Isolation and characterization of a mutant of Tetrahymena thermophila

blocked in secretion of lysosomal enzymes

PETER HUNSELER, GERDA SCHEIDGEN-KLEYBOLDT and ARNO TIEDTKE*

Institute (if '/.ouloiiy, I 'mveisity of Minister, Schloflplatz 5, D-4400 Minister, FRG

• Author for correspondence

Summary

The development of a sensitive screening pro-cedure for mutants of Tetrahymena thermophilablocked in secretion of lysosomal enzymes isdescribed.

By means of this procedure a mutant blockedin secretion of lysosomal enzymes has been iso-lated. This sec~ mutant, MS-1, is constitutivelyblocked in release of at least six lysosomal en-zymes, under both nutrient and non-nutrientconditions. MS-1 possesses, bound within thecell, the same amount of active lysosomal en-zymes as the wild type. During starvation in

media of low ionic strengh MS-1 develops ahighly vacuolated phenotype. This phenotype iscaused by the sec~ allele. It is reversed to anormal cell shape when the mutant is transferredto isotonic medium.

The sec~ mutant MS-1 contains mucocysts andis capable of inducing exocytosis of these se-cretory organelles, suggesting that Tetrahymenapossesses at least two independent protein-secret-ing organelles.

Key words: Tetrahymena thennoplula, lysosomal enzymes,protein secretion, secretory mutants.

Introduction

Several eukaryotic cells are known to secrete lysosomalhydrolases into the surrounding medium. Mammaliancells in culture (for review see von Figura & Hasilik,1986) and lower eukaryotes like Dictyostelium discoid-eum (Diamond et al. 1981), Acanthamoeba castellanii(Hohmann & Bowers, 1984), Leishmania donovani(Gottlieb & Dweyer, 1981), Saccharomyces cerevisiae(Field & Shekman, 1980) and Tetrahymena thermo-phila (Miiller, 1972) share this remarkable feature.Muller showed that the secreted acid hydrolases of theciliated protozoon TetraJiymena are lysosomal in originand that high amounts of these enzymes are releasedconstitutively into both nutrient and non-nutrientmedium.

The biological significance of secretion of lysosomalhydrolases is unknown. Some species of the genusTetrahvmena, however, live as histophages or as facul-tative parasites in insect larvae (Corliss, 1973) in asituation in which the ability to secrete acid hydrolasesmay be of vital importance for feeding. Other unsolvedproblems concern the route of the secreted lysosomal

Journal of Cell Science 88, 47-55 (1987)Printed in Great Britain © T h e Company of Biologists Limited 1987

hydrolases through the cell, the mode of sequestrationfrom other secretory products, and the cellular site ofrelease.

T. thermophila possesses some unique features thatmake it an attractive model for studies on the biology ofsecretion. First, it contains both secretory lysosomesand mucocysts (Tokuyasu & Scherbaum, 1965; Allen,1967), and this provides the opportunity to followsynthesis, segregation, transport and release of differ-ent secretory products in the same cell. Second, recentadvances in genetic techniques for T. thermophila(Orias & Bruns, 1976; Orias et al. 1979; Bruns &Brussard, 1981fl,6), with respect to mutant isolationand genetic analysis, facilitate genetic dissection ofthese secretory systems with the help of mutantsaffected in one or the other of these processes. Mutantsblocked in exocytosis of mucocysts have been describedrecently by Orias et al. (1983) and Maihle & Satir(1985). Here we report on the development of asensitive screening method for the isolation of mutantsblocked in secretion of lysosomal hydrolases. In ad-dition, we describe the phenotype of a mutant blockedin secretion of lysosomal hydrolases.

47

Materials and methods

StrainsWe used the strains CU399 (Chx/Chx; cy sens., VI), afunctional heterokaryon derived from inbred strain B ofTetrahymena lliennophila (Orias & Bruns, 1976) and C*,which is a vegetative derivate of inbred strain C (Allen,1967). Both strains were kindly provided by Dr P. Bruns,Cornell University, Ithaca, NY.

