FEMS Microbiology Leucrs 69 (1990) 7-I2 7 Published by Elsevier

FEMSLE 03973

Fluid-phase endocytosis in yeasts other than Saccharomyces cerevisiae

Niev~;s Fernandez . Pilar Puente and F e r n a n d o Leal

Departamenlo de MnerobJologia, Gendtica. Medlcina Prevenliva y Salad Pubhca and Insrttuta de Mic~bialag|a Bioqu~mico- Universidad de S~lamanca /Consejo Superior de lm,esltgaci~ne.~ Cwntlficas, Salamanca, .~;pam

Received 21 December lqg9 Accepted 23 December 1q89

Key ~ords: Endocytosis. fluid.phase: Yeast: FITC-dextran

1. SUMMARY

A FITC-dextran internalization assay with S,c- charomyces cerevisiae as positive control was used to determine whether fluid-phase endocytosis is a general characteristic of yeasts. $chizosaccharo- myces pombe, Pk'hia polymorpha, Khqveromyces phaseolosporus, Yarrowia li?olytica and Candida albicans were clearly positive, whereas results ob- tained with Debaryomyces ,~arama were inconclu- sive. In all cases internalized FITC-dextran was found to be localized in the vacuoles and the process was always time- and temperature-depen- dent. Lo~,er euearyotes, particularly yeasts, appear to have the abi!i~y ~o incorporate substances from the extracellular medncr, z through fluid-phase en- docytosis.

2. INTRODUCTION

Most euearyotic cells are able to internalize extracellular molecules by ewiocytosis. Important

C~rrespc~ldence 1o: F. Leal, Dplo. Microbiologia, Facuhad de Biologla. Universidad de Salamanca. 37071 Salamanca. Spain.

nutrients, toxins, effector molecules (growth [ac- tors. hormones, antibodies) and enzymes are among the most usually endocytosed substances. Internalization takes place through receptor-medi- ated endocytosis [1,2], a mechanism which is saturable and that can concentrate ligand in- tracellularly. Concurrent with receptor-mediated endocytosis, parl of the extracellular fluid is also ;-lternalized into the endosome. This process may also take place independently [1] when it is desig- nated as fluid-phase endocytosis, however it is not saturable and is proportional to the concentration of molecules in the extracellular fluid. Probably both kinds of endocytosis have a common mecha- nism.

To detect fluid-phase endocytosis cells are ex- posed to an impermeable solute th.,~ d,;e~ ,~ot bind to the plasma membrane. Useful cytologic markers are: electron-dense materials, such as ferritins [3,4] and some colloidal gotd preparations [5]. enzymes, such as horseradish peroxidase, and fluo- resceinated dextrans [6],

Recently, Riezman [7] and Makarow [8] have described the internalization of fluorescent markers (Lucifer Yellow CH or FITC-dextran) into bakers" yeast by fluid-phase endocytosis. The uptake of a-amylase by whole cells and of en-

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veloped viruses by spheroplasts of the same yeast has also been reported [9]. Since the endocytotic apparatus in Saccharomyces cerevisiae had been identified, we were prompted to attempt to dis- cover whether this is a general characteristic of yeasts.

3. MATERIALS AND METHODS

3.1. Culture conditions and yeast strains Yeast cells were cultured in YED medium (1%

yeast extract, 1% glucose). All experiments were performed with exponentially growing cells (A660 -0.8-1.0). The yeast strains used were: Sac- eharomyces cerevisiae X2180-1A (Yeast Genetic Stock Center, Berkeley, CA, U.S.A.), Schizosac- charomyees pombe 972 h- (P. Nurse, Imperial Cancer Research Fund, Oxford, U.K.), Pichia polymorpha (A.T.C.C. 48432), Kluyveromyces phaseotosporu~ (Dpt. of Food Sci,~nce and Tech- nology, Davis, CA), Candida albicans (A,T.C.C. 10231), Yarrowia Iipolytica CX 39 74(= (J. Basset, Berkeley, CA) and Debaryomyces marama (from Spanish country-cured hams, kindly provided by E. Monte, University of Salamanca, Spain),

croscope with an EF-A epifluorescence device and an HBO 100 W/2 light source.

