The molecular basis of an osmotically reparable mutant of Neurospora crassa producing unstable glutamate dehydrogenase

J. Mol. Biol. (1977) 110, 627-642

The Molecular Basis of an Osmotically Reparable Mutant of Neurospora crassa Producing Unstable Glutamate Dehydrogenase J . R . S. F n ~ c ~ A ~ t AND A. J . BARON

Department of Genetics, University of Leeds Leeds LS2 9 J T , England (Received 20 September 1976) M u t a n t 14 in the am gene of Neurospora crassa, coding for NADP-specific glutam a t e dehydrogenase (E.C.1.4.1.4), has been previously shown to complement positively with several other am m u t a n t s a n d negatively with the wild type, b u t to produce no detectable g l u t a m a t e dehydrogenase protein under normal conditions of growth. W e show in this p a p e r t h a t amld is p a r t i a l l y repaired b y 1.0 to 1-5 M-glycerol, glucose, mannitol or sorbitol, or 0.5 to 0-75 M-KC1 in the growth m e d i u m so as to produce highly unstable g l u t a m a t e dehydrogenase. Two kinds of pseudo-wild r e v e r t a n t s from am14 produce p a r t i a l l y stabilized g l u t a m a t e dehydrogenase varieties on normal m e d i u m b u t their enzyme formation is still increased b y glycerol in the growth medium. The formation of the enzyme in am14 a n d in both t y p e s of r e v e r t a n t is highly temperature-sensitive. Amino acid sequence analysis of the enzyme varieties formed b y the two r e v e r t a n t t y p e s shows the replacement Leu20 --> His in one case (R5) a n d Leu20 --> T y r in the other (R1). I t is deduced b y coding considerations t h a t the p r i m a r y replacement in am14 itself m u s t be Leu20 --> His, a n d t h a t R5 has a p a r t i a l l y compensating replacement a t a second unknown residue, while R1 has a second change in the same residue, Leu20 -* His -* Tyr. The properties of the am14 enzyme are a t t r i b u t e d to unstable q u a t e r n a r y structure due to the repacement of a hydrophobic side chain (leucine) b y a polar one (histidine) a t or near a p o i n t of contact between monomers in the enzyme hexamer. I t is speculated t h a t the relative stabilization brought a b o u t in growth media of high osmolality is due to the reduction on the effective concentration of water molecules surrounding the protein, with a consequent reduction of the disruptive effect of the polar side chain. 1, Introduction M u t a n t s in t h e am ( a m i n a t i o n ) gene o f Neurospora crassa (the s t r u c t u r a l gene for N A D P - s p e c i f i c g l u t a m a t e d e h y d r o g e n a s e E.C.1.4.1.4) i n c l u d e s e v e r a l w h i c h s h o w allelic c o m p l e m e n t a t i o n ( P i n c h a m & S t a d l e r , 1964). All e x c e p t one o f t h e complem e n t i n g m u t a n t s each p r o d u c e s a p r o t e i n , in some cases w i t h c o n d i t i o n a l G D H a s e $ a c t i v i t y , r e l a t e d t o t h e w i l d - t y p e G D H a s e a m i n o a c i d sequence ( H o l d e r et al., 1975) b y a single r e s i d u e r e p l a c e m e n t ( B r e t t et al., 1976). T h e sole e x c e p t i o n , am14, h a s n o t hitherto been found to produce any GDHase-related protein detectable either by f r a c t i o n a t i o n or b y i m m u n o l o g i c a l a s s a y for cross-reacting m a t e r i a l ( R o b e r t s , 1971). ~fPresent address: Department of Genetics, University of Edinburgh, West Mains Road, Edinburgh EH9 3JN, Scotland. :~Abbreviation used : GDHase, glutamate dehydrogenase (E.C. 1.4.1.4). 41 627

628

J.R.S.