MediaCells were grown to late log phase in 1 % proteose peptonemedium (PP) supplemented with 0 1 % yeast extract (Y) and0-003% sequestrene (S): PPYS medium.

For initiation of conjugation the cells were washed into oneof the following starvation media: 10mM-Tris- HC1, pH 7-4,or Dryl's solution (Dryl, 1959).

Mutagenesis

Late log-phase cells of CU399 were diluted to2X 10s cells ml"1 with fresh PPYS medium and then treatedfor 4h at 30°C with the mutagen A-methyl-A'-nitro-A"-nitrosoguanidine (NG) at a final concentration of10/igNG ml"1. At the end of the mutagen treatment the cellswere washed twice with, and kept in, PPYS medium for4 -6ha t 30°C.

Generation of isozygotis inutagenizecl pmgenyIn order to produce whole-genome-homozygous (=isozy-gous) progeny of the mutagenized strain CU399 we used theprotocol of short-circuit genomic exclusion as developed byBruns el al. (1976). In short: the mutagenized strain CU399and the untreated strain C* were washed into Dryl's solution,kept overnight at 30°C in this solution at cell concentrationsadjusted to 1 5 x lO'cellsml"1 and 30X 10s cells ml"' , re-spectively. Equal volumes of both strains were then mixed topermit conjugation. To prevent further conjugation (secondround of genomic exclusion), 14h after mixing an equalvolume of double concentrated PPYS medium was added.Sucessful short-circuit genomic exclusion progeny were posi-tively selected 28 h after mixing by adding cycloheximide at afinal concentration of lS^gml" ' ; S6h after mixing thesurviving cells were distributed to 96-well microtitre plates(0' 1 ml/well) at a cell concentration calculated to give anaverage of one isozvgous survivor per well. Under theseconditions according to the Poisson distribution about 37%(l/e) of the wells are expected to have monoclones. Thevegetative progeny of these clones were screened for theirability to secrete lysosomal hydrolases.

Screening for mutants unable to secrete IvsosornalhydrolasesClones growing in microtitre wells were replicated with a 48-pronged replicator from master microtitre plates into cyclin-drical holes of agar plates filled with 40;<l of PPYS-medium.The agar plates were prepared as follows: 20ml of anautoclaved solution of 1 5 % (w/v) agar were poured intosterile Petri dishes, 125cm in diameter. By means of acustom-made metal puncher, 48 holes were made in thesolidified agar reflecting the pattern of half a microtitre plate.

The punched agar cylinders, 5 mm in diameter, were aspir-ated and the remaining cylindrical holes were filled with 40 fdof PPYS medium with an eight-channel dispenser. Thereplicated clones were grown for 2 days at 37CC in order todetect conditional and temperature-sensitive mutants at thesame time. Growth of the clones was scored under a dark-field dissecting microscope. The cell suspensions of the agarholes were aspirated. Remaining cells were removed by twowashes, one with distilled water, the second with 0-1 M-citratebuffer, pH 4-6. The empty agar holes were filled with 20;/l ofa 0-3mgml~' solution of methylumbelliferyl-A'-acetyl-/}-D-glucosaminide, a fluorogenic substrate for /3-hexosaminidase.A parallel Petri dish was similarly prepared with the fluoro-genic substrate for acid phosphatase(methylumbelliferylphosphate). After lOmin at 37CC, the substrate solutionswere removed and the plates scored under ultraviolet (u.v.)light for the presence or absence of a fluorescent halo in theagar surrounding a hole. The absence of a halo of fluorescingumbelhferol indicated the presence of a potential mutantclone unable to secrete /3-hexosaminidase and/or acid phos-phatase.

Preparation of secreted and total acid hydrolasesSecreted acid hydrolases were obtained from cell-free fluidsof clones either grown in PPYS medium, or starved in Dryl'ssolution. To obtain cell-free fluids the cells were sedimentedat 400g for 3min into a cushion of 10%(w/v) Ficoll(Pharmacia). The supernatants were aspirated and thenpassed through 0-2/mi sterile filters. The total activities weredetermined from parallel samples of cell suspensions whosecellular acid hydrolases were liberated by freeze—thawing andsonication in the presence of 0-1 % Triton X-100. Insolublecell debris was removed by centnfugation at 20 000 g- for20 mm.

