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Partial Correction of Structural Defects in Alcohol Dehydrogenase Through Interallelic Complementation in Drosophila melanogaster


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Partial Correction of Structural Defects in Alcohol Dehydrogenase Through

Interallelic Complementation in



Hope Hollocher' and Allen R. Place'

Department of Biology, Leidy Laboratories, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Manuscript received October 16, 1986

Revised copy accepted March 10, 1987


Alcohol dehydrogenases (ADH) from the F1 progeny of all pairwise crosses between 12 null-activity

mutants and crosses between these mutants and four active variants, ADH"' ADHF, ADHD and ADHS,

were analyzed for the presence of active or inactive heterodimers. Gels were stained for ADH enzyme

activity, and protein blots of duplicate gels were probed with ADH-specific antibody to detect cross-

reacting material. Crosses between the three major electrophoretic variants. ADHF, ADHS and ADHD,

all produced active heterodimers. Four mutant proteins (ADH"', ADHn4, ADH"" and ADH"") did

not form heterodimers with any other ADH subunit tested. Of the 28 crosses involving the remaining

null activity mutants, 22 produce heterodimers. Twelve of these exhibit partial restoration of enzyme

activity. In five cases of active heterodimers from null-activity crosses, Adh"" supplied one of the

subunits. In two crosses involving the active variant ADHD, the null activity mutant subunits (ADH"'

and ADH"') destabilized the heterodimer sufficiently to cause inactivation of the ADHD subunit. In

the cross between AdhF and Adh"', the activity of the ADHF subunit was also greatly reduced in

association with the ADH"' subunit. Two crosses (Adh"' X Adh"" and Adhn5 X Adh"12) result in partial restoration of one of the homodimeric proteins (ADH"' and ADH"", respectively), as well as forming active heterodimers.

NTERALLELIC complementation is the complete


or partial restoration of enzyme activity through

the noncovalent interaction of defective subunits in a

multisubunit protein (ZABIN and VILLAJERO 1975;

ZABIN 1982). It is a phenomenon that allows for new protein phenotypes to result from protein/protein

interactions, and does not require any alteration or

recombination of the defective genes. It is also a

phenomenon that, despite extensive research (FIN-

CHAM 1966,1977; ZABIN and VILLAREJO 1975; ZABIN 1982), is not well understood.

CRICK and ORCEL (1 964) proposed that partial en-

zyme activity is regained by correcting the misfolding

of a mutant subunit through association with a "com- plementing'' subunit which is usually defective in some

other manner. SCHWARTZ (1975), on the other hand,

argued that, ". . . the phenomenon results from insta-

bility o r abnormal maturation of the mutant homodi-

mer rather than correction of configurational defects

in mutant heterodimers."

Originally a tool of the geneticist for studying the nature of a gene and its product, interallelic comple- mentation has begun to receive interest from investi- gators of protein structure and folding. When the X- ray crystallographic structure of an enzyme is available and the exact amino acid substitution in the defective

' Present address: Department of Biology, Washington University, St.

* To whom all correspondence should be addressed. Louis, Missouri 6 3 130.

Genetics 116: 265-274 (June, 1987)

protein is known, then structural mechanisms respon- sible for the repair can be inferred from the interal-

lelic complementation data (SCHACHMAN et al. 1984).

If detailed information o n the protein structure is not

available, as it is not available for Drosophila ADH, the pattern of interallelic complementation is useful for grouping the mutant proteins according to shared

structural properties (SCHWARTZ and SOFER 1976b).

T w o mutant protein subunits can also interact in manners that d o not result in complementation. T h e two subunits may become unstable in combination and not be able to form heterodimers (no heterodimer formation), o r the mutant subunits may form a het- erodimer yet remain inactive (neutral heterodimer formation). In a cross between an active allele and an inactive allele, the resulting heterodimer also may be inactive. In this case, the active subunit has become deactivated in combination with the inactive subunit (negative complementation).

