Large and small invertases and the yeast cell cycle

113

Biochimica et Biophysica Acta, 475 (1977) 113--122 © Elsevier/North-Holland Biomedical Press

BBA 98848 LARGE AND SMALL INVERTASES AND THE YEAST CELL CYCLE PATTERN OF SYNTHESIS AND SENSITIVITY TO TUNICAMYCIN

GILAD

G A L L I L I a and J. O L I V E R

LAMPEN

b

a Laboratory of Applied Microbiology, The Hebrew University, Jerusalem (Israel)and b Waksman Instituteof Microbiology, Rutgers University, The State University of N e w

Jersey, New Brunswick, N.J. 08903 (U.S.A.)

(Received August 30th, 1976)

Summary We have examined the pattern of synthesis of the glycoprotein form o f invertase and of the smaller carbohydratefree form in synchronous culture to obtain further information concerning their biosynthetic relationship. Saccharomyces m u t a n t 1710 was chosen since its invertase production is almost completely derepressed during growth in 0.1 M mannose medium. The large enzyme, unlike the small form, binds to concanavalin A-Sepharose, and on this basis the two types can conveniently be separated for analysis. Large invertase was produced throughout the cell cycle. Synthesis of the small invertase was periodic; the single burst occurred at or close to the budding stage. Tunicamycin, which inhibits the synthesis of external glycoproteins, halted formation of the large enzyme but not of the small form, and there was no accumulation of invertase activity with the properties of the small enzyme. Hence, it is unlikely that the small form is a precursor of the large one. Despite marked differences in their amino acid compositions, the two enzymes have m a n y similarities. They axe probably, in part, the products of the same gene(s), and the differences between them may largely reflect differences in post-translational processing.

Introduction There are two predominant forms of invertase (/3-D-fructofuranoside fructohydrolase, EC 3.2.1.26) in Saccharomyces cells [1,2]. The large enzyme, which accounts for most of the invertase activity of derepressed cells, is a glycoprotein about 50% of which is mannan and 3% glucosamine [3,4]. Most of it is located in the periplasmic space and within the wall matrix [5--7]; the small a m o u n t of internal large enzyme is associated with the cell vacuole [8]. The small enzyme lacks carbohydrate [9] and is reported to be present mainly in the cytosol [8].

114 Despite differences in their amino acid composition and in their electrophoretic mobilities and pH-stability curves [2], there are several reasons to believe that the t w o invertases axe related: the molecular weight of the small invertase approximates that of the protein moiety of the large form; the enzymes cross-react immunologically; mutants unable to ferment sucrose lack b o t h large and smll invertases; and the two enzymes have the same substrate specificity and kinetic properties [2,4]. The possible biosynthetic relationship of the two invertases has been considered b y several investigators, and at least four hypotheses have been suggested: (a) the enzymes are aggregates of subunits one (or more) of which is present in both enzymes and carries the active site [2]; (b) the small enzyme is a precursor of the large form [10,11]; (c) the small invertase is a step in the breakdown of large invertase b y removal of the carbohydrate chains and a portion of the amino acids [12]; and (d) the small invertase is a side product released into the cytosol during synthesis and secretion of the large invertase [13]. All these proposals (most of them not mutually exclusive) are based on the assumption that there is at least one structural gene which is required for the formation of both enzymes. No conclusive evidence has been obtained for any of these theories. Since the pattern of synthesis throughout the cell cycle is a characteristic of an individual enzyme, a comparison of the patterns for t w o enzymes can provide information concerning their biosynthetic relationship. This led us to carry out the experiments presented here on the formation of the large and small invertases in synchronous cultures of Saccharomyces mutant 1710. The glucosamine-containing antibiotic tunicamycin [14] blocks the synthesis of secreted glycoprotein enzymes in yeast protoplasts [15], probably b y preventing the formation of their N-acetylgiucosamine-containing mannan side chains [16,17]. Since the large invertase is a glycoprotein, tunicamycin might selectively block its synthesis without interfering with that of small invertase. An accumulation of the small invertase in the presence of tunicamycin would suggest that the enzyme is a precursor of the large invertase. Materials and Methods