Enzyme assaysThe enzyme activities of acid glycosidases and of acidphosphatase were assayed at 37°C, using the appropriate />-nitrophenyl substrates (Tiedtke, 1983). Usually, a sample of100/<1 was added to lOOjil of lOmM-p-nitrophenyl substratedissolved in 0 1 M citrate buffer, p H 4 6 , supplemented withsodium azide (0-04%) and bovine serum albumin (0-2%).After incubation for 10-30 min, 1 ml of 04M-glycine- NaOHbuffer, pH 10-4, was added to stop the reaction. The liber-ated £-nitrophenol was determined photometrically at405 nm. The enzyme activity of DNase was determined by amodification of the method of deDuve el al. (1955). Insteadof nuclei, DNA dissolved in 0'lM-sodium acetate buffer,p H 5 0 was used. One unit is defined as the amount ofenzyme that releases 1 fimo\ of p-nitrophenolmin~ . Thevolume activity is expressed as the number of units ml" ' orper 10s cells and the specific activity as the number of unitsmg~' protein. The protein content was determined by themethod of Lowry el al. (1951).

Capsule sheddingThe ability of the wild type (CU399) and the secretorymutant (MS-1) to form a capsule was tested as described byTiedtke (1983, 1985a). In short, cells from the late logarith-mic phase of growth were washed into Wagner's solution andthen incubated at 30°C without shaking. For composition of

48 P. Hiinseler et al.

Wagner's solution see Tiedtke (19856). Samples of 1 ml werewithdrawn and capsule formation was induced by addingAlcian Blue 8GS (Chroma, Stuttgart) to a final concentrationof 0'2 %. The cells were fixed with 2 % neutral formol and theamount of capsule shedding was calculated from the ratiobetween the number of cells encaged in a capsule plus emptycapsules and the total number of cells (encaged in capsulesand without capsules).

Results

Screening for mutants blocked in secretion oflysosotnal hydrolases

The purpose of this study was to isolate Tetrahymenamutants affected in secretion of lysosomal hydrolases.First we had to develop a screening procedure enablingus to detect the different phenotypes that might havebeen affected along the multiple steps leading from thesynthesis of lysosomal hydrolases to their final releaseat the cell membrane. Our screening procedure, illus-trated below, covered this purpose, since it selected atthe final step, i.e. for the presence or absence of activelysosomal hydrolases in the cell-free medium. Itsprinciple is based on our observations, that lysosomalhydrolases secreted by wild type cells diffuse into theagar surrounding a culture hole, in amounts highenough to become visualized by their enzymic activityagainst fluorogenic substrates. When the cells wereremoved from the culture hole and a specific enzymesubstrate conjugated to methylumbelliferol was added,a bright fluorescent halo of enzymically liberatedumbelliferol surrounding the culture hole showed upunder u.v. irradiation. Mutant clones unable to secreteactive forms of lysosomal hydrolases would be recog-nized by the absence of such a fluorescent halo.

The screening procedure as finally used is illustratedin Fig. 1. The holes in the agar plate not surrounded bya fluorescent halo, arranged in an M-shaped pattern,indicate mutant phenotypes blocked in secretion oflysosomal hydrolases. The holes surrounded by brightfluorescent halos indicate wild-type cells (CU399) ableto secrete lysosomal hydrolases. Actually, only the fiveholes of the right arm of the dark 'M' contained denselygrowing cells of a mutant clone (MS-1) unable tosecrete lysosomal hydrolases. The four holes connect-ing the right and left arm of the 'M' contained neitherwild-type nor mutant clones but were incubated withthe fluorogenic substrate for /3-hexosaminidase alone.They served as a control for the stability of theumbelliferyl substrate. The five holes of the left arm ofthe 'M' contained wild-type cells, but were incubatedwithout a fluorogenic enzyme substrate after the cellswere removed. They excluded autofluorescence as asource for fluorescent halos.