Complementation at the Drosophila Adh locus has

been investigated previously (GRELL, JACKSON and


CIA and COOPER 1984). T o add to the existing studies,

we used the information obtained from our earlier characterization of the inactive proteins (HOLLOCHER

and PLACE 1987) to predict the locations of the het-

erodimers o n polyacrylamide isoelectric focusing gels, which are more sensitive to charge differences than



above. In addition to staining the gels to detect cata-

lytically active heterodimers, we investigated other

types of subunit interaction by using protein blotting

and an immuno-detection procedure to identify inac-

tive heterodimers, and to ensure that the activity seen

upon staining the gels could be directly attributed to

ADH cross-reacting material (CRM). By this means,

we have guarded against attributing nonspecific stain- ing t o the action of heterodimers and have distin-

guished subunits that do not dimerize from those

subunits that dimerize yet remain inactive. I n the case of interactions between inactive and active subunits, we have also detected those subunits that dimerize yet diminish the catalytic ability of a previously active subunit.

O u r findings corroborate and extend those of the

earlier investigations. We find, in addition, some un-

expected results, such as partial restoration of activity in a previously inactive homodimer and inactivation of a fully active subunit in the heterodimer. We discuss the behavior of these ADH phenotypes in light of the

recently described phenomenon of “conformational

drift” (WEBER 1986). We also address whether our data helps distinguish between the contrasting expla- nations given above for the structural basis of inter- allelic complementation.


Drosophila strains: See LINDSLEY and GRELL (1 967) for a more complete description of these second chromosome mutants.

AdhS carries the electrophoretic “slow” variant of ADH in the Schenk Forest strain.

w;AdhF (WEP) carries the X-linked gene white eyes and the naturally occurring electrophoretic “fast” protein var-

iant of ADH. The ADH of AdhF differs from that of AdhS

by a threonine amino acid change at residue 192 (THATCHER

AdhD pr cn carries an electrophoretic variant of ADH that migrates slightly faster than ADHF. It is an active variant

derived from the Samarkand stock of AdhF by EMS muta-

genesis (GRELL, JACOBSON and MURPHY 1968). AdhD differs

from AdhF by a glycine to glutamate amino change at residue

232 (SCHWARTZ andJORNvALL 1976).

Adh”’ through Adhn3 are three null activity ADH strains

generated by GRELL, JACOBSON, and MURPHY (1968) from

the Canton S AdhS strain.

Adhn4 and are two strains derived from AdhD

(GRELL, JACOFSON and MURPHY 1968). Adhn4 does not pro-

duce an active ADH protein (SCHWARTZ and SOFER 1976b).

Adh”’ is a temperature sensitive strain which produces ADH with residual activity under the conditions used for these

experiments (VIGUE and SOFER 1974).

are inactive ADH strains generated in AdhF b cn ug and selected using the pentenol procedure

(GERACE and SOFER 1972).

Flies were cultured at 22” on Drosophila Medium, Blue 4-24. Flies were aged 4-10 days prior to being quick-frozen

in liquid N2 and stored at -70”.

Antibody production: Goat anti-ADH antibody was pro- duced by immunizing a young female goat according to

SPIELMAN, ERICKSON and EPSTEIN (1 974). The goat antisera


Adhn6 through

was purified by ammonium sulfate fractionation and antigen

affinity chromatography as described by PELLICCIA and SO-

FER (1982). The anti-ADH antibody constituted approxi-

mately 1 % of the IgG fraction.

Protein extraction: Soluble protein was extracted by

homogenizing flies in 0.02 M Na-phosphate buffer (pH 7.5)

with 1 mM EDTA, 5 mM 0-mercaptoethanol, and 15% (v/

v) glycerol using a motorized glass pestle designed to fit

microcentrifuge tubes (1.5 ml). The crude homogenate was filtered by centrifugation through a glass-fiber filter. Native

proteins were extracted on ice. Typically, 0.1 to 0.5 fly

equivalents were analyzed. Each sample was then serially diluted threefold for internal standardization of the band intensities recorded from the polyacrylamide gels and nitro-

cellulose transfers (KLEBE 1975).

Gel method: Horizontal native polyacrylamide gel isoe- lectric focusing (PAGIEF) was performed using the Phar- macia Fine Chemical Co. Flatbed apparatus FBE 3000. The

gels (75 mm X 230 mm) consisted of 5.0% acrylamide, 0.3%

bis-acrylamide, 13.3% glycerol, 1 % ampholytes (a 2: 1 mix-

ture of pH range 3-9.5 and pH range 5-8), 0.0152% ammonium persulfate and 0.05% TEMED. Prefocusing was carried out at 10 watts (constant wattage) for 1 hr. Samples were applied to the surface of the gel and focused for an additional hour at 10 watts (constant wattage). The gel temperature was regulated by using a water bath set at 4” to circulate through the cooling plate under the gel. Samples were loaded in duplicate sets with half the gel designated to be stained for activity and the other half to be transferred onto nitrocellulose for antibody probing.