Yeast strain and growth conditions Saccharomyces m u t a n t 1710 [18] was produced by double irradiation of Saccharomyces 303-67 which contains a single gene for invertase production (SUC2). (The function of this gene will be discussed later). Mutant 1710 has a high invertase content during growth in marginally repressive conditions and, thus, a relatively low susceptibility to repression by hexoses. Under the conditions of the synchronous growth experiments, mutant 1710 grows as small cell clusters as a result of the inability of the daughter cells to separate from the mother cells. The related mutants FH4C and 1016, which are more resistant to repression, grow as irregular clumps with many incomplete septa [18] and the cell number cannot be determined. Stock cultures were maintained on slants of 0.3% Bacto peptone (Difco), 0.2% Bacto yeast extract (Difco), and 1% glucose agar. For batch growth 250ml flasks containing 50 ml of 0.3% peptone and 0.2% yeast extract (YEP me-

115 dium) and 0.1 M sugar (as indicated) were incubated at 28°C with rotary shaking at 250 rpm. For production of single cells a 5 liter fermenter (New Brunswick Scientific Co.) containing 3 liters of YEP medium with 0.1 M glucose was employed. The agitation speed was 400 rev./min and aeration was one half liter of air per min per liter of medium. A 3% inoculum of an exponential phase culture was used in all experiments.

Chemicals All chemicals were of reagent quality. [8-~4C]Adenine (40 to 60 Ci/mol) and ~4C-labeled amino acids mixture were purchased from New England Nuclear Corp., Boston, Mass. Tunicarhycin was a gift of Professor G. Tamura of the University of T o k y o , Japan. Determinations Synthesis of nucleic acid was estimated by the incorporation of [14C]adenine. At the beginning of a growth experiment [14C]adenine (50/~Ci in 0.5 ml) was added to 200 ml of culture, and duplicate samples (0.5 ml) were taken throughout the synchronous growth cycle. One sample was mixed with 0.5 ml 1 N NaOH, held at room temperature for 16 h to hydrolyze the RNA, and precipitated with one ml of cold 20% trichloroacetic acid. After 1 h at 0°C the precipitate was collected on glass fiber paper (Whatman G F / A ) which was then washed twice with 5 ml of cold 5% trichloroacetic acid and placed in ascintillation vial along with 0.2 ml distilled water and 10 ml of Aquasol (New England Nuclear). The radioactivity was determined in a Packard Tri-Carb liquid scintillation spectrometer. The other sample (0.5 ml) was used to determine the incorporation of [~4C]adenine into total nucleic acids by omitting the 16 h hydrolysis step. R N A was calculated by difference. Protein synthesis was estimated by the incorporation of a 14C-labeled amino acids mixture into hot trichloroacetic acid-precipitable material. 1 ml of 14Clabeled amino acids mixture (100 pCi) was added to a 200 ml culture at the beginning of synchronous growth. Samples (1 ml) were mixed with one ml of 10% trichloroacetic acid and incubated at 90--95°C for 20 min to hydrolyze the nucleic acids. The precipitates were collected on glass fiber filters and washed twice with 10 ml portions of cold 5% tricholoracetic acid. The filters were placed in scintillation bottles with 0.5 ml distilled water and 10 ml Aqua° sol and radioactivity measured. Cells were counted using a Petroff-Hauser chamber and a phase contrast microscope. As soon as a bud was visible, it was counted as a cell. Measurement of invertase formation Both large and small invertases were assayed by colorimetric determination of the glucose released b y the enzymatic hydrolysis of sucrose [4]. The total concentration of invertase was determined after the cells (in 0.05 M potassium phosphate, pH 7.0) were lysed with the enzyme Zymolase (550 units/g; obtained from Dr. Y. Yamamoto, Kirin Brewery Co., Takasaki, Gunma, Japan). For determination of the small invertase, the lysate was clarified by centrifugation and the large invertase was removed b y absorbtion on concanavalin A-Sepharose. In this procedure a mixture of 0.5 ml cell lysate, 0.3 ml of 0.05 M

