signifi-cance level of 0.05. For cases where the mutant sample variance isconsiderably larger than the Ax3 sample variance, there is anincreased probability of erroneously assigning that strain amutant phenotype. To lessen this problem, known as the Beh-ren's-Fischer problem, we used Welch's approximate t. Growthsecretion averages were also compared with Ax3 averages usingStudent's i-test, but, because of the small Ax3 sample size, thistest was not valid for mutants with a small sample size. For thesecases, an arbitrary decision was made on the basis of the mutantaverage and the standard deviation. For cases where only onesample existed, the mutant average was considered significantlydifferent if it was greater than two standard deviations from thewild-type average.

Statistical classification, such as used here, is limited by thenatural variability of living organisms. Some strains have a muchhigher variance than others, reducing the significance of thedifference from Ax3. We have intentionally chosen to stay true tothe statistical test, even if the average percentage secretion wouldseem to indicate that the strain may be assigned a differentmutant phenotype.

Isoelectric focusingIsoelectric focusing gels (0.8% agarose-EF, LKB, Rockville, MD)were prepared and run as described (Dimond et al. 1983; Green etal. 1986). Samples of crude cell extracts containing equal amountsof enzyme activity (2 units of iV-acetylglucosaminidase or acidphosphatase, or 0.5 unit of cr-mannosidase) and Bromphenol Bluetracking dye were loaded onto the gel. Electrophoresis wascontinued until the tracking dye reached the acidic wick. Theenzymes were not allowed to reach equilibrium since this wouldbe at the extreme edge of the anode wick (Knecht et al. 1985).Conditions were established to maximize the separation betweenlysosomal enzymes of the known modification mutant, M31, andlysosomal enzymes of wild-type cells.

Histochemical stainingIsoelectric focusing gels were stained using the protocol ofDimond et al. (1983). The enzyme activities were visualized using0.1 % Fast Garnet GBC salt and 0.2% of the appropriate naph-thyl-derivatized substrate, naphthol-AS-BI-iV-acetyl-;S-D-glucosa-minide, l-naphthyl-<*-D-mannopyrannoside (Koch-Light Labora-tories Ltd, Colnbrook Bucks, England), or cv-naphthyl phosphate,and incubating at 37°C. The glycosidases were stained in 0 . 1 Macetate buffer, pH5.0, while acid phosphatase was stained in0.18M acetate buffer, pH5.0, containing 10mM EDTA.

ThermolabilityTo test for the thermolability of enzyme activities, cell extracts

were incubated with the appropriate assay buffer at elevatedtemperature for 0 and 60 min, then placed on ice. ForiV-acetylglucosaminidase, the cell extracts were incubated at55°C with the addition of 1.7% proteose peptone no. 2 (Difco,Detroit, MI). For a--mannosidase, extracts were incubated at 62 °C.For /S-glucosidase and acid phosphatase, cell extracts were incu-bated at 45°C, but for /S-glucosidase the extracts contained 1 %proteose peptone no. 2. After incubation, the appropriate sub-strate was added to assay the remaining activity. If more than30% of the remaining activity was lost the procedure wasrepeated using 0, 10, 30, 60 and 100 min time points. Enzymeactivities were considered unstable if they repeatedly lost morethan 50% of their activity by 100 min.

In vivo instabilityWe determined the average percentage loss or gain of lysosomalenzyme activity during standard secretion conditions by dividingthe total activity present at any time point by the total activitypresent at the Omin time point. We compared this statisticallywith the average percentage loss or gain of Ax3 lysosomal enzymeactivities.

Results

Characterization of secretion under starvation conditionsWe have previously described the secretory characteristicsof the wild-type strain, Ax3 (Dimond et al. 1981). Thestandard secretion assay has been repeated many timessince then, allowing us now to present a statistically moreaccurate picture of the wild-type secretory characteristics(Fig. 1). Consistent with previous results, Ax3 secretediV-acetylglucosaminidase, a'-mannosidase and /3-glucosi-dase to a greater extent and much more rapidly than acidphosphatase.

On the basis of their behavior under standard secretionconditions, 57 of the 74 mutants studied fell into one offour statistically distinguishable secretory classes (Fig. 2and Table 1). The first class comprises 19 strains thatsignificantly undersecreted the three glycosidases as wellas acid phosphatase (for example, see Fig. 3A). The secondclass includes 17 strains that undersecreted the glycosi-dases, with the secretion of acid phosphatase being affec-ted to a minor but not statistically significant extent (forexample, see Fig. 3B). The third class contains five strainsthat oversecreted acid phosphatase (for example, see

1 2 3 4 5 6Time (h)

Fig. 1. Percentage of enzymes secreted by Ax3 under standardsecretion conditions. The amount of extracellular activity isgiven as a percentage of the total activity, both extracellularand intracellular, at each time point. The standard error foreach time point is within the dimensions of the symbol.iV-acetylglucosaminidase (O); a'-mannosidase (D); /5-glucosidase(A); acid phosphatase (•).