FINCHAM AND A. J. BARON

Sundaram & Fincham (1968) were able to isolate, though in low yield, the enzymaticaUy active product of complementation between am14 and am3, a m u t a n t which b y itself produces a stable GDHase variety which is inactive under physiological conditions. Freezing and thawing of this supposedly hybrid complementation product in the presence of 0-1 M-NaCI, a procedure which promotes a near-random redistribution of monomers between hexamers of native DGHase, led to the recovery of apparently pure m u t a n t GDHase of the am3 type; the putative am14 component disappeared, presumably as insoluble denatured material. I t was also shown t h a t a heterokaryon formed between am14 and wild type produced GDHase which was heterogeneous with respect to heat stability, much of it being much more labile than the wild-type enzyme. All of these observations suggested that am14 produced a GDHase monomer which was grossly defective in its formation of stable hexamers, although capable of contributing normal GDHase activity in more or less stabilized mixed hexamers formed with other mutant monomers. The failure to detect crossreacting material in am14 extracts, even using antibodies against denatured GDHase (Roberts, 1971), is probably most reasonably explained as the result of proteolysis of denatured polypeptide. Stadler (1966) obtained four classes of prototrophic revertants from am14, distinguishable on the basis of thermal stabilities at two different p H values. One of these, represented b y only one revertant, appeared to be identical to standard wild type. The other three classes produced GDHase varieties which were, to different extents, abnormally thermolabile. I t appeared reasonable to regard these non-wildtype revertants as representing different degrees of stabilization, through further mutational change, of the unstable am14 GDHase structure. In this paper we show t h a t am14 is, in fact, capable of forming active GDHase by itself when grown in medium of high osmotic pressure and at relatively low temperatures. Furthermore, we have re-isolated two revertant classes (Stadler's original revertants having been lost), and shown that their formation of GDHase is temperature-sensitive and stimulated b y glycerol, though they are much less extreme in both these respects than is am14 itself. Sequence analysis of the GDHase varieties formed by representatives of these two classes permits us to deduce the primary amino acid replacement in the am14 polypeptide chain. Osmotically reparable mutants are a well known and plentiful class of missense (and usually allelically-complementing) mutants in the yeast 8accharomyces cerevi. siae (Hawthorne & Friis, 1964). One mutant, a temperature-conditional lethal, has also been shown to be osmotically reparable in N. crassa (Metzenberg, 1968). No investigation of the nature of the enzyme defects in such mutants has, however, been reported.

2. Materials and Methods (a) Neurospora strains and isolation of revertants T h e am14 strain used throughout was amld-6-1a, a derivative of the original mutant isolated after 6 generations of repeated crosses to the standard wild type ST4A. The am1 strain aml-6-3a had a similar derivation. The prototrophic revertants amld R1 to R5 were selected following ultraviolet irradiation of amld-6-1a conidia (various doses, giving survival of between 2 and 90%) and plating on Vogel's (1956) 0-2% sucrose-l.0~o sorbose medium supplemented with 0.02 M-glycine to restrict the usual "leaky" growth of am mutants (Fincham, 1950). Five apparently revertant colonies were picked and the mutations responsible were purified by crossing to the parent mutant in the opposite mating type

(amldA).

N o attempt was made to determine the reversion frequency.