116

Tris HC1 buffer, pH 7.5, and 0.2 ml of concanavalin A-Sepharose (Con A-Sepharose, Pharmacia Fine Chemicals) was incubated at 30°C for 1 h with vigorous shaking. The concanavalin A-Sepharose (and large i n v e r t a s e ) w a s removed b y filtration through Whatman G F / A glass fiber paper. The small invertase was n o t bound, and its activity could be measured in the filtrate. Results

Synthesis of small and large invertases under repressive and derepressive conditions Saecharomyces m u t a n t 1710 grows rapidly on glucose or fructose media and invertase formation is Tepressed. Mannose utilization is somewhat impaired in this mutant: in 0.1 M mannose medium the growth rate was a b o u t two-thirds of that on fructose (Table I), and more than 50% of the initial level of mannose was still present after 300 to 400 min of incubation (Gallili, G. and Lampen, J.O., unpublished data). As might be expected, mannose was only weakly repressive even at 0.1 M. This situation made it possible to maintain a relatively constant derepression of invertase synthesis during several cycles of synchronous growth. To determine the relative sensitivity of formation of the t w o invertases to repression b y hexoses, a batch culture of mutant 1710 was grown to exponential phase in YEP medium containing 0.1 M fructose (invertase formation repressed) and the cells were washed and suspended in fresh medium containing either fructose or mannose. Table I shows the levels of lm'ge and small invertase after 1 h of exponential growth. Large invertase activity was a b o u t 20 times greater in the derepressed than in the repressed cells while small invertase activity increased only 2.4 fold. Thus, as with other yeast strains [1,9,11], the amplitude of the derepression was much greater for the large glycoprotein enzyme than for the carbohydrate-free small form.

Isolation o f single cells The tendency of m u t a n t 1710 to grow in clusters necessitated a special technique to produce the individual cells required for synchronized growth. When the m u t a n t was grown in a 5-liter fermenter under the conditions described in Materials and Methods, single cells became detached from the clumps near the end of the active growth phase and did not initiate new buds. A large number

TABLE I I N V E R T A S E F O R M A T I O N BY M U T A N T 1 7 1 0 U N D E R R E P R E S S I V E A N D D E R E P R E S S I V E C O N D I TIONS E x p e r i m e n t a l c o n d i t i o n s f o r b a t c h g r o w t h are given in Materials and M e t h o d s . Sugar

R e l a t i v e g r o w t h rate (rng c e t l s / m l p e r h)

Large invertase ( m u n i t s / m g cells)

Small i n v c r t a s e ( m u n i t s / m g cells)

M a n n o s e , 0.1 M

0.24

1863

M a n n o s e , 0.01 M

0.24

1954

F r u c t o s e , 0.1 M

0.34

90

26 11

(% of t o t a l ) 1.4 12

117 of such cells with essentially derepressed levels of invertase were present after 24 h. To isolate the cells, the culture (3 liters) was sedimented for 5 min at 5000 × g and the supernatant fluid discarded. The pellet was suspended in 300 ml of water and centrifuged 3 min at 550 × g. The supernatant fluid was retained and the pellet (mostly cell clusters) was recycled through the two steps. The resulting supernatant fluid was combined with that from the original cycle and subjected to an additional sedimentation cycle to yield the final suspension of single cells. The cells could be stored in water at 4°C for 24 h without impairing their ability to grow synchronously on subsequent transfer to fresh medium.