Lysosomal enzyme secretory mutants 493

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Table 1. Secretion of unstable enzyme activities

Class

Wild type

I

n

IV

Strain

Ax3

HL242HMW500HMW501HMW506HMW507HMW511HMW516HMW517HL240HL241HMW520HMW523HMW526HMW527HMW534HMW568HMW570

* Average % enzyme secreted

N-acetylglucosaminidase

Time (h) % Secreted*

1 26.7±1.6 (63)2 38.6±1.8(63)4 47.4±2.0 (54)

4 54.1 (1)4 58.5±13.6(3)

Time (h)

124241

1112242111

a--Mannosidase

% Secreted

36.1±1.6(59)51.7±1.9 (59)59.5±1.9 (52)8.3±2.2 (3)7.3±0.9 (2)2.5 (1)

9.3±3.8 (3)14.1±0.8(2)1.8±0.9 (3)

26.7±3.1 (3)25.5±4.6 (3)27.5±4.4 (2)24.1±2.3 (2)11.3±2.4 (3)3.9±1.9(3)4.9±1.3 (4)

at time indicated (±standard error). The number of trials is indicated in parentheses

Time (h)

1241

414

1112

21

/3-Glucosidase

% Secreted

39.5±2.2 (44)55.3±1.9 (44)60.6±2.1 (41)4.0±2.3 (4)

21.3±8.9 (3)16.8±7.5 (3)15.2±3.9 (3)

3.2±2 0(3)14.3±0.9 (3)12.4±2.8 (3)5.2±2.1 (3)

18.7±5.2 (4)1.3±1.3 (3)

1 2 3 4 5 6 1 2 3 4 5 6

Fig. 3. Percentage of enzymes secreted by the mutants understandard secretion conditions. The percentage of enzymeactivity secreted was calculated as for Fig. 1. The standarderror is represented by vertical bars. If there is no bar, thestandard error is within the dimensions of the symbol. Brokenlines represent wild-type secretion as in Fig. 1. Symbols are asin Fig. 1. A. Class I strain, HMW500. B. Class II strain,HMW522. C. Class III strain, HMW540. D. Class IV strain,HMW568.

Fig. 3C). One strain, HMW543, accumulated a significantamount of /3-glucosidase activity over the 6h secretiontime course. At 6h, on the average, this strain has 249.8±48.9 % of the activity present at the 0 h time point. Thisquite likely contributed to the apparent oversecretion of/J-glucosidase. The fourth class consists of 12 strains whoselysosomal enzyme secretion was not significantly differentfrom the secretion of Ax3 lysosomal enzymes (for example,see Fig. 3D). While secretion was normal under standard

secretion conditions, all of these class four strains werealtered in secretion under growth conditions. The remain-ing 21 strains had a variety of multiple or single enzyme-secretory defects.

A basic assumption of the secretory analysis was thatthe total enzyme activity for each of the mutant strainswould remain stable over the 6h time course. However,this was untrue for a number of strains (see below). Toavoid errors in the interpretation of our data due toselective loss of activity either inside or outside of the cell,we compared the percentage of extracellular activity fromthe wild-type strain and some mutant strains at an earliertime point when there was no significant loss of activity(Table 1). This comparison, and not the comparison at the6h time point, was used to place the strain in theappropriate secretion class.

Characterization of secretion under growth conditionsAx3 also secretes a significant level of lysosomal activityduring axenic growth (Fig. 4). Secretion is under complexregulation during growth (Burns et al. 1981), but thepercentage of extracellular activity was fairly constantduring exponential growth between the cell concen-trations of 5xlOB and 5xl06cellsml~1 (data not shown).Therefore, all wild-type and mutant strains were sampledbetween these concentrations.

Thirty-nine of the 74 mutant strains fall into four majorclasses according to their growth secretion phenotype.Fig. 4 presents 40 of the more significantly altered strains.The largest class (Class A) comprises 22 strains, all ofwhich undersecreted the glycosidases. It was not possibleto identify mutants that undersecreted acid phosphataseusing our statistical method, since acid phosphatase se-cretion from the wild-type was already very low. Nonethe-less, 14 of these 22 strains also undersecreted the glycosi-dases in our standard secretion system. Class B is made upof strains that undersecreted the glycosidases, but have aslight although statistically significant increase in acidphosphatase secretion. The second largest class (Class C)contains eight strains that oversecreted acid phosphatase.HMW545 also oversecreted only acid phosphatase understandard secretion conditions.

