YEAST

VOL.

6: 483490 (1990)

An Assay of Relative Cell Wall Porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe JOHANNES G. DE NOBEL*, FRANS M. KLIS, TEUN MUNNIK, JAN PRIEM A N D HERMAN VAN DEN ENDE

Department of Molecular Cell Biology, Biotechnology Center, University of Amsterdam, Kruislaan 318, 1098 S M Amsterdam, The Netherlands Received 6 February 1990; revised 7 April 1990

We have developed a new assay to determine relative cell wall porosity in yeasts, which is based on polycation-induced leakage of UV-absorbing compounds. Polycations with a small hydrodynamic radius as measured by gel filtration (poly-L-lysine) caused cell leakage independent of cell wall porosity whereas polycations with a large hydrodynamic radius (DEAE-dextrans) caused only limited cell leakage due to limited passage through the cell wall. This allowed the ratio between DEAE-dextran- and poly-L-lysine-induced cell leakage to be used as a measure of cell wall porosity in Saccliaromyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe. Using this assay, we found that the composition of the growth medium affected cell wall porosity in S. cerevisiae. In addition, we could show that cell wall porosity is limited by the number of disulphide bridges in the wall and is dependent on cell turgor. It is argued that earlier methods to estimate cell wall porosity in S. cerevisiae resulted in large underestimations. KEY WORDS

~

Cell wall porosity: permeability; polycation assay; cell wall structure.

INTRODUCTION The cell wall of Saccharomyces cerevisiae consists of glucans, mannoproteins and a small amount of chitin (Cabib et al., 1982; Ballou, 1982). The glucans determine the rigidity of the cell wall (Zlotnik et al., 1984)and the mannoproteins determine its porosity (Zlotniketal., 1984; DeNobeletaI., 1989). Porosity is an important property of the cell wall, because it limits the secretion of homologous (e.g. periplasmic proteins) and heterologous proteins (De Nobel et d., 1989). It might also affect the efficiency by which yeast cells are transformed by heterologous DNA (Brzobohaty and Kovac, 1986). Scherrer et al. (1974) stated that only molecules smaller than 700 Da can pass the wall, but numerous examples of secretion of larger molecules into the medium are known. such as the heterologous proteins prochymosin (60 kDa; Smith et al., 1985) and Ig chains (28 and 63 kDa; Wood et al., 1985). In earlier work we used endocytosis of FITC-dextrans and the release of the periplasmic enzyme invertase to estimate the porosity of yeast cell walls (De Nobel e f al., 1989). On the basis of these results we concluded that yeast *Addressee for correspondence. 0749-503X/90/06048348 $05.00 1990 by John Wiley & Sons Ltd

C '

cell walls are, in principle, permeable to globular proteins with a molecular mass up to 400 kDa. Also the susceptibility of yeast to pl-3 glucanase has been used as a measure of cell wall porosity (Zlotnik et al., 1984). Since these methods are laborious, we have developed a simple assay to determine the porosity of cell walls which is based on cell leakage induced by polycations interacting with the cell membrane. We also show that our assay can equally well be used in other yeasts such as K. lactis and S. pombe. MATERIALS AND METHODS Yeast strains and growth Saccharomyces cerevisiae strain X2 180-1A was obtained from the Yeast Genetic Stock Center, Berkeley, California, U.S.A. Kluyveromyces lactis (MATa lac4-1 trpl IysS-1) was obtained from Dr B. Zonneveld, University of Leiden, The Netherlands. Schizosaccharomyces pombe (972hp) was obtained from Dr P. Nurse, Oxford University, U.K. All strains were grown at 28°C in YPG medium [ 1% (w/v) yeast extract, Gibco; 1 YO(w/v) bactopeptone,

484 Difco; 3% (w/v) glucose]. In specified cases cells were grown in minimal medium [0.67% (w/v) Yeast Nitrogen Base without amino acids, Difco; 2% (w/v) glucose]. Cells were harvested in early exponential phase (absorbance at 530 nm = 2; this corresponds with 1 mg fresh weight per ml). Polycation assay