MOLECULAR BASIS OF OSMOTIC REPARABILITY

629

(b) Conditions of culture and growth tests Experiments to determine the effects of various supplements on growth employed Vogel's (1956) minimal m e d i u m with 2% sucrose, and where appropriate, solidified with 1-5~o agar; in some experiments the " l e a k y " growth of am m u t a n t s was suppressed b y the addition of 0.02 M-glycine. Small scale cultures for tests of GDHase formation under various conditions were grown in 50-ml lots of Vogel's medium, appropriately supplemented, in 250 ml Erlenmeyer flasks from conidial inocula, usually without shaking. Large scale cultures of R1 a n d R5 for GDHase isolation were grown in the modified Vogel's m e d i u m described b y A s h b y et al. (1974), with reduced a m m o n i u m a n d increased nitrate. The t e m p e r a t u r e sensitivity of GDHase formation in these revertants m a d e it inappropriate to use the usual shaking incubator since this could not be conveniently m a i n t a i n e d below 28~ I n the case of R5, batches of R o u x bottles and F e r n b a c h flasks, each containing 150 ml of medium at a depth of a b o u t 1 cm, were used unshaken a t room t e m p e r a t u r e (up to 25~ Growth from h e a v y conidial inocula was continued for 56 to 65 h. The yield from each bottle or flask was a b o u t 5 g of d a m p (blotted) mycelium. I n the case of R1 a 10-1 New Brunswick bench-top fermenter (Microferm) was used with water cooling to m a i n t a i n the t e m p e r a t u r e at 23 to 25~ a n d with mechanical stirring a n d forced aeration. I n c u b a t i o n starting with a h e a v y conidal inoculum for 22 h yielded about 280 g of d a m p mycelium from each 10-1 batch. Mycelium was freeze-dried and stored at --20~ until use for GDHase preparation. (c) Measurement of GDHase activities in small scale cultures Mycclial pads, grown as described in section (b) above, were washed, blotted a n d ground in a m o r t a r with a roughly equal weight of glass beads and 5 vol. (made after grinding to 10 vol.) of 0-05 M-sodium phosphate (pH 7-4), with 0-001 M-ethylene diamine tetraacetate. Homogenates were centrifuged a t 15,000 revs/min for about 20 rain. Protein in the supern a t a n t s was measured b y the micro-Biuret method. E n z y m e activities were assayed at 35~ in the reductive amination system (system A) of Coddington et al. (1966) and specific activities were expressed in units of ~O.D.34o • 100/rag protein. (d) Purification of GDHase; isolation and characterization of tryptic peptides Purification of GDHase from am14 R1 a n d am14 R5 was b y the procedure of A s h b y et al. (1974). The R1 p r e p a r a t i o n s t a r t e d with 1200 g of d a m p mycelium (freeze-dried before extraction) and the yield of purified GDHase was 48 mg. I n the case of R5 the yield was 39 mg from 970 g of d a m p mycelium. S - c a r b o x y m e t h y l a t i o n a n d t r y p t i c digestion of the protein, column fractionation a n d p a p e r chromatographic characterization of peptides, and amino acid sequence determination were carried out b y the adaptions of essentially conventional methods described b y W o o t t o n et al. (1975). Amino acid analyses were performed using a R a n k - H i l g e r Chromaspek amino acid analyzer.

3. Results (a) Osmotic repair of growth G r o w t h o f am14 on l i q u i d or solid m i n i m a l m e d i u m was s t i m u l a t e d b y glycerol a t c o n c e n t r a t i o n s b e t w e e n 0.5 M a n d 1.5 M, t h e o p t i m u m being a b o u t 1.0 to 1.2 M. W i l d t y p e is p r o g r e s s i v e l y i n h i b i t e d b y g l y c e r o l o v e r this r a n g e o f c o n c e n t r a t i o n , a n d t h e g r o w t h o f am14 r e l a t i v e to wild t y p e i n c r e a s e d u p t o 1.5 M-glycerol, a t w h i c h concent r a t i o n t h e g r o w t h o f wild t y p e a n d m u t a n t were n e a r l y e q u i v a l e n t on a g a r m e d i u m . T h e s t i m u l a t o r y effect o f g l y c e r o l on am14 g r o w t h is n o t specific. A c o m p a r a b l e effect is s h o w n b y m a n n i t o l or KC1, b o t h in l i q u i d m e d i u m (Table 1) a n d on a g a r p l a t e s (Fig. 1) a n d o t h e r e x p e r i m e n t s , t h e results o f which a r e n o t d e t a i l e d here, h a v e d e m o n s t r a t e d t h a t glucose a n d s o r b i t o l a r e as effective as m a n n i t o l or e v e n m o r e so. E t h y l e n e glycol was also s o m e w h a t effective b u t was m o r e difficult t o