Lysosomal enzyme secretory mutants 495

LU

A'-Acetylglucosaminidasea-Mannosidase/3-GlucosidaseAcid phosphatase

Strain:

Class:

x x x x x x x x x

100 T

Strain:

Class:

20

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Fig. 4. Classification of mutantstrains by growth secretioncharacteristics. The percentage ofenzyme activity secreted undergrowing conditions wascalculated as for Fig. 1. Thestandard error is represented byvertical lines. Strain HMW516lacks /?-glucosidase activity andstrain UM1 lacks a-mannosidaseactivity.

Screening for variants altered in modificationMutations affecting post-translational modification of pro-teins can have a variety of effects on the protein. Themutations may alter the total charge on the protein(Freeze and Miller, 1980), decrease the stability of theprotein (Wang and Hirs, 1977), or increase the susceptibi-lity of the protein to proteolytic degradation (Olden et al.1978; Schwarz et al. 1976).

Owing to the large amount of charge on wild-type D.discoideum oligosaccharides, mutations in the modifi-cation system are likely to result in altered isoelectricpoints for lysosomal proteins. Mutational alterations in 18of 72 strains slowed the migration of at least one of threeenzymes on nonequilibrium isoelectric focusing gels(Fig. 5). Mutant strains HMW528 and HMW563 gave

variable results but have shown altered migration inprevious experiments. Six strains that are known modifi-cation mutants showed a marked decrease in mobility onthis gel system. Of these six strains, HL240 throughHL244 lack a sulfated antigen common to all D. discoid-eum lysosomal enzymes (Knecht et al. 1984), while totalglycopeptides from mutant strain M31 contain threefoldless sulfate and 15 % as much phosphate as Ax3 glycopep-tides (Freeze and Miller, 1980).

Carbohydrate modifications contribute to the in vitrostability of many proteins. One way to measure stability ofproteins is to test for enzyme inactivation at elevatedtemperatures. Only two mutant strains contained un-stable enzyme activities at elevated temperatures in vitro.HMW568 cell extracts lost 100% of their AT-acetyl-

496 D. L. Ebert et al.

/V-Acetylglucosaminidase Table 2. In vivo stability of lysosomal enzymes, . ' • - . " ? • • - . •

a-Mannosidase

. ' >J W V* '•

A A AAcid phosphatase

•

i

*r ** *» x ' r r g c g ^ x

X XX I55555 555IIXXI XXI

Fig. 5. Non-equilibrium isoelectric focusing of mutantlysosomal enzymes. Extracts were prepared from axenicallygrowing cells. Equal units of enzyme activity were loaded intoeach well and gels were run as described in Materials andmethods. Enzyme activities were visualized using histochemicalstains for iV-acetylglucosaminidase, a'-mannosidase or acidphosphatase.

glucosaminidase activity within lOmin at 55 °C, whileAx3 extracts maintained 92 % of their activity after 1 h.HMW517 cell extracts lost 60% of their a--mannosidaseactivity by 2 h at 62 °C; Ax3 extracts lost no a-mannosidaseactivity under these same conditions. Under standardsecretion conditions, on the average, Ax3 neither syn-thesizes nor loses lysosomal enzyme activity (Dimond et al.1981). An in vivo loss of activity under these conditionsand no loss of activity in the in vitro assay may beindicative of increased susceptibility to proteolytic degra-dation. A total of 25 strains lost a significant amount ofactivity for at least one enzyme (Table 2).

Strain

Ax3

HL240HL241HL242HL243HL244HMW500HMW501HMW506HMW507HMW511HMW516HMW517HMW520HMW523HMW526HMW527HMW534HMW547HMW548HMW561HMW579HMW580HMW585HMW590HMW592

% Activity

cr-Mannosidase

106.2±3.056.1±4.255.1±1.349.2±5.964.2±7.9

WTt65.2±10.066.0±8.1

WTWT

62.3±3.947.7±13.432.2±5.971.2±16.867.8±24.872.9±15.265.2±5.871.0±6.163.7±15.3

WT57.869.5±22.355.070.1±11.461.0±7.071.7±15.5

(58)

(3)(3)(4)(3)

(2)(3)