Freshly harvested cells were washed three times withdistilled water. Washed cells (3 mg fresh weight; 9 x lo7 cells) were incubated at 30°C for 30 min in 1 ml 10 mM-Tris-HC1, pH 7.4, containing 5 pg DEAE-dextran or 10 pg poly-L-lysine, and shaken at 250rpm. As a control, cells were incubated in buffer without polycations. After incubation, the cells were pelleted at 10 000 x g for 2 min, and the supernatant was collected and again centrifuged at 10 000 x g for 2 min. Subsequently, the supernatant was filtered (Nihon Millipore HV 0.45 pm, Kogyo KK) and its absorbance at 260 nm measured. Relative porosity was defined as (A,,,DEAE-dextran A,,,buffer) x 1OO/(A,,,poly- L-lysine - A,,,buffer). In specified cases, the polycation assay was performed in the presence of sorbitol. HP geljiltration

A TSK G4000SW column (7.5 x 600 mm; LKB) eluted with 100 mM-Na,SO,, 20 mM-sodium phosphate buffer, pH 6.8, at a flow rate of 0 2 5 ml min-’ was used for gel filtration of poly-L-lysines. Elution of poly-L-lysine was monitored as absorbance at 206 nm. A Superose 6 column (10 x 300 mm; Pharmacia) eluted with lOmM-Tris-HC1, pH 7.4, at a flow rate of 0.25 ml min-’ was used for gel filtration of DEAE-dextran. Fractions of 0.5ml were collected and assayed for dextran with the phenolsulphuric acid method (Ashwell, 1966). The collected fractions were also assayed for polycation activity by adding 100 p1 of each fraction to yeast cells (3 mg fresh weight) in 1 mi 10 mM-Tris-HC1, pH 7.4. After incubation at 30°C for 30 min, release of UV-absorbing compounds from the yeast cells was measured as described above. Zymolyase assay

Washed cells (1 mg fresh weight; 3 x lo7 cells) were incubated at 20°C in 1 ml 10 mM-Tris-HC1, pH 7.4, containing 1.5 units of Zymolyase 20T (Kirin Brewery), and the decrease in A,,, was followed in time. To allow comparison of different

J. G . DE NOBEL ETAL.

treatments, the time needed for 50% reduction in A,,, was determined graphically. This is called the Zymolyase resistance of the cells. Sulphydryl determination with DTNB (Ellman’s reagent) Intact cells (80 mg fresh weight) were incubated at 4°C for 30 min in 1 ml 10mM-Tris-HC1, pH 7.4, with or without 20 mM of the disulphide-reducing agent dithiothreitol (DTT) or 20mM of the sulphydryl-oxidizing agent sodium tetrathionate. Subsequently, the cells were washed three times in 10 mM-Tris-HC1, pH 7.4. Washed cells (1 5 mg fresh weight) were incubated at 20°C for 10 min in 1 ml 50 mM-potassium phosphate buffer, pH 7.5, containing 10 pM-DTNB (5,S-dithiobis [2-nitrobenzoic acid]; Ellman’s reagent). The reaction mixture was centrifuged at 10 000 x g for 2 min and the absorbance at 412 nm of the supernatant measured. According to Ellman (1959), the A,,, of the supernatant is a measure of the number of reduced sulphydryl groups. DTT was used as a standard. The increase in reduced sulphydryl groups after DTT treatment compared to control treatment was considered to be a measure of the number of disulphide bridges in intact cells. Viability Cell viability was determined by plating a dilution series of cells on YPG-agar (2%, w/v, agar in YPG medium). After 2 days of growth at 28”C, colonies were counted. Chemicals Poly-L-lysines (hydrobromide) with molecular masses of 3.8, 8.8, 25-0, 50.0, 100.5, 249.2 and 378.0 kDa were from Sigma. Poly-L-arginine (HCI; 1 15 kDa), poly-L-glutamic acid (sodium salt; 5 1 kDa), poly-L-aspartic acid (sodium salt; 42-5kDa), L-arginine (HCl), DEAE-dextran (chloride form; 500 kDa; Lot 48F-0518), DTT and sodium tetrathionate were also from Sigma. L-lysine (HCl) was obtained from BDH and DTNB was purchased from Boehringer.