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J . R . S. F I N C H A M

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assess because of its strong inhibition of the wild type; we suspect an inhibitory impurity in the batch of ethylene glycol used. None of these solutes had any stimulatory effect on a second am m u t a n t (am1) which produces an unconditionally inactive GDHase which fails to bind coenzyme (J. C. Wootton, B. Ashby & M. Brett, unpublished results). The data of Table 1 suggest that aml4 is slightly "leakier" on unsupplemented minimal agar medium than am1, an effect which had not been previously recognized but which we have now confirmed in several experiments. (b) Glycerol repair of GDHase formation The effects of glycerol in the growth medium on the levels of GDHase activity were investigated for am14 and for two representative revertants, am14 R1 and am14 R5. In one experiment on am14 in comparison with wild type, cultures were grown both with and without a glutamate supplement to make growth independent of GDHase; in a second experiment which included R1 and R5 in the comparison and in which a range of temperatures were tested, DL-alanine (equivalent to glutamate in supporting growth of am mutants) was present in all media. The different cultures were not closely comparable within either experiment because of the different growth rates and times of incubation on the different media, but neverthless the main effects on levels of GDHase were clear. Table 2 summarizes the data which show that glycerol promotes GDHase formation in am14 whether glutamate is present or not, but that the effect is temperature-dependent, being negligible at 30~ or above and stronger at 20~ than at 25~ Table 2B also shows that GDHase formation in the two am14 revertants R1 and R5 is also markedly temperature-sensitive, little active enzyme being formed in either revertant at 30~ and still less at 37~ glycerol protects GDHase formation at the higher temperatures to some extent, especially in R1. In view of the effect of glycerol in the growth medium on GDHase formation in am14, the possible effect of including 1.0 H-glycerol in the extracting buffer was tested but no significant increase in the yield of GDHase in am14 extracts was obtained by this means. (c) Thermal stabilities of a m l 4 and revertant enzymes An experiment in which crude extracts in 0-07 M-sodium phosphate (pH 6.5) were heated at 57~ showed that all five revertants produced GDHase which was less stable than wild type. Whereas the wild-type enzyme lost no significant activity under these conditions the revertants fell clearly into two groups; the enzyme activity in R1 and R2 decayed with a half-life of 1.2 minutes and in R3, R4 and R5 with a half-life of 3-3 minutes. The results of a further experiment in which the stabilities of the R1 and R5 enzymes were compared with that of the GDHase formed in am14 grown with 1.25 M-glycerol are shown in Figure 2. The half-lives of the R1 and R5 enzymes were similar within experimental error to those found in the previous experiment, while the am14 enzyme was virtually completely inactivated within two minutes at 57~ Even at 50~ at p H 6.5, conditions under which the R1 enzyme is almost completely stable, the am14 GDHase decayed with a half-life of about five minutes. I t is clear that the different degrees of thermolability displayed b y the revertant enzymes represent partial repair of a much greater instability in the original mutant.

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Fro. 2. T h e r m a l i n a c t i v a t i o n o f G D H a s e in c r u d e e x t r a c t s o f wild t y p e , am14, a m 1 4 R l a n d amldR5. C u l t u r e s were g r o w n for e n z y m e e x t r a c t i o n as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s , am14 for 88 h a t 20~ w i t h 1.25 M-glycerol in t h e m e d i u m a n d t h e o t h e r s for 48 h a t 25~ o n p l a i n m i n i m a l m e d i u m . E x t r a c t s in 0"05 M - s o d i u m p h o s p h a t e + 0.001 M - E D T A ( p H 7.4) were a d j u s t e d to p H 6.5 t h r o u g h t h e a d d i t i o n of 0.83 vol. o f 0.1 M-NaH2PO4. P r o t e i n c o n c e n t r a t i o n s d u r i n g h e a t i n g were v e r y similar : 2'5, 2'8, 3'4 a n d 3.2 m g / m l for am14, R 1 , R 5 a n d wild t y p e , r e s p e c t i v e l y . S a m p l e s were d i s p e n s e d into s m a l l s i m i l a r t u b e s , p l a c e d t o g e t h e r in t h e w a t e r - b a t h a t t h e a p p r o p r i a t e t e m p e r a t u r e , a n d i n d i v i d u a l t u b e s were t r a n s f e r r e d to a n i c e - b a t h a t t h e t i m e s i n d i c a t e d for s u b s e q u e n t a s s a y .