(3)(2)(3)(2)(2)(2)(3)(4)(2)

(1)(2)(1)(3)(2)(2)

remaining*

/3-Glucosidase

112.1±6.4

53.2+16.041.0±14.417.7±2.948.9±10.639.6±8.3

WT47.9±3.659.1±1.860.6±7.358.2±4.0

NAt37.6±11.344.7±6.4

WT56.7±14.152.7±2.9

WT46.7±5.240.5±9.345.1

WT28.9

WT44.0

WT

(49)

(3)(3)(4)(3)(3)

(3)(3)(4)(3)

(3)(2)

(2)(3)

(2)(2)(1)

(1)

(1)

* Percentage of the total activity at the 0 h time point that isremaining after 6h under standard secretion conditions, ±the standarderror. The number of trials for each strain is indicated in parentheses.No mutants had significantly unstable acid phosphatase activity.HMW568 and HMW570 had only 67.1±6.8 and 67.9±19.1 %,respectively, of their W-acetylglucosaminidase activity remaining. Ax3values for N-acetylglucosaminidase and acid phosphatase were103.4±3.2 (60) and 97.7±3.6 (51), respectively.

t Percentage of activity remaining not significantly different from Ax3;WT, wild type.

$ No /3-glucosidase activity.

Discussion

The high frequency at which secretory mutants occurredand the wide variety of secretory classes described in thispaper indicate there are a large number of genes involvedin the lysosomal secretory process in D. discoideum. Weestimate that a minimum of three sets of genes arerequired for directing secretion of lysosomal enzymesduring starvation and growth, and a fourth set is requiredfor the proper secretion of lysosomal enzymes only duringgrowth. This conclusion is based on the three standardsecretion mutant classes that distinguish between generallysosomal enzyme secretion (Class I), glycosidase secretion(Class II), and acid phosphatase secretion (Class III), andthe fourth mutant class for which secretion was onlyaltered during growth (Class IV). There are many otherfactors involved in the secretion of other combinations ofenzymes as well as individual enzymes, as evidenced bythe 17 strains and 10 different secretory phenotypes of thesecretory mutants that could not be classified.

Gene products that have been altered in Class I strainsmost likely are involved in aspects of lysosomal enzymesecretion that are common to all lysosomal enzymes. Forexample, secretion of all enzymes tested from mutantstrains HMW500 (Fig. 3A) and HMW501 was depressedalmost to the levels of/S-galactosidase-2 secretion from thewild-type strain (<10%). This relative inability to secretelysosomal enzymes could indicate a defect in the regulat-ory machinery that normally triggers secretion uponstarvation. Alternatively, the defect could be in the transit

Lysosomal enzyme secretory mutants 497

of the lysosome to, or the fusion of the lysosome with, theplasma membrane. Hunseler et al. (1988) speculated thattheir T. thermophila lysosomal enzyme secretory mutant,MS-1, may be defective in this stage of the secretorypathway. Whatever the mechanism, these mutations donot discriminate between various lysosomal enzymes.

On the other hand, gene products altered in Class IImutant strains apparently play a much more significantrole in glycosidase secretion than in acid phosphatasesecretion. In fact, the secretory characteristics of mutantstrain HMW522 (Fig. 3B) would argue that there is atleast one gene product that is essential for normal se-cretion of the glycosidases, but is not important for thesecretion of acid phosphatase. Conversely, the Class IIImutant, HMW540 (Fig. 3C), supports the argument thatthere is also a gene product that plays a major role in acidphosphatase secretion, but not glycosidase secretion.These mutants provide genetic evidence for the earlierphysiological observations of Dimond et al. (1981) thatdemonstrated three functional classes of lysosomes understandard secretion conditions, those characterized by therapid secretion of the glycosidases, those characterized bythe slower secretion of acid phosphatase, and those that donot secrete lysosomal enzymes.

The fourth class of mutants had secretory character-istics that did not differ significantly from the character-istics of Ax3 under standard secretion conditions. How-ever, each of these strains was altered in some aspect ofsecretion during growth. Since our standard secretionassay, in the absence of additional enzyme synthesis,measures secretion from preformed lysosomal vesicles,Class IV mutant strains are most likely not altered intheir ability to direct lysosomal enzymes from the lyso-some to the plasma membrane, but may rather be alteredin the regulation of this event or in the direction oflysosomal enzymes from the point of synthesis to thelysosomal vesicles. In fact, one of the class IV strains,HMW570, secretes greater than 80% of its cv-mannosidaseand jS-glucosidase precursors constitutively duringgrowth, targeting less than 20 % to lysosomes (Ebert et al.19896). The 20% that is directed to lysosomes is secretednormally.