Activity detection: ADH activity was detected by staining in 0.02 M Na-phosphate buffer (pH 7.5), containing 0.18%

NAD, 0.1% (w/v) nitro blue tetrazolium, 0.004% (w/v)

phenazine methosulfate, and 3.6% (v/v) 2-butanol at 27”

for 50 min. After staining, the gels were fixed in 7% acetic acid overnight, and then air-dried on GelBond away from direct light. The linear range of formazan deposition was

between 0.0 to 0.12 enzyme units.

Protein blotting: The GD4 Destainer apparatus and the Destainer power supply by Pharmacia were used to perform

protein blotting as described by TOWBIN, STAEHELIN and


(1980) using 0.375 M Tris-HC1 (pH 8.8). The proteins were

transfered at 12 V onto nitrocellulose paper (0.45 pm pore

size) from Schliecher and Schuell. Whatman 3 M M chro-

matography paper was used to ensure complete contact between the gel and the nitrocellulose.

Immunodetection: Protein blots were probed using the

general procedure described by TOWBIN, STAEHELIN and

GORDON (1 979). Specifically, after transfer, the filters were

incubated in 3% (v/v) BSA in 0.01 M Tris-HC1 (pH 7.6),

0.9% (w/v) NaCl and 0.01 % (w/v) NaNs for a minimum of

1 hr. The filters were washed with Tris-saline buffer (de- scribed above) for 30 min and then incubated in 10 ml of

1.5% (w/v) BSA containing 10% (v/v) horse serum and 1.4

pg/ml of goat anti-ADH antibody for 3 hr. After rinsing for

30 min, the sheets were incubated in 10 ml of 2 pg/ml of

rabbit anti-goat horseradish peroxidase conjugated antibody for 3 hr. Peroxidase-mediated deposition of 3,3 ’-diamino- benzidine tetrahydrochloride (DAB) was performed in 0.15

M phosphate-citrate buffer (pH 5.0) containing 0.04% (v/v)

H40P and 0.2% (w/v) DAB. To intensify the color of the precipitate, 3 mg of nickel sulfate and 3 mg of cobalt

chloride were added to each 25 ml of incubation solution

(DEBLAS and CHERWINSKI (1983). The reaction was stopped

by rinsing thoroughly in distilled-deionized H20. Nitrocel- lulose sheets were air-dried away from direct light.


paratus) was used to quantitate band intensities. The dried

gels stained for enzyme activity were scanned directly from

the GelBond. The nitrocellulose sheets probed for cross

reacting material were first clarified using microscopic cedar

oil (optical density 1.515) and then placed between two

pieces of GelBond before scanning. T h e chart speed of the

recorder was set to match the speed of the scan at 0.5 mm/

sec. The areas under the traced peaks were measured to the

nearest 0.1 mm using a digitizing tablet (GTCO bitpad).

Materials: Bis-acrylamide, B-mercaptoethanol, TEMED, DAB, rabbit anti-goat peroxidase conjugated IgG, 30%

hydrogen peroxide, nitro blue tetrazolium, phenazine meth-

osulfate, NAD+, and Amberlite MB-I were purchased from

Sigma Chemical Co. Acrylamide of 99.9% purity was pur-

chased from Bio-Rad. Carrier ampholytes for PAGIEF were

the Ampholine brand from LKB. Nitrocellulose and What-

man 3 MM paper were from Schliecher and Schuell. Gel- Bond was obtained from the FMC Corporation. Microscopic cedar oil was from Fritzche Brothers, Inc. Drosophila melan- ogaster stocks were supplied by WILLIAM SOFER.


For a dimeric protein, like the Drosophila ADH, three distinct forms, two homodimers and a single heterodimer, are expected in extracts from a hetero- zygous individual. However, because the Drosophila

ADH has the capacity to form adduct-bound isozymes

in vitro as well as in vivo (JOHNSON and DENNISTON 1964; URSPRUNC and LEONE 1965; JACOBSON, MUR-

PHY and HARTMANN 1970; JACOBSON et al. 1972;

KNOPP and JACOBSON 1972