Synchronous growth When a suspension of the single cells was inoculated into YEP medium with 0.1 M glucose or mannose, growth and budding were synchronous as illustrated schematically in Figs. 1 and 3. Since the cells did not separate after budding, they were counted in doublets in the second generation and in tetrads in the third. The first budding occurred after about 110 min. During this adaptation period the cells grew appreciably in volume. The second cycle of dubbing began 75 min after the completion of the first. Fig. 2 illustrates the increase in DNA, RNA and protein during growth, as determined by the incorporation of radioactive precursors. DNA synthesis was periodic; the DNA content of the cells increased shortly before budding. RNA and protein synthesis showed linear patterns [19]. In synchronous culture the appearance of large invertase activity seems to be linear or linear exponential (Fig. 3). In contrast, the synthesis of small invertase is clearly periodic with the bursts of enzyme formation occurring close to or at the budding stage (Figs. 3 and 4), except for the first burst which began about 40 to 60 min after bidding had started. During the first two and a half hours most cellular activities may be directed toward growth, with little of either enzyme form being synthesized

100

80

tD

o x 01 ..J -J hi (J

60 C3o

•

m

./. I I00

I 150

I 200

I 250

TIME (rnin)

Fig. 1. Cell c o u n t during s y n c h r o n o u s g r o w t h o f Saccharomyces 1710. A s u s p e n s i o n o f single cells w a s a d d e d t o Y E P m e d i u m eontalning 0.1 M m a n n o s e a n d i n c u b a t e d a t 30°C w i t h shaking. A n e w b u d w a s c o n s i d e r e d a cell.

118

E Ol2 r

--

o RNA •

4 -=9 I-

20

DNA

•

CELL

A

PROTEIN

CYCLE

6 15 O~ .E

?1-

/

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//-

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_.x

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-7

b 0

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TIME (rain)

200

~,00

Fig. 2. P a t t e r n o f i n c o r p o r a t i o n o f [ ! 4 C ] a d e n i n e i n t o n u c l e i c a c i d s a n d o f 14C.labele d a m i n o acids m i x t u x e i n t o p r o t e i n s d u r i n g s y n c h r o n o u s g r o w t h o f Saccharomyces 1 7 1 0 . G r o w t h c o n d i t i o n s as in Fig. 1. I n c o r p o r a t i o n o f r a d i o a c t i v e p r e c u r s o r s i n t o D N A , R N A a n d p r o t e i n w a s m e a s u r e d as d e s c r i b e d in Materials a n d M e t h o d s . T h e p o i n t s (filled t r i a n g l e s ) o n t h e cell c y c l e cttrve i n d i c a t e s c h e m a t i c a l l y t h e b e g i n n i n g a n d e n d o f t h e b u d d i n g stages as d e t e r m i n e d b y p h a s e c o n t r a s t m i c r o s c o p y . T h e initial cell n u m b e r w a s approximately 107/ml.

•

CELL CYCLE

~

|

o LAROE INVERTASE • SMALL INVERTASE

1

c:C"~; /

a:Z> E US w US

~

US 00

-I

0 [ IO0

I

I 200

I

~ 300

Ji~

TIME (rain) Fig. 3. I n c r e a s e of s m a l l a n d large i n v e r t a s e a c t i v i t i e s d u r i n g s y n c h r o n o u s g r o w t h o f Saccharom~cces 1 7 1 0 . G r o w t h c o n d i t i o n s as in Fig. 1. S m a l l a n d large i n v e r t a s e s w e r e m e a s u r e d as d e s c r i b e d in t h e t e x t . T h e cell c y c l e w a s d e t e r m i n e d as in Fig. 2.

119 T A B L E II EFFECT OF TUNICAMYCIN ON THE SYNTHESIS OF LARGE AND SMALL INVERTASES EXPONENTIAL-PHASE CULTURE OF MUTANT 1710

IN AN

Cells g r o w n o n Y E P m e d i u m w i t h 0 . 2 M g l u c o s e w e r e t r a n s f e r r e d t o 0.1 M m a n n o s e - Y E P m e d i u m a n d i n c u b a t e d 3 0 r a i n a t 3 0 ° C . T u n i c a m y c i n (TM; 1 pg/ml) w a s t h e n a d d e d ( z e r o t i m e ) t o o n e h a l f o f t h e c u l ture and incubation continued for 90 rain. Incubation (rain)

Invertase formed (munits/ml) Large

0 30 90

Small

--TM

+TM

--TM

+TM

15 45 275

15 40 60

1.1

1.1

4.1

3.8

until after the initial budding. Only small amounts of invertase were released into the medium under the conditions used.