RESULTS Efect of polybasic molecules on yeast cells The polycations, poly-L-lysine, poly-L-arginine and DEAE-dextran released UV-absorbing compounds from intact s. cerevisiae cells, in contrast to

SACCHAROMYCES CEREVISIAE, KL UYVEROMYCES LACTIS AND SCHIZOSACCHAROMYCES POMBE

485

Table 1 . Release of UV-absorbing material from S . cerevisiue after treatment with positively or negatively charged poly-amino-acids

Pol y-amino-acid Pol y-~-lysine Poly-L-arginine Poly-L-aspartic acid Poly-~-glutamicacid

Molecular mass (kW 50 115 42.5 51

A260

0.137 Ifr 0.004 0.1 19+0,030 n.d. n.d.

(TI = 30) (n=4)

Exponential-phase yeast cells (3 mg fresh weight in I ml 10 mM-Tris-HC1, pH 7.4) were incubated at 30°C in the presence of 100 bg poly-amino-acid for 30 min. The release of ultravioletabsorbing compounds was measured as A,, of the supernatant and corrected for release during incubation in the absence of poly-amino-acids. n.d. =not detectable.

the polyanions, poly-L-glutamic acid and p o ly - ~ aspartic acid (Table 1). The released material had an absorption maximum at 260 nm. The free amino acids L-lysine and L-arginine were incapable of causing leakage of UV-absorbing compounds from yeast cells (highest concentration tested: 100 pg ml- I ) . Time-course experiments showed that the release of UV-absorbing compounds was complete with 20 min (Figure la). Similar results were obtained with poly-L-lysines fom 25 kDa up to 378 kDa. When cells were incubated at 30°C for 30 min at different concentrations of poly-L-lysine (molecular mass 50 kDa), maximal release of UV-absorbing compounds was reached at 5 pg poly-L-lysine per ml (Figure 1 b). Yeast cells were treated with poly-L-lysines of different molecular mass to determine whether the yeast cell wall acts as a barrier to poly-L-lysine. All poly-L-lysines with a molecular mass above 25 kDa were able to lyse yeast cells to the same extent. Only the shortest poly-L-lysines (3.8 and 8.8 kDa) hardly released any UV-absorbing compounds (Figure Ic). The tested poly-L-lysines (50 up to 378 kDa) reached their half-maximal effect all at the same concentration (between 1.6 and 2.0 pg ml-I), indicating that not low-molecular-mass impurities but the high-molecular-mass poly-L-lysines themselves were responsible for cell leakage. This implies that the high-molecular-mass poly-L-lysines were capable of passing the cell wall with the same efficiency as the smaller poly-L-lysines. To explain this, we determined the hydrodynamic behaviour of different poly-L-lysines by gel filtration. All tested poly-L-lysines eluted at the same volume, behaving like globular proteins of about 12 kDa. This is probably because poly-L-Iysine lacks a defined tertiary

structure and is capable of so-called reptation (Creighton, 1983). Hence, poly-L-lysines of different molecular masses can freely pass the cell wall. All further experiments were performed at 30°C for 30 min with 1Opg poly-L-lysine (molecular mass 50 kDa) in 1 ml 10 mM-Tris-HCI, pH 7.4, and 3 mg fresh weight yeast cells. These conditions reduced cell viability by 99% within 10 min.

Eflect of DEAE-dextran on yeast cells Durr et a f . (1975) have shown that DEAEdextran is capable of lysing yeast spheroplasts by interaction with the plasma membrane. We show here that DEAE-dextran is also capable of releasing UV-absorbing compounds from intact cells. DEAE-dextran eluted in the range of 5 to 380 kDa, as defined by globular proteins, and released UVabsorbing compounds (up to 20% of the poly-Llysine control) from intact yeast cells over the whole range of molecular masses (Figure 2a). Interestingly, the high-molecular-mass DEAE-dextrans had a higher specific activity, i.e. they released more UV-absorbing compounds per pg of glucose in DEAE-dextran (Figure 2b). In order to determine whether a polycation with a hydrodynamic radius larger than poly-L-lysine (assessed by gel filtration) was retarded by the cell wall, we compared the cell-lytic effect of DEAEdextran with that of poly-L-lysine. Cell leakage due to DEAE-dextran levelled off after incubating at 30°C for 30 min with 10 pg DEAE-dextran per ml (Figure 3a and b). A half-maximal effect was obtained with 5 pg DEAE-dextran per ml. Under standard conditions, the release of UV-absorbing compounds from exponentially growing yeast cells

486

J. G. DE NOBEL ETAL.

0.3

0.04

(a)

0.8

,

0.03 0.02

v)

co

0

U

N

U

0.01

4

0.00 I

I

0.0

I

I

-0.01

Incubation Time [min]

0.3

0.2 0 (D

nJ

a

5 0.1

0.0 0

20

60

40

80

PoI y- L-Iys i ne Conce nt rat io n

0.0 0

10

15

20

25

Elution volume [ml]

100

200 ~. .