Preliminary tests were carried out to determine whether the inclusion of 1.0 M-glycerol in the heating buffer would stabilize the wild type, am14 and R1 revertant GDHase varieties. The p H in this case was 7-4, and all enzyme varieties were considerably more stable t h a n t h e y were at p H 6.5 while maintaining the same stability relationship wild >> R1 >> am14. I t was clear t h a t glycerol had a stabilizing effect on the am14 and R1 enzymes (increased half-lives b y about three times at 54~ and 60~ respectively) but the wild-type enzyme at 60~ was significantly stabilized b y the glycerol also. A more detailed investigation would be required to make the case t h a t glycerol has a special effect in protecting am14 GDHase against heat inactivation which it does not exert on the wild-type enzyme. (d) Kinetic properties of the mutant GDHase varieties A series of determinations of approximate Miehaelis constants for all five substrates (glutamate, NADP, ~-oxoglutarate, N H + and NADPH2) were carried out on crude extracts of wild type, am14, am14 R1 and am14 R5. The results, which will not be reported in detail here, showed no clear differences between any of the m u t a n t s and the wild type. l~or was there a n y indication of pronounced activation effects or lags in reaction rates, such as are found with m u t a n t GDHase (such as t h a t of am3; Fineham, 1962) with disturbed allosteric properties.

(e) Analysis of amino acid replacements in R1 and R5 The glycerol-promoted GDHase of am14 itself is too unstable and produced in too small amounts to permit its purification. Attention was therefore concentrated on the enzymes produced b y the two representative revertants R1 and R5. The R5 GDHase, being the more stable of the two, was analyzed first.

MOLECULAR BASIS OF OSMOTIC R E P A R A B I L I T Y

635

The p a p e r chromatographic analysis of the tryptic peptides from S-carboxym e t h y l a t e d R5 enzyme, after a preliminary fractionation on AG50 X 4 resin, showed a conspicuous difference from the normal wild-type pattern: an absence of the tyrosine-positive spot corresponding to the standard peptide T2. This peptide has a relatively high RF and emerges fairly early from the AG50 • 4 column in the gradient of increasing pH. A candidate for a modified T2 was seen as a tyrosine-positive spot at an unusual position in the profile, considerably later off the column and with a lower R F value. Figure 3 shows the position on the chromatogram of this new peptide in relation to the normal position of T2. I t was purified b y a further fractionation on a column of Sephadex G25 and, on analysis, proved to have an amino acid composition similar to t h a t of T2 except t h a t it contained one equivalent of histidine and appeared to have only two equivalents of leucine instead of three. The peptide was sequenced b y the manual densyl-Edman method and gave a completely clear endgroup at each step. The second leucine of the normal T2 sequence (Leu20 in the whole chain) was replaced b y histidine. Table 3 summarizes the data. A similar chromatograph analysis of the tryptic peptides obtained from S-carboxym e t h y l a t e d R1 protein again failed to reveal a n y tyrosine-positive spot in the normal position of T2. Neither was there a n y sign of the same altered peptide as had been identified in the R5 digest. A search of the profile revealed a rather streaky tyrosinecontaining component emerging from the A G 5 0 • 2 column slightly later t h a n standard T2 and with a somewhat lower but not accurately determinable Rr.