The effect of tunicamycin on invertase formation Tunicamycin has been shown to block the formation and secretion of large invertase in protoplasts of Saccharomyces 1016 without causing an accumulaA

SMALL INVERTASE (NO TM)

•

SMALL INVERTASE

o

LARGE INVERTASE (NO TM)

(+ TM)

•

LARGE INVERTASE

V

CELL CYCLE (NO TM)

( + TM)

•

CELL CYCLE (+ TM)

E E

TM

.1

E 3

|

.J -I ,4

,4 .J

I

0 I00

~

IO0

0 200 TIME (rain)

250

Fig. 4. E f f e c t o f t u n i c a m y c i n ( T M ) o n t h e f o r m a t i o n o f l a r g e a n d s m a l l i n v e r t ~ e a a n d o n s y n c h r o n o u s g r o w t h o f Saccharomyces 1 7 1 0 . G r o w t h c o n d i t i o n s as i n F i g . 1. T h e c u l t u r e w a s d i v i d e d i n t o t w o p o r tions after 180 rain of synchronous growth (arrow); one received 1 ~g tunieamycin/rnl, the other no antib i o t i c . T h e cell c y c l e w a s d e t e r m i n e d as i n Fig. 2.

120

tion of the small carbohydrate-free form (as measured by a step-wise elution from DEAE-Sephadex [15]). Before testing its effects on synchronously growing cells, we added the antibiotic to a logarithmic phase culture of Saccharomyces 1710. As with the protoplasts, tunicamycin at 1 pg/ml caused an almost complete cessation of large invertase formation after a b o u t 30 min (Table II). The production of small invertase was not inhibited nor was there an accumulation of activity associated with this form. The invertase level of the culture fluid did n o t increase in the presence of tunicamycin even though the cells had b e c o m e swollen and round after 30 min. The effects of tunicamycin at 1 pg/ml on synchronously growing Saccharomyces 1710 cells are illustrated in Fig. 4. After the addition of the antibiotic the increase in large invertase activity began to slow down almost immediately and stopped after 40 min. There was no effect on small invertase synthesis for 20 min; the increase in activity then halted for 30 min and resumed later though at a slower rate than in the control without drug. The treated cells gradually became swollen and did n o t form new buds. Discussion

There are at least six different genes for sucrose fermentation (SUC1-6) in Saccharomyces which individually are sufficient to allow the production o f a large and a small form of invertase [20]. The enzymes formed in the presence of the various genes are very similar although they appear to differ in some kinetic properties [21]. It is n o t clear whether these are regulatory or structural genes. Mutants unable to ferment sucrose (or slow fermenters ) lack b o t h large and small invertases, and revertants regain b o t h forms (refs. 2, 22 and Zimmermann, K.F., personal communication). Thus the available genetic information does n o t specify the biosynthetic relationship between the t w o enzymes. We therefore examined the production of large and small invertases throughout the cell cycle of Saccharomyces m u t a n t 1710 which carries the SUC2 gene for sucrose fermentation. This mutant is relatively resistant to repression by hexoses and has a budding pattern that permits one to follow cell counts through at least three cycles (Figs. 2 and 4). Synchronized growth of m u t a n t 1710 was accomplished b y the isolation of single cells formed during fermentation, followed by suspension of these cells in a rich medium. The parameters of growth (RNA, DNA, and protein synthesis) which were followed during the first three generations, gave patterns similar to those previously reported for synchronous growth of yeast [23,24]. The small invertase was formed in a step-wise manner with the burst of activity occurring close to or during budding. In contrast, formation of large invertase was continuous during the cell cycle with the rate of formation approximately doubling during each generation. There is no previous report of the pattern of synthesis of the t w o invertases during the cell cycle, b u t the production of total invertase activity has been examined b y Gorman et al. [25] w h o found that invertase synthesis b y a hybrid Saccharomyces yeast occurred in steps. This result is seemingly in disagreement with ours, since their culture was at least partially derepressed and in such cells the large enzyme accounts for much of the total invertase.