300 _..

100

[ug/m I]

400

Mr Poly-L-lysine [kDa] Figure 1. Effect of incubation time, concentration and molecular mass on the release of UV-absorbing compounds from yeast cells by poly-L-lysine. Exponential-phase cells (3 mg fresh weight in 1 ml 10 mM-Tris-HCI, pH 7.4) were incubated at 30°C in the presence of (a) 40 pg poly-L-lysine (25 kDa) for different incubation times; (b) different amounts of 50-kDa poly-L-lysinefor 30 min; (c) 10 Fg poly-L-lysine of different molecular masses for 30 min. After incubation with poly-L-lysine the release of UVabsorbing compounds from the cells was determined as A,,, of the cell-free solution.

Figure 2. Gel filtration of DEAE-dextran on a Superose 6 column. (a) Collected fractions were assayed for carbohydrate content, shown as A,,, (W), and cell-lytic activity, shown as A,, (0);thyroglobulin (T, 670 kDa), IgG (I, 158 kDa), ovalbumin (0, 44 kDa), myoglobulin (M, 17 kDa) and vitamin B-12 (V, 1.35 kDa) were used as molecular markers. (b) The specific activity of DEAE-dextrans of different molecular mass was calculated as amount of released UV-absorbing compounds per pg glucose in DEAE-dextran. The void volume and total volume of the column were at 8.9 ml and 24.2 ml, respectively.

induced by DEAE-dextran was about 30% of the release induced by poly-L-lysine (Table 2); cell viability dropped to 10%. In order to determine whether the ratio between DEAE-dextran and poly-L-lysine effect increased at higher cell wall porosities, we treated the cells with DTT or EDTA. These treatments increase cell wall porosity (measured by the increased release of the periplasmic enzyme invertase and increased uptake of FITC-dextran) without affecting cell viability (De Nobel et al., 1989). Table 2 shows that none of the treatments resulted in an increase of poly-Llysine-induced cell leakage of UV-absorbing compounds compared with control cells; however, DEAE-dextran-induced leakage of UV-absorbing compounds increased. This demonstrates that the sensitivity of yeast cells to DEAE-dextrans depends on cell wall porosity. The ratio between DEAEdextran-induced cell leakage and poly-L-lysine-

SACCHAROMYCES CEREVISIAE, K L UYVEROMYCES LACTIS AND SCHIZOSACCHAROMYCES POMBE

487

resistance to pl-3 glucanase (Zlotnik et al., 1984), can be used as a tool to study wall porosity. Eflect of growth medium on yeast cell wall porosity 0 W N

U

Incubation Time [mln]

0 (D

N

a

I

0.00 0

I

I

I

I

20

40

60

80

DEAE-dextran

100

[uglmi]

Figure 3. Effect of incubation time and concentration on the release of UV-absorbing compounds from yeast cells by DEAEdextran. Exponential-phase cells (3 mg fresh weight in 1 ml 10 mM-Tris-HCI, pH 7.4) were incubated at 30°C in the presence of (a) 10 pg DEAE-dextran for different incubation times; (b) different amounts of DEAE-dextran for 30 min. After incubation with DEAE-dextran, the release of UV-absorbing compounds was determined as A,, of the cell-free solution.

Rothstein et al. (1984) showed that secretion of alpha-amylase into the medium by yeast cells was more efficient when cells were grown in rich medium than when cells were grown in minimal medium. Penttila et al. (1988) showed the same effect for the secretion of cellobiohydrolases. We tested the influence of different growth media on Zymolyase resistance and DEAE-dextran sensitivity. Table 3 shows that cells grown to early exponential phase (absorbance at 530 nm = 2) in minimal medium showed an increase in Zymolyase resistance compared to growth in YPG medium. This indicates a decrease in cell wall porosity. An alternative explanation could be a change in the ratio between p 1-3 and pl-6 glucan in the cell wall which would result in a higher Zymolyase resistance. This seems unlikely in view of the observation that relative DEAE-dextran sensitivity also decreased in minimal medium (Table 3). Since poly-L-lysine-induced cell leakage did not change with growth in different media, a changed susceptibility of the plasma membrane to polycations can be excluded. This leads to the conclusion that the reduced DEAE-dextran sensitivity of cells grown in minimal medium is due to a reduction in cell wall porosity. Eflect of osmotic pressure on cell wall porosity