Fig. 1. Photographs of an agar plate illustrating theselection of mutants affected in secretion of lysosomalhydrolases. The holes surrounded by bright fluorescenthalos indicate the wild type able to secrete lysosomalhydrolases. The holes missing a fluorescent halo arearranged in an M-shaped pattern and indicate the presenceof a mutant clone unable to secrete lysosomal hydrolases.

Isolation of mutants affected in secretion of lysosomalhydrolases

With the help of the described mutagenesis and screen-ing procedures we obtained three mutants affected insecretion of lysosomal hydrolases. The frequency ofmutant clones obtained using our selection scheme waslow, probably less than 1 per 10 mutagenized homo-zygous clones. Two of the mutant clones could not bepropagated on PPYS medium and died. The third one(MS-1) is blocked constitutively in secretion of lyso-somal enzymes known to be secreted by the wild type.It grows well in the temperature range of 20°C to 37°Con PPYS medium (Fig. 2) and in the basic syntheticmedium (data not shown).

Secretion characteristics of the wild type and the see"mutant of MS-l

First we had to show whether the mutant phenotype ofMS-1 reflects a block in secretion of lysosomal hydro-lases, or impaired hydrolytic activity of the two en-zymes tested during the screening for mutants. Asshown in Table 1 the mutant phenotype of MS-1 isindeed caused by a block in secretion: the specificactivities of A'-acetyl-^S-D-hexosaminidase (/3-hexosami-nidase) and acid phosphatases are equivalent to orhigher in the total system (cells plus medium) in M S-1,than in the wild-type strains (CU399 and BVII),whereas the mutant released only background amountsof these enzymes into the medium. The wild-typestrains CU399 and BVII secreted 77% and 89%,respectively, of their /J-hexosaminidase activities, and43 % and 49%, respectively, of their acid phosphatase

Secretory mutants of Tetrahymena 49

activities during 4h of starvation. These results indi-cate that the mutant phenotype of MS-1 is caused by ablock in secretion of intracellularly active lysosomalenzymes. We therefore propose the designation sec~for the mutant and sec+ for the wild-type allele.

30Culture age (h)

Fig. 2. Kinetics of growth of the wild type (CU399) andthe see' mutant (MS-1) in PPYS medium at 30°C. Meanvalues of four independent counting are shown. Thestandard deviation was below 10 % for each time point(• • ) CU399; (O O) MS-1.

Table 1. Comparison of extracellular and totalactivities of N-acetyl-fi-D-hexosaminidase and acidphosphatases of the secretory mutant MS-1 and thewild-type clones CU399 and BVII of T. thermophila

iY-acctyl-/3-D-hc.\osaniinidase Acid phosphatases

Clonein units nig ' m units nig ' m units mg ' m units mg 'extracellular* total systenvf extracellular* total systemf

MS-1 3-4± 0-1CU399 63-5 ±2-2BVII 63-1 ±3-8

81-8±2-782-0 ±4-470-7 ±0-7

5-1 ± 1-542-6 ±2-355-5 ± 1-9

134-9 ±6-1102-1 ±3-4113-2 ± 1-0

Samples of cells from the same phases of growth were adjustedto 10' cells ml ' and incubated in starvation buffer at 30°C for 4h.Mean and standard deviation of four independent measurementsarc shown.

* Extracellular, enzyme activities found in the cell-free medium.f Total system, enzyme activities found in cells plus medium.

We also tested whether secretion of additional lyso-somal enzymes was affected by the mutation. As seen inFig. 3, neither jS-glucosidase nor DNase was secretedby the mutant, but they were both released in theamounts expected by the wild type. Other lysosomalenzymes like cr-mannosidase, O"-glucosidase, RNaseand acid phospholipases are not secreted by the mutant(data not shown), again indicating that a general step inthe secretion of lysosomal enzymes is affected in MS-1.