121 The addition of tunicamycin (1 pg/ml) to an exponential phase culture of mutant 1710 rapidly blocked the formation of large invertase but did not affect the rate of accumulation of the small form (Table II). Similar results had been obtained by Kuo and Lampen [15] in their study of the action o f t u n i camycin on yeast protoplasts. In our synchronous cultures, the production of large invertase was similarly blocked by tunicamycin while the synthesis of the small form continued, although the rate and timing of its formation had changed. This shift in timing might be attributable to the unbalanced growth which is a delayed effect of the antibiotic [15,17]. There was a slow accumulation of the small form under these conditions. The results presented here make it unlikely that the small enzyme which accumulates in the cytosol is a direct precursor of the large glycoprotein form. If the small enzyme were converted to the large form in a direct process, the periodic increase in the level of small invertase should have been followed by a decrease as conversion occurred. This was not observed. Moreover, one would have expected tunicamycin to produce an accumulation of small invertase beyond that which occurs in its absence. The slow continued formation of the small enzyme in the presence of tunicamyci~l might occur either by degradation of pre-existing large enzyme or by an independent route. For example, its appearance in small amounts near the budding stage may reflect some temporary alteration or abbrerration in the processes leading to the production of large invertase. Our results are most easily rationalized by proposing that the large and small enzymes are produced independently in the sense that both may be derived from a common polypeptide precursor(s) but neither enzyme type, once formed, is converted to the other. Final resolution of the biosynthetic pathways must await clarification of the chemical relationships between the large and small enzymes (and the intermediate forms; Moreno et al. [11]), and location of the several stages in their synthesis within the yeast cell [8,26]. The time when an enzyme is produced during a growth cycle is often considered as the time at which transcription of the appropriate gene occurs. On this basis one could suggest that there is continuous transcription of the gene(s) for large invertase and periodic transcription f o r small invertase. This explanation appears tenuous in light of the known similarities and interrelations between the two forms and the likelihood that both are composed of subunits (Gascon, S., Abrams, B.B. and Lampen, J.O., unpublished results) which individually may not be active. The difference between the patterns of synthesis of the large and small enzymes during the growth cycle can be explained, without specifying the timing of transcription, if the mRNA for the large glycoprotein invertase has a sufficiently long half life that this enzyme activity is continuously produced. Evidence that the total invertase mRNA in protoplasts has an apparent half life of about 20 minutes has been presented by Lampen et al. [13]. This half life, which is based on the continuing increase in invertase activity following the addition of an inhibitor of mRNA synthesis, does not appear to be long enough to support the continuous synthesis of large invertase through a cell cycle of about 75 min (as observed here) but it seems too long to be easily reconciled with the short synthetic periods detected by Gorman et al. [25].

122

Mechanisms controlling enzyme synthesis at the translational level were recently detected in the fission yest Schizosaccharomyces pombe [27,28] and there is indirect evidence that catabolite repression of invertase formation in Saccharomyces protoplasts can occur at the translational stage [ 13]. In spite of the nearly maximal derepression of invertase synthesis in mutant 1710 under our conditions for synchronous growth, some translational control must still exist. In addition, invertase molecules of intermediate size (presumably only partially glycosylated) have been demonstrated in Saccharomyces yeasts [11] and are reported to be localized in the plasma membrane [26]. Hence, the glycosylation process may also be rate-limiting under the proper circumstances. Appropriate combinations of these factors and a m R N A of relatively long half life [27] could probably yield either the essentially continuous production of large invertase seen in our system or a step-wise formation as reported by Gorman, et al. [25].