Hypertonic solutions do not induce plasmolysis but induce shrinkage of the entire yeast cell envelope (Morris et a/., 1986). We were interested whether osmotically induced shrinkage would affect DEAEdextran sensitivity. In the presence of increasing concentrations of sorbitol, the cells decreased in size, and concomitantly, the relative DEAE-dextran sensitivity decreased (Figure 4). The decrease in A,,, of 20% is an underestimation of the actual decrease in cell size, since microscopical observations showed a twofold decrease in cell size.

induced cell leakage (relative DEAE-dextran sensitivity) might therefore be used as a relative measure of cell wall porosity. Treatment with DTT resulted in an opening up of disulphide bridges (measured as an increase in free sulphydryl groups) in the cell wall (Table 2). These disulphide bridges are located in the cell wall, since the membrane of S. cerevisiae is impermeable to DTNB (data not shown) as has also been demonstrated with other cells (Miyakawa et al., 1985; Comparison of diflerent species of yeast Reglinski et al., 1988). Opening up of disulphide bridges by DTT was accompanied by a twofold deThe polycation assay was also performed with crease in Zymolyase resistance of the cells. When the Kluyveromyces lactis and Schizosaccharomyces cells were treated with tetrathionate, the number of pombe. Table 4 shows that they, like S. cerevisiae, disulphide bridges increased, together with the were also differentially sensitive to poly-L-lysine and resistance of the cells to Zymolyase. These data DEAE-dextran. This indicates that our assay might indicate that resistance to Zymolyase, just like the be generally useful for yeasts.

488

J. G . DE NOBEL ET AL.

Table 2. Effect of different pretreatments on yeast cell wall porosity as determined with the polycation assay and the Zymolyase assay Cell leakage ('4260)

[I12

Free sulphydryl groups

DEAE-dextran

Poly-~-lysine

("/I

(min)

(%)

0.043

0.139

0.054

0.140

7.87 f0.10 (n = 3) n.d.

100

+ 100 mM-EDTA + 20 mM-DTT

n.d.

0.060

0.142

+20 mM-Na,S,O,

n.d.

n.d.

30.8 f 1.8 (n = 8) 38.2f 1.1 (n = 8) 42.4 f 1.8 (n = 8) n.d.

4.05 f0.06 ( n = 3) 9.85 0.09 (n =4)

200 24 (n = 6) 81 f 3 (n=4)

Pretreatment

10 m~-Tris-HCl, pH 7.4

Relative DEAE-dextran sensitivity

Zymolyase resistance

Pretreatment took place at 4°C and lasted 30 min. The cells were washed and assayed for polycation sensitivity, Zymolyase resistance and sulphydryl content of cell wall mannoproteins. Relative DEAE-dextran sensitivity is presented as the ratio between release of UVabsorbing compounds as induced by DEAE-dextran and poly-~-lysine.Sulphydryl content is presented as a percentage of control cells; 100% free sulphydryl groups corresponds to 90 nmol/mg fresh weight. n.d. =not determined.

Table 3. Effect of growth medium on cell wall porosity of exponentiaIly growing yeast cells as determined with the polycation assay and the Zymolyase assay

Medium

DEAE-dextran sensitivity (Yo)

Zymol yase resistance [I,, (min)

Minimal YPG

2.6f1.5(n=6) 28.8+1.5(n=16)

17.3f1-7(n=3) 7.8f1.4(n=3) 0.0

0.2

0.4

0.6

0.8

1.0

DISCUSSION Polycations but not polyanions caused leakage of UV-absorbing compounds from yeast cells (Table 1). The polyanions contained less than 1 mMsodium as counter ions, making it unlikely that these were responsible for the lack of effect of polyanions. This was confirmed by the observation that the poly-L-lysine effect was not inhibited by sodium chloride concentrations up to 25 mM. As shown by Schlenk and Dainko (1965) and Yphantis et al. (1 967) the released compounds probably represent the nucleotide and coenzyme pool. The smallest poly-L-lysines (3.8 and 8.8 kDA: Figure 3c) and free amino acids were incapable of causing cell leakage. This demonstrates that a minimal degree of polymerization of the polycation is also a prerequisite for cell leakage. This is supported by the fact that