Secretion during growth parallels secretion under stan-dard secretion conditions for only 16 strains. This is takinginto consideration that we are unlikely to identify strainsthat undersecrete acid phosphatase during growth. Se-cretion during growth and secretion under standard se-cretion conditions hold in common that aspect of secretioninvolved in directing the lysosomal enzymes outside thecell. Therefore, these mutants, unlike the Class IV mu-tants, are most likely defective in an aspect of secretionafter lysosomal localization. Furthermore, these mu-tations would not be subject to differential regulationunder nutrient-rich conditions versus nutrient-deprivedconditions. For example, HL242, is able to process proteo-lytically both cv-mannosidase and /J-glucosidase to matureforms and, presumably, direct these enzymes to lysosomalvesicles (unpublished observations), but secretes verylittle, if any, of these mature forms during growth or understandard secretion conditions.

The lack of correlation between secretion under the twoconditions for strains other than those in Class IV issomewhat surprising. However, the rate of secretion understandard secretion conditions is tenfold greater than dur-ing growth (Dimond et al. 1981). Therefore, the magnitudeof the effect any particular mutation has on the rate ofsecretion is likely to be considerably less during growth

than during the standard secretion assay. Furthermore,secretion during growth is not only a function of therelease of lysosomal enzymes from lysosomal vesicles, butalso all steps prior to lysosomal localization. This includesenzyme synthesis, transport from the endoplasmic reticu-lum and theGolgi, and the relative partitioning betweenthe precursor secretion pathway and the lysosomal path-way. Strain HMW570 has already been mentioned to bedefective in the partitioning of lysosomal enzymes. StrainM31, a known a--glucosidase-II mutant (Freeze et al.19836) that undersecretes a--mannosidase during growth(unpublished observations), is known to be relatively slowin transporting o'-mannosidase from the rough endoplas-mic reticulum to the Golgi, but has relatively normalsecretion of this enzyme under standard secretion con-ditions (Ebert et al. 1989a).

Since nearly half of the secretory mutants are likelyaltered in some aspect of post-translational modification,there is apparently a correlation between secretion andmodification. However, for individual strains we do notknow what role, if any, post-translational modification ofthe lysosomal enzyme plays in lysosomal enzyme se-cretion. Defective post-translational modification may bethe result or the cause of altered subcellular targeting andsecretion. We are currently investigating these possi-bilities for a number of mutant strains.

Future studies will be directed at genetic analysis of themutants to determine the number of double mutants thatmay exist in the mutant population as well as the numberof loci that are represented by the mutants. We do notthink that the existence of double mutants presents aserious problem, however. Initial screens were all forsingle enzyme-secretory defects. Assuming the frequencyat which a secretory mutation affecting a single enzymearose from the initial population is equal to the frequencyat which a secondary mutation affecting another enzymemight occur in the mutant population, there should be nomore than one double mutant. It will still be necessary tocarry out complementation analysis, since the secretoryclasses do not represent the number of genes involved inthe secretion of lysosomal enzymes. This will also give usinformation concerning the amount of allelic variationthat occurs at each locus.

Overall, our results are consistent with there being agenetic basis for the functional heterogeneity of thelysosomal system. These mutants will be useful tools indeveloping an understanding of the generation and regu-lation of this heterogeneous system, as well as the move-ment of lysosomal vesicles to the plasma membrane andthe release of mature lysosomal enzymes from the cell.Finally, these mutants may also prove useful in establish-ing a role for the lysosomal system in the developmentalphase of the D. discoideum life cycle. More than 80 % of thestrains we isolated were unable to aggregate, the first stepin the differentiation of this organism from the single cellstate to the multicellular state. In contrast, only 5.5 % ofthe total mutagenized population were unable to aggre-gate, indicating that lysosomal enzyme secretion may bean essential process in development.

We thank the following people for assistance in the characteriz-ation of individual strains: Melissa Anderson, Rebecca Borst, TomDay, Joan De Witt, Jay Farnsworth, Deborah Galuska, CathyHahn, Ann Marie Herrien, Anne Janzer, Jamie Lewis, MaryO'Connor, Joseph Richards and Lynn Wunderlich. Special thanksgo to Eric Green, Cathy Judelson and Chris Mlot. This work wassupported by the College of Agriculture and Life Sciences,

498 D. L. Ebert et al.

University of Wisconsin-Madison and by the National Institutesof Health under grants GM29156 and GM31181.

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