Kinetic studies of enzyme secretion under prolongedstarvation confirmed that the secretion deficiency in themutant MS-1 remained in principle unchanged(Fig. 4). While the wild type secreted more than 70%of /J-hexosaminidase and about 50 % of its acid phos-phatases into the starvation medium during 7-5 h, themutant released less than 5 % of either into thismedium. After 24 h of starvation not more than 7 % ofthese enzymes had been released into this medium(data not shown).

It has been shown by Miiller (1972) that T. thermo-phila releases lysosomal enzymes into both non-nutri-ent and nutrient media. We therefore measured thesecretion of /J-hexosaminidase and acid phosphatase ingrowing and dividing wild-type and mutant cell cul-tures during their exponential phases of growth. Asshown in Fig. 5 the total activities on a per cell basis arelower in MS-1 than in the wild type, due to the smallersize and lower protein content, on average, of thegrowing mutant cells. The secretory deficiency ofMS-1 remained as low as in starving cells. About 45 %of /3-hexosaminidase and 21 % of acid phosphataseaccumulated during 4 h in the medium of the wild type,while only 1-5% and 2-2%, respectively, of theseenzymes were found in the medium of MS-1. Thelower percentages of enzymes secreted into this me-dium may be explained by the replacement of theenzymes in growing cells.

Morphological characteristics of the secretory mutantMS-1

The mutant MS-1 exhibits wild-type morphologywhen growing in PPYS medium. However, whenstarved in Dryl's medium or other starvation media oflow osmolarity it exhibits a dramatic change in cellmorphology. While the starved wild-type cell is charac-terized by a slimcell body with only a few food vacuoles(Fig. 6B), the sec~ mutant MS-1 develops after about4h of starvation up to 10 large vacuoles, whichprogressively enlarge and, finally, after 5-12h fuse toform a single big vacuole (Fig. 6A). In this final stagethe cell resembles a big vacuole surrounded by a rim ofcytoplasm and organelles.

When the starved mutant cells were transferred tofresh nutrient medium or to a starvation medium of

50 P. Hiinseler et al.

higher osmolarity (e.g. lOOmM-Tris- HC1, pH7-4),the cells retained their normal shape over 4h.

Capsule shedding

We also tested whether the sec~ mutant MS-1 wasaffected in exocytosis of mucocysts. For this purposewe used the capsule shedding test as described byTiedtke (1983; see also pp. 240-241 in the review byHausmann, 1978). Cells able to secrete mucocysts

•>

II:: E

•— C

if

respond to challenge with the secretagogue Alcian Blue(8GS) by simultanous exocytosis of mature mucocysts.The secreted constituents of the mucocysts bind toAlcian Blue and form a readily visible blue capsulearound a cell. The see" mutant MS-1 and the wild-typeCU399 were both able to form capsules. Other secreta-gogues like dibucaine induced exocytosis of mucocystsas well. We therefore conclude that the sec~ mutantMS-1 is wild type with respect to exocytosis of muco-cysts.

160

120

40

Acid phosphatase DNase

MS-1

CU-399

1

•

m

CU-399

MS-

JL80

60

40

20

N-acetyl-^-glucosaminidase

CU-399 MS-1

1

f _

110

8

6

4

2

0-Glucosidase

MS-r n . i399

Fig. 3. Secretion of lysosomal hydrolases by the wild type (CU399) and sec mutant (MS-1) of T. tliennophila during 4hof starvation in Dryl's solution. Open bars: total activity; dotted bars: extracellular activity. Means and standard deviationsof four independent experiments are shown.

90

« 70o

:E so

u

10

A'-acetyl-/5-glucosaminidase Acid phosphatase

•

0 1 2 4 6 7-5 1 2 4Time (h)

Fig. 4. Kinetics of secretion of lysosomal hydrolases during prolonged starvation. (<^» Wild type (CU399);mutant (MS-1). Means and standard deviations (bars) of four independent experiments are shown.