Sorbitol

[MI

Figure 4. Effect of medium osmolality on cell volume and cell wall porosity. Cells were cultured in YPG in the absence of sorbitol. Cell wall porosity (0)was determined with the polycation assay in the presence of different concentrations of sorbitol and presented as relative DEAE-dextran sensitivity. The effect of sorbit01 on cell volume ( 0 )was monitored at 530 nm and presented as a percentage of the value found in the absence of sorbitol.

the smallest DEAE-dextrans were less efficient in causing membrane leakage than the larger ones (Figure 2). Increased cell wall porosity resulted in a higher sensitivity to DEAE-dextran but not in a higher poly-L-lysine sensitivity (Table 2). Apparently, the passage of poly-L-lysine through the wall is not limited by wall porosity. This is further supported

SACCHAROMYCES CEREVISIAE, K L UYVEROMYCES LACTIS AND SCHIZOSACCHAROMYCES POMBE

489

Table 4. Effect of polycations on different yeast species Cell leakage (AZbO) Yeast S . cerevisiae K. lactis S. pombe

Relative DEAEdextran sensitivity

DEAE-dextran

Poly-L-lysine

("/I

0.043 0. I58 0.035

0.140 0.253 0.194

30.5 0.6 (n =4) 62.5 0.5 (n=4) 18.0f0.5 (n=4)

S. cerevisiae, K . luctis and S. pombe were all harvested at an OD,, of approximately 2.

by the observation that the cell wall of yeast does not constitute an effective barrier to cytochrome c, which is a basic globular protein of 12 kDa and also releases UV-absorbing compounds from yeast cells (Svihla et al., 1969). Figure 2 shows that intact yeast cells are, in principle, sensitive to DEAE-dextrans that in gel filtration elute at the same position as globular proteins of molecular mass 380 000. This is in agreement with earlier reported results about internalization of FITC-dextrans by yeast cells, which suggested that their walls were permeable to globular proteins up to 400 kDa (De Nobel et al., 1989). Cell wall porosity varies. It is affected by the composition of the growth medium (Table 3), by the content of disulphide bridges in the wall, which in itself depends on the local oxygen concentration (Table 2 ) , and by the turgidity of the cells (Figure 4). Cells shrunken in volume in hypertonic solutions showed a significant reduction in cell wall porosity (Figure 4). This probably explains why Morris et al. ( I 986) found an increased resistance to enzymatic digestion by Novozym 234 with cells fixed in hypertonic solutions compared with cells fixed in hypotonic solutions. In addition, Schwencke et al. (1971) showed that cells transferred from a medium with 1.5 M-mannitol to a medium without mannitol, release a considerable amount of their periplasmic enzymes into the medium. These data demonstrate that the turgidity of the cell significantly affects cell wall porosity. Since isolated cell walls are not stretched at all, this implies that porosity determinations on isolated cell walls, as done with s. cerevisiar (Gerhardt and Judge, 1964) and Neurospora crassa (Trevithick et al., 1966), result in an underestimationofcell wall porosity in vivo. Scherrer et al. (1974) determined that only molecules up to 700Da were capable of passing S. cerevisiae cell walls. They worked, however, with stationary-phase cells in the presence of polyethylene glycol which