4. RESULTS AND DISCUSSION

Fhiid-phase endocytosis has recently been shown to occur in Sa¢¢haromyces cerevisiae. Con- sequent ly this microorganism offers an excellent control system to confirm the existenee of the process in other yeasts.

As previously reported by other authors [81, we found that early-tog phase S. cerevisiae cells took up FITC.dextran efficiently (Fig. 1). Fluorescent label became localized in the vacuoles and inter. nalization was both time- and temperature.depen- dent. The reaction proceeded rapidly at 370C (Fig. 1A,B), but was totally inhibited at 4°C (Fig. 1C,D). FITC-dextran was added to exponentially growing cultures of Schizosaccharomyces pomhe (Fig. 2A,B), Pichia polymorp,~a (Fig. 2C,D), Kluyveromyces phaseolosb, oeus (Fig. 2E.F), Candida albicans (Fig, 3) and Yarrowia lipolytica (not shown) under identical conditions. In all cases the fluorescence became localized in the vacuole and the process was time- and temperature-dependent, but was undetectable at 4 ° C. The intensity of the

3.2. Uptake of FITC, dexlran We followed the protocol described by

Makarow [8] with slight variations: yeast cells, extensively washed in YED medium buffered with 20 mM Hepes (Sigma, U.S.A.) pH 7.2, were resus- pended (10 ~ ceils/ml) in the same raedium supple- mented with 100 mg/ml of Fluoreseein-isothio- eyanate labeled dexlran (FITC-dextran 70S, Sigma, U.S.A.), and incubated ~i!h:r at 4°C or 37 ° C, usually for 40 rain. After incubation, sam- ples were placed on ice and diluted in 1 ml of cold phosphate buffered saline (PBS), washed three times with I ml o[ cold PBS, and finally lcsus- pended into 100/~l of mounting medium (25 mM sodium barbital, 107 mM NaCl, pH 8.6). 5 #1 of this suspension was applied to microscope slides pre-treated overnight with 0,5 mg/rai of con- canavalin A (Sigma, U,S.A.) to immobilize the living cells. Internalization was followed by ob- servation through a Nikon Optiphot XFR,H mi-

: t=

Fig. I, Uptake of FlTC-dextran into Saccharomyces cerevisiae cells. Cells were incubated, as described in ~m*Eat̂ LS Arid M~HODS [or 40 rain in the presence of FITC.dextran at 37°C (A,B) or 4°C (C,D). Phase-contrast (left) and fluorescence

(right) photomicrographs, Magnification 530)<.

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Fig. 2. Uptake of F1TC-dextran into different yeast cells. Cells were incubated (see UA'rI~RIAt.S ANO MtTMODS) far 40 mia in the presence of FITC-dextran a137* C. Phase-eonlrast (upper) and fluorescence (Iow.,r) photomicrographs of Sebi:o~accharomvces pombe

(A,B), Pichia polymorpha (C,D) and Kluylwrom)'t'es phaseolo~por~cr ~ E,F). M~gaification 1060 ×.

label varied with different yeasts and was always less marked lhan for the S, cerevisiae positive control. Debaryorn),ces marama, another yeast species, save inconclusive results (not shown). The standard incubation time was 40 rain to allow for maximal FITC-dextran internalization, but times as short as 10 rain were long ¢nough to delect fluorescence in the vacuoles. Even shorter times (1-5 rain) revealed fluorescence in small vesicles

(SV) distributed across the cytoplasm (Fig. 4). This localization probably corresponds to tran- sient states in the movement to the vacuoles. Con- sistent with this interpretation, Makarow et al, [12] have described the existence in S. cerevisiae of an 'endosome-like' compartment in which the accumulation of fluorescent marker took place in

Fig, 3. Uptake of FITC-dexttan into eandida albicans cells, Cells were incubated (see materials and melh~ls for 40 mill in the pre~nce of FITCodexlran tl! 37°C. Fluorescence nnicrr,-

graph, Magnification t150 ×.