6 7-5

Secretory mutants o/Tetrahymena 51

iV-acetyl-0-D-glucosaminidase

CU-399_ i _

r+-l

MS-116

12

8

4

Acid phosphatase

•

•

CU399_ L MS-

> -

l iSo-?2 5

UJ

Fig. 5. Secretion of lysosomal hydrolases by wild type(CU399) and see' mutant MS-1 cells of T. ihennophiladuring 4 h of exponential growth in PPYS medium. Openbars: total activity; dotted bars: extracellular activity.Means and standard deviations of four independentexperiments are shown.

Discussion

We have screened for phenotypes unable to secreteactive lysosomal enzymes into the cell-free medium.Because we selected at a final event, namely fusion ofsecretory vesicles with the cell membrane and release oflysosomal enzymes into the surrounding medium, wecould expect to find different mutants corresponding tothe multiple steps leading from biosynthesis to se-cretion of lysosomal enzymes. In theory, mutants ofthe following classes could be isolated. (1) Mutantswith defects in the structure genes leading to non-functional lysosomal enzymes. (2) Mutants affected inproper sorting of lysosomal enzymes during theirpassage through the Golgi apparatus. In this class ofmutants either the 'sorting domain' on N-linked carbo-hydrates or a specific polypeptide sequence necessaryto establish a sorting signal could be affected (Creek &Sly, 1984; Schwaiger et al. 1982). Alternatively,thereceptor recognizing the sorting signal could be altered.(3) Mutants affected in membrane proteins that facili-tate the fusion of the secretory lysosomes with specificsites at the cell membrane. This third class of mutantscould be of particular interest, because it could uncoverthe underlying molecular mechanism and this couldprovide information on how this process operates.Although the membrane proteins of the secretorylysosomes in the wild-type and mutant MS-1 have notbeen studied, circumstantial evidence (discussed be-low) enabled us to suggest that MS-1 may belong tothis third type of mutants. Before discussing thecharacteristic features of the mutant MS-1, we want topoint out, that the block in secretion of lysosomal

enzymes is caused by a recessive single gene mutation.We have called this locus sec and have been able to mapthe sec~ allele on chromosome 4 of the haploid 5chromosomes of T. thetviophila (Hiinseler & Tiedtke,unpublished data).

The secretory mutant MS-1 is constitutively blockedin secretion of all the lysosomal enzymes tested to date.The cells contain the same specific activities of theseenzymes as the wild type. Since we observed no releaseof lysosomal enzymes, even in continuously growingcultures of MS-1, the question arises of how the mutantregulates the behaviour of its lysosomal enzymes.Answers to this question may come from studies on thebiosynthesis of lysosomal enzymes. Results on thebiosynthesis of /J-hexosaminidase (Hiinseler et al.1987) show no striking differences in the biosynthesisand modification of this lysosomal enzyme between thewild-type and the mutant MS-1. While radioactivelylabelled ^3-hexosaminidase is secreted almost quantitat-ively into the medium by the wild type, no enzyme isimmunoprecipitable from the cell-free medium of themutant. However, a gradual degradation of /3-hexosa-minidase was observed in the mutant cell starting 2hafter synthesis of the enzyme. On the basis of theseresults we conclude that MS-1 is blocked in secretion oflysosomal enzymes.

As reported, we observed lysosomal enzyme activi-ties in the range of 5 % in media of growing andstarving mutant cells. This could be explained by celllysis occurring preferentially in starved and tran-sitional-phase MS-1 cells, where we observe the highlyvacuolized phenotype of the mutant. Alternatively,Tetrahymena could possess other (minor) source(s)and route(s) for release of lysosomal enzymes inaddition to secretory lysosomes. The mucocysts, se-cretory organelles of unknown function, remain candi-dates for this route(s). Mucocysts are membrane-bound organelles docked at predictable sites on the cellmembrane (Satir et al. 1973). Although physiologicalstimuli for their secretion are not known, dockingbehaviour and condensation of their contents suggestthat they belong to the type of regulated secretoryvesicles (Kelly, 1985). If we assume that the mucocystproteins are synthesized on membrane-bound ribo-somes (for review see Allen, 1978) as shown forlysosomal enyzines, then errors in sorting could resultin occasional delivery of lysosomal enzymes to muco-cysts. Analogies exist in mammalian cells, where lyso-somal enzymes without the sorting signal, mannose6-phosphate, are not delivered into the lysosomes, butbecome secreted together with other secretory productsof the cell (for review see Creek & Sly, 1984). This mayexplain the fact that discharging mucocysts of Tetraliy-inena occasionally stain for acid phosphatase activity(Tiedtke & Gortz, 1983).