causes osmotic shrinkage. Since stationary-phase cells also show a considerable reduction in cell wall porosity compared with exponentially growing cells (De Nobel et al., 1990), we conclude that the value obtained by Scherrer et al. (1974) is an underestimation of in vivo wall porosity of exponentially growing cells. ACKNOWLEDGEMENTS We are indebted to Martijn Rep, Roy Montijn and Said El Abouti for carrying out preliminary experiments. REFERENCES Ashwell, G. (1966). New colorimetric methods of sugar analysis. Meth. Enzymol. 8,85-95. Ballou,C. E. (1 982). The yeast cell wall and cell surface.In Strathern, J. N., Jones, E. W. and Broach, J. R. (Eds), The Molecular Biology of the Yeast Saccharomyces. Metabolism and Gene Expression. Cold Spring Harbor Laboratory, New York, pp. 335-360. Brzobohaty, B. and Kovac, L. (1986). Factors enhancing genetic transformation of intact yeast cells modify cell wall porosity. J. Gen. Microbiol. 132,3089-3093. Cabib, E., Roberts, R. and Bowers, B. (1982). Synthesis of the yeast cell wall and its regulation.Ann. Rev. Biochem. 51,763-193. Creighton, T. E. (1983). Proteins: Structures and Molecular Properties. Freeman and Company, New York. De Nobel, J. G., Dijkers, C., Hooijberg, E. and Klis, F. M. (1989). Increased cell wall porosity in Saccharomyces cerevisiae after treatment with dithiothreitol or EDTA. J. Gen. Microbiol. 135,2017-2084. De Nobel, J. G., Klis, F. M., Priem, J., Munnik, T. and vanden Ende, H. (1990). Theglucanase-solublemannoproteins limit cell wall porosity in Saccharomyces cerevisiae. Yeast 6,491499. Diirr, M., Boller, T. and Wiemken, A. (1975). Polybase induced lysis of yeast spheroplasts; a new gentle method for preparation of vacuoles. Arch. Microbioi. 105,319-327. Ellman, G. L. (1959). Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82,70-77.

490 Gerhardt, P. and Judge, J. E. (1964). Porosity of isolated cell walls of Saccharomyces cerevisiae and Bacillus meguterium. J. Bacreriol. 87,945-9s 1. Miyakawa, T., Kaji, M., Yasutake, T., Jeong, Y. K., Tsuchiya, E. and Fukui, S. (1985). Involvement of protein sulfhydryls in the trigger reaction of Rhodotorucine A, a farnesyl peptide mating pheromone of Rhodosporidium toruloides. J . Bacteriol. 162,294-299. Morris, G. J., Winters, L., Coulson, G. E. and Clarke, K. J. (1986). Effect of osmotic stress on the ultrastructure and viability of the yeast Saccharomyces cerevisiae. J. Gen. Microbiol. 129,2023-2034. Penttila, M. E., Andre, L., Lehtovaara, P., Bailey, M., Teeri, T. T. and Knowles, J. K. C. (1988). Efficient secretion of two fungal cellobiohydrolases by Succharomyces cerevisiae. Gene 63, 103-1 12. Reglinski, J., Hoey, S., Smith, W. E. and Sturrock, R. D. (1988). Cellular response to oxidative stress at sulfhydry1 group receptor sites on the erythrocyte membrane. J . Biol. Chem. 263, 12360-12366. Rothstein, S. J., Lazarus, C. M., Smith, W. E., Baulcombe, D. C. and Gatenby, A. A. (1984). Secretion of a wheat alpha-amylase expressed in yeast. Narure 308,662-665. Scherrer, R., Louden, L. andGerhardt, P. (1974). Porosity of the yeast cell wall and membrane. J . Bacteriol. 118, 534-540.

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Schlenk, F. and Dainko, J. L. (1965). Action of ribonuclease preparations on viable yeast cells and spheroplasts. J . Bacteriol. 89,428436. Schwencke, J., Farias, G. and Rojas, M. (1971). The release of extracellular enzymes from yeast by ‘osmotic shock’. Eur. J. Biochem. 21,137-143. Smith, R. A., Duncan, M. J. and Moir, D. T. (1985). Heterologous protein secretion from yeast. Science 229,1219-1224. Svihla, G., Dainko, J. L. and Schlenk, F. (1969). Ultraviolet micrography of penetration of exogenous cytochrome c into the yeast cell. J. Bucteriol. 100,498-504. Trevithick, J. R., Metzenberg, R. L. and Costello, D. F. (1966). Genetic alteration of pore size and other properties of the Neurospora cell wall. J. Bacteriol. 92, 101 6 1020. Wood, C. R., Boss, M. A., Kenten, J. H., Calvert, J. E., Roberts, N. A. and Emtage, J. S . (1985). The synthesis and in vivo assembly of functional antibodies in yeast. Nature 314,44&449. Yphantis, D. A., Dainko, J. L. and Schlenk, F. (1967). Effect of some proteins on the yeast cell membrane. J . Bacteriol. 94, 1 509-1 5 15. Zlotnik, H., Fernandez, M. P., Bowers, B. and Cabib, E. (1984). Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. J . Bacteriol. 159, 1018-1026.