Acknowledgements This research was supported by a U.S. Public Health Service grant (AI04572) from the National Institute of Allergy and Infectious Diseases. We thank Professor G. Tamura of the University of Tokyo, Japan, and Dr. Y. Yamamoto, Kirin Brewery Co., Japan, for generous gifts of tunicamycin and Zymolase, respectively. References 1 2 3 4 5 6 7 S 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Gascon, S. and Ottolenghi, P. (1967) Compt. Rend. Trav. Lab. Carlsberg 36, 85--93 Gascon, S., Neu man n, N.P. and Lampen, J.O. (1968) J. Biol. Chem. 243, 1 5 7 3 - - 1 5 7 7 Neuman n, N.P. and Lampen, J.O. (1967) Biochemistry 6 , 4 6 8 - - 4 7 5 Goldstein, A. and Lampen, J.O. (1975) in Methods E n z y m o l . 42, 504--511 Friis, J. and Ottolenghi, P' (1959) Compt. Rend. Tray. Lab. Carlsberg 3 1 , 2 5 9 - - 2 7 1 Sutto n, D.D. and L a m p e n , J.O. (1962) Bioehim. Biophys. Acta 56, 303--312 Arnold, W.N. (1972) J. Baeteriol. 112, 1 3 4 6 - - 1 3 5 2 Meyer, J. and Matile, P. (1975) Biochem. Physiol. Pflanzen 1 6 6 , 3 7 7 - - 3 8 5 Gascon, S. and Lampen, J.O. (1968) J. Biol. Chem. 243, 1 5 6 5 - - 1 5 7 2 Beteta, P. an d Gascon, S. (1971) FEBS Lett. 1 3 , 2 9 7 - - 2 9 9 Moreno, F., Ochoa, A.G., Gascon, S. and Villanueva, J.R. (1975) Eur. J. Bioehem. 50, 571--579 Tkacz, J.S. (1971) Ph.D. Thesis, Rutgers University, The State University of N.J., New Brunswick, New Jersey Lamp en, J.O., Kuo, S.-C. and Liras, P. (1963) in Third I n t e r n a t i o n a l S y m p o s i u m on Yeasts, O t a n i e m i / Helsinki (Suomalainen, H. and Waller, C., eds.), Part 2, pp. 129--147 Takatsuki, A., Arima, K. and Tamura, G. (1971) J. Antibi ot . 24, 215--233 Kuo, S.-C. and Lampen, J.O. (1974) Biochem. Biophys. Res. C o m m u n . 5 8 , 2 8 7 - - 2 9 5 Tkacz, J.S. and Lampen, J.O. (1975) Bioehem. Biophys. Res. C o m m u n . 65, 248--257 Kuo, S.-C. and Lampen~ J.O. (1976) Arch. Biochem. Biophys. 172, 574--581 Montenecourt, B.S., Kuo, S.-C. and Lampen, J.O. (1973) J. Bacteriol. 1 1 4 , 2 3 3 - - 2 3 8 Mitchison, J.M. (1969) Science 1 6 5 , 6 5 7 - - 6 6 3 Ottolenghi, P. (1971) Compt. Rend. Tray. Lab. Carlsberg 38, 213--221 Ottolenghi, P. (1971) Eur. J. Biochem. 18, 544--552 Hackel, R.A. (1975) Molec. Gen. Genet. 140, 3 6 1 - - 3 7 0 w i n i a m s o n , D.H. and Scopes, A.W. (1960) Exp. Cell Res. 20, 338--349 Mitchison, J.M. a n d Creanor, J. (1969) J. Cell Sci. 5 , 3 7 3 - - 3 9 1 Gorman, J., Tauro, P., La Berge~ M. and Halvorson, H.O. (1964) Biochem. Biophys. Res. C o m m u n . 15, 43--49 Holbein, B.E., Forsberg, C.W. and Kidby, D.K. (1976) Can. J. Microbiol. 2 2 , 9 8 9 - 9 9 5 Fraser, R.S.S. (1975) Eur. J. Biochem. 6 0 , 4 7 7 - - 4 8 6 Creanor, J., May, J.W. and Mitchison, J.M. (1975) Eux. J. Biochem. 60, 487--493