52 P. Hiinseler et al.

Secretion of mucocysts may not account for thewhole amount of released lysosomal enzymes, sinceMS-1 shows wild-type phenotype with respect tosecretion of mucocysts. MS-1 forms capsules whenexocytosis of mature mucocysts is induced by a secreta-gogue (Alcian Blue 8GS). It also released decondcnsed

mucocyst matrices (Tiedtke, 19856) into the surround-ing medium. From these observations we are temptedto predict that single gene mutants affected in assemblyof mucocyst components or exocytosis of these organ-elles (as isolated by Orias et al. 1983) are wild type withrespect to secretion of lysosomal enzymes.

Fig. 6. Micrographs (dark phase-contrast) of living, slightly compressed Tetrahymetia. The cells were starved for 6h inDryl's solution. (A). The secretory mutant MS-1; and (B), the wild-type CU399. n\ contractile vacuole; mac,macronucleus; inic, micronucleus. Bar, 10/Jm.

Secretory mutants of Tetrahymena 53

The secretory mutant MS-1 exhibits a rather dra-matic change in morphology when it is starved ininorganic buffers of low osmolarity like Dryl's solutionor 10mM-Tris-HC1. Vesicles of unknown identityenlarge and fuse gradually until the cell contains a hugevacuole. In this final stage the cytoplasm and organellesare compressed to a small rim surrounding thevacuoles. We know from the breeding analysis of MS-1(Hiinseler & Tiedtke unpublished data), that thevacuolization is caused by the sec~ allele. Preliminaryobservations reveal that the osmoregulatory organelle(contractile vacuole) of the mutant pulses as frequentlyand enlarges as much as in the wild type, where we donot observe this vacuolization. Mutant cells transferredinto buffer of higher osmolarity, e.g. 100 raM-Tris- HC1, retain their normal phenotype over 4 h. Thevacuolization also takes place when MS-1 is grown innutrient medium to stationary phase of growth. Tetra-hymena develops autophagic vacuoles during thisphase, and while starving in inorganic buffers (forreview see Nilsson, 1979). While the wild type hassecreted most of its lysosomal enzymes, under theseconditions, these enzymes are kept cellularly bound bythe mutant. In the latter, fusion between autophagicvacuoles and vesicles containing lysosomal enzymescould result in the building up of an osmotic pressurethat cannot be counteracted sufficiently by the osmore-gulatory contractile vacuole of the cell.

The route of the secreted lysosomal enzymesthrough the cell, and the site of fusion between thesecretory vesicles and the cell membrane, are stillunknown. Since secretion of lysosomal enzymes is aconstitutively occurring dynamic process, cytologicaldemonstration of the route is difficult to establish in thewild type. In the mutant MS-1 and other sec~ mutants(to be isolated) the route should become traced bycombined biochemical and immunoelectron-micro-scopic techniques.

We have been able to grow MS-1 on defined medium(Rasmussen & Modeweg-Hansen, 1973), on PPYS-medium and on bacteria. In all cases we found similargrowth rates in the wild type and the mutant. Weconclude from these observations that the lysosomesmust be functionally active in the mutant. This mayindicate that the product of the sec allele is needed foreither transport to, or insertion of, secretory lysosomesinto the correct domains on the cell membrane.

We thank Dr L. Rasmussen for critical reading of and formany helpful comments on the manuscript. Support fromthe Deutsche Forschungsgemeinschaft (Ti 89/5-3) is grate-fully acknowledged.

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(Received 7 April 1987 - Accepted 7 May 1987)

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