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Unfortunately, this peptide was in precisely the same fractions as the well known standard peptide T13, and refractionation successively on Sephadex G25 and Biogel P4 failed to separate the two, though most other contaminating material was removed. Amino acid analysis suggested the presence of about 60 nmol of T13 and about 37 nmol of a peptide with a general resemblance to T2 but with a clear deficiency of leucine and too much tyrosine in relation to the phenylalanine. Table 4 shows the calculation. Rather than risk the small quantity of peptide in further fractionation by another ion exchange procedure, it was decided to sequence the putative altered T2(R1) in mixture with T13, the sequence of which was known. The results are shown in Table 5. The interpretation of the thin layers was complicated b y a very evident one-residue lagging in the degradation of T13, so that at each step two endgroups attributable to this peptide were seen. A third major spot, not as strong as the two from T13, was seen after each round and this corresponded in each case to the residue expected for T2 except that in the sixth position (i.e. after 5 rounds of degradation) tyrosine was seen instead of leucine. In order to confirm the tyrosine at position 6 in the altered peptide T2(R1), samples of the peptide mixture were taken for amino acid analysis after five and six rounds of degradation. The results of the analyses are shown in Table 6. I t was evident that T2(R1), the minority component at the start, had, for some reason, become even smaller in quantity relative to T13 after five rounds of the E d m a n procedure. The picture was also complicated by the evident persistence of the significant amount of peptide (some of it contaminant) which appeared to be resistant to degradation. These features did not, however, obscure the main result which was that three amino acids decreased significantly in quantity relative to leucine as a result of the sixth E d m a n degradation. Of these, aspartic acid and phenylalanine are the ones expected at the N-termini of the two versions of the lagging T13, while the third, tyrosine, confirms the conclusion that this is the residue present at position 6 of T2(R1).

4. Discussion We conclude from the tryptic peptide analysis of the two revertants am14 R1 and am14 R5 that residue 20 of the GDHase chain, normally leucine, has been replaced in each. In R5 the identification of histidine at residue 20 was completely clear. In R1, although the analysis was less clear because of the recovery of the relevant peptide in mixture with another known peptide and a minor amount of other contaminating material, we are satisfied that our several lines of evidence (composition, sequence analysis and residual analysis of the mixture before and after the critical E d m a n round) establish tyrosine with reasonable certainty as the replacing residue in this case. The finding that Leu20 is replaced in each of the two independent revertants and, more conclusively, the fact that the R1 replacement Leu --> T y r could not, on the basis of the normal coding rules, have taken place in only one mutational step, points to residue 20 as the one altered in the primary m u t a n t a m l 4 . This conclusion is in accord with the position of the am14 site in the genetic map (Smyth, 1973), to the left (N-terminal side) of all other sites so far identified with amino acid changes (Brett et al., 1976). I t is strengthened by our recovery from the R5 tryptic digest of peptides (with the exception of T2) representing the entire normal amino acid analysis up to residue 92, and again from residue 102 to 121; T11, covering residues 93 to 101,