Fig. 4. Uplake of F[TC-dextran into Sacchar~myces cereui.*iue cells. Cells were incubated (see MA'fEItlALS AND MI~'qI'HOD$) fol" 4 rnin in the presence of F[TC-dextran at 37°C. Fluorescence micrograph. Magnification 860×. (V= Vacuoles, SV = Small

Vesicles)

10

the presence of NaF as metabolic inhibitor and in the absence of glucose in the medium, Release from inhibition resulted in normal transit of the marker to the vacuole,

Non-specific binding to the cell walt or plasma membrane was not observed. Only Candida al- bicans and Yarrowitt lipolytica showed, in addition to the vacuolar fluorescence, a bright halo along the cell wall (Fig, 3) which persisted, although to a lesser extent, at 4°C. A similar staining pattern was obtained by Riczman [7] in S. cerevtsiae using Lucifer Yellow CH as internalization marker. The small size of this marker (MW 457.25) and its negative charge might explain the non-specific bi- nding. Adsorption of FITC-dextran to C albicans and Yarrowia lipolytica (former Candida lipolytica ) could be indicative of a particular cell wall com- position or architecture not present in the other yeasts tested, Consistent with this s~gestion was the observation that FITC-dextran treated Candida cells lost a great part of their previous ability to adsorb onto ConA pre-treated microscopic slides, perhaps as a result of non-specific binding of the marker to the wall.

We were unable to apply the a-amylase inter- nalization assay, previously described [8], because some of the yeasts used in our study were strongly amylolytic.

There has been some controversy about using FITC-dextran as a fluid-phase internalization marker. A recent report [13] has suggested that endocytosis of FITC-dcxtran by S, cerevisiae only reflects the upt::tk¢ of low molecular weight impur- ities - - not dextran - and has proposed diffusion as the mechanism whereby FITC alone enters the yeast vacuole. We have no experimental data available to distinguish between the entrance of FITC or F1TC-dextran into yeast vacuoles. In any ease, if diffusion were the mechanism responsible, fluorescence should be distributed uniformly throughout the cell, at least after short internaliza- tion times, and this is not consistent with our data (see Fig. 4), Furthermore, Makarow et al. have demonstrated [12] the even distribution of fluores- cence in heat-permeabilized cells by contrast with localized accumulation of the marker in an endo- some-like compartment in the presence of NaF in normal yeast ceils. Even more convincing are data

showing that quinacrine, a weak base that diffuses across membrane.s and accumulates in acidic com- partments, is localized in yeast vacuoles, like FITC-dextran. Although quinacrine accumulation is totally inhibited by the lysosomotropic salt am- monium acetate, which raises intravacuolar pH in yeasts, FlTC-dextran localization was not affected [14], In addition, the degradation of FITC-dextran after internalization in the vacuole has also been reported by the same authors. This could account for the FITC found in yeast extracts after incuba- tion with FITC-dextran.

Several phylogenetically well separated yeast species are able to take up molecules actively from extracellular medium. This observation indicates that fluid-phase endocytosis could be a general mechanism acting to a variable extent in all yeasts. Nevertheless the biological significance of this phenomenon is still obscure. Whether the process acts 'in vivo" under natural circumstances remains to be demonstrated. Perhaps the other more specific pathway described for S. cerevisiae, i.e. receptor-mediated endocytosis, is also present in these other yeasts. However, as long as specific receptors remain unidentified, the existence of such a pathway cannot be confirmed.

ACKNOWLEDGEMENTS

We wish to thank Carlos Bclinchon for photo- graphic services and Dr. Angel Duran for critical reading of the manuscript. We are grateful to Nicholas Skinner fo- revis:,ng the English manuscript.

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