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J. R. S. F I N C H A M

AND

A. J. B A R O N

was seen in its normal position on the chromatogram but was lost during later fractionation. I t thus seems very unlikely that there is, either in R5 or, by implication, in am14 itself, any amino acid replacement other than at residue 20 in the N-terminal 121 residues of GDHase; from the genetic mapping data it is extremely unlikely that the am14 replacement falls outside this region. Examination of the genetic code shows, as mentioned above, that L e u - + T y r requires two mutational steps. Furthermore, the only possible two-step route, which will also account for His in R5, is a "knight's move" via His. In terms of RNA codons the two successive changes would be CU~ (Leu) --> CA~ (His) --> UA~ (Tyr). It thus appears that am14 must be the same as R5 in having histidine at residue 20, and t h a t whereas in R1 the partial restoration of normal GDHase properties has been brought about by a second change in the same codon, in R5 it must be due to a compensating replacement elsewhere in the polypeptide chain. This postulated second-residue replacement is still being sought; as noted above it does not appear to be within 100 residues of the primary replacement. Two previous examples of distant secondresidue compensating mutations have been described for the Neurospora GDHase system (Brett et al., 1976). All the known properties of a m l g GDHase, and of the partially normalized enzymes in the revertants, are consistent with the conclusion that the effect of the replacement of Leu20 is to destabihze the quaternary structure of the enzyme. This is most strongly suggested by the experiment of Sundaram & Fincham (1968) which showed that dissociation and reassociation of the complementation product formed in a heterokaryon between am14 and am3 led to apparently complete loss of the am14 component and formation of pure am3 mutant enzyme. Furthermore, an unpublished experiment shows that am14 GDHase formed as a result of osmotic repair is completely inactivated (presumably denatured) by the same procedure (freezing and thawing with 0.1 M-NaC1) as brings about efficient hybrid formation with wild type and other mutant GHDase varieties. So far as we have determined, am14 GHDase seems to be quite normal in its enzyme properties so long as it can be stabilized. This is in line with its ability to complement any other m u t a n t which can supply a stable quaternary structure. Hexamers containing am14 monomers are, however, not completely normal in stabihty, as shown by the "negative complementation" shown by am14 and wild type (Sundaram & Fincham, 1968). It is likely that the case of m u t a n t enzyme repair described in this paper is an example of a rather general mechanism. "Osmotically remedial" mutants, which we prefer to call osmotically reparable, are rather common in yeast and respond to a very similar range of solutes, over the same range of concentrations, as we found to be effective for am14 (Hawthorne & Friis, 1964). In Neurosl~ora, although such mutants have not been systematically looked for, at least one other m u t a n t with very similar repair requirements is known (Metzenberg, 1968). The association of osmotic reparabihty with temperature sensitivity, noted in this study, is common in the yeast mutants. The enzymic basis of the phenotype has not been worked out in any of these other cases, but they all resemble the one described here in that virtually any non-toxic solute of sufficient solubihty can effect the repair. We suggest t h a t a common factor is involved in all these cases and that this factor is water activity. Activating effects of the solute molecules per se seem very unlikely in view of their diverse nature; KCI and various non-electrolytes are about equally effective at the same osmolality. We do not know the intracellular concentrations achieved b y

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glycerol and the other solutes used in our experiments. They all appear to penetrate the plasma membrane fairly readily since plasmolysis, ff it occurs at all, is followed b y recovery and good, though somewhat inhibited growth. Glycerol and glucose can both be used as carbon sources, while both glucose (Scarborough, 1970) and potassium ion (Slayman, 1970) are known to be taken up by hyphae b y specific transport systems. Whether the concentration of solute within the hyphae comes to equal t h a t in the growth medium or not we would expect water to be equilibrated across the plasma membrane, and the water activity within the cells to equal that outside. In the ease reported here, a possible effect of water activity is suggested b y the nature of the amino acid replacement in the m u t a n t enzyme. I f Leu20 is at or close to a point of hydrophobic contact between adjoining monomers in the hexameric enzyme its replacement b y the polar histidine residue is likely to have a markedly destabilizing effect. The histidine will not necessarily be ionized in the "closed" hexamer but it would be much more prone than leucine to associate with water molecules and thus to promote the unfolding and/or dissociation which would allow this association to occur. Any reduction in the availability of water molecules would, on this view, reduce the tendency to dissociate. Given the co-operative nature of oligomerization (especially so if the oligomer is a hexamer as in our case) it would not be surprising if a large change in the yield of oligomers were to occur over a narrow range of water activity. I t is perhaps harder to accept that this critical range is commonly as high as 0-97 to 0-98 (corresponding to solute molalilities of 1.0 to 1.5), The only previous investigators of the effects of water activity, as such, on Neurospora were Charlang & Horowitz (1971) who found that water activities in the range 0.92 to 0-94 caused loss from conidia of factors necessary for germination (later identified as siderochromes: Horowitz et al., 1976). The effect on conidia was attributed to membrane damage, but the relatively low water activities responsible for this did not prevent growth completely, though they did very greatly inhibit it. Thus the repair of the m u t a n t GHDase in the am14 strain appears to be brought about b y reductions in water activity which are small compared with those which are needed to cause extensive damage to other cell components. An implication of our hypothesis is t h a t am14 is representative of a class of mutants in which normal hydrophobic interactions are weakened b y single amino acid replacements, but that this weakening is not usually so great that it cannot be rather readily repaired. In the case of am14 the marginal nature of the defect is suggested by the slight "leakiness" of the phenotype, and the repair is by no means complete. I t m a y be true rather generally t h a t the stability of oligomeric proteins depends on multiple contacts between monomers and t h a t a single amino acid replacement, even one of a rather drastic character, is seldom sufficient to cause extreme and irreparable disruption. In the present study we were not able to make any experimental distinction between the conditions necessary for the initial assembly of the nascent monomer into oligomers and those necessary for the stability of the ohgomer once formed. The distinction m a y nevertheless be an important one to make; we suspect that it is the assembly of the oligomer rather than its stability once assembled which is the more critically affected by water activity and temperature. We acknowledge the contribution of Mr Alan Boyd who made the initial observations on the osmotic repair of a m l d as part of an undergraduate project in the Department of

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Biochemistry, University of Leeds. W e also t h a n k Mrs Margaret Burnley for all the amino acid analyses. The greater p a r t of the work was supported b y a g r a n t from the Science Research Council. A few experiments were carried out in the Division of Biology, California I n s t i t u t e of Technology, a n d the senior author (J. R. S. F.) is indebted to D r N. H. Horowitz for the use of his laboratory. REFERENCES Ashby, B., Wootton, J. C. & F i n c h a m , J. R. S. (1974). Biochem. J . 143, 317-329. Brett, M., Chambers, G. K., Holder, A. A., Wootton, J. C. & Fincham, J. R. S. (1976). J. Mol. Biol. 106, 1-22. Charlang, G. W. & Horowitz, N. H. (1971). Proc. Nat. A c ~ , Sci., U.S.A. 68, 260-262. Coddington, A., Fineham, J. R. S. & Sundaram, T. K. (1966). J. Mol. Biol. 17, 503-512. Fincham, J. R. S. (1950). J . Biol. Chem. 182, 61-73. Fincham, J. R. S. (1962). J . Mol. Biol. 4, 257-274. Fincham, J. R. S. & Stadler, D. R. (1964). Genet. Res. Camb. 6, 121-129. Hawthorne, D. C. & Friis, J. (1964). Genetics, 56, 829-839. Holder, A. A., Wootton, J. C., Baron, A. J., Chambers, G. K. & Fincham, J. R. S. (1975). Biochem. J. 149, 757-773. Horowitz, N. H., Charlang, G., Horn, G. & Williams, N. P. (1976). J. Bact~riol. 127, 135-148. Metzenberg, R. L. (1968). Arch. Biochem. Biophys. 125, 532--541. Roberts, D. B. (1971}. J. Gen. Microbiol. 69, 143-144. Scarborough, G. A. (1970). J. Biol. Chem. 245, 1694-1698. Slayman, C. W . (1970}. Biochim. Biophys. Acta, 211, 502-512. Smyth, D. R. (1973). Austr. J. Biol. ,.~ci. 26, 1355-1370. Stadler, D. R. (1966}. Genet. Res. Camb. 7, 18-31. Sundaram, T. K. & Fincham, J. R. S. (1968). J. Bacteriol. 95, 787-792. Vogel, H. J. (1956). Miorob. Genet. Bull. 13, 42. Wootton, J. C., Taylor, J. G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975). Biochem. J. 149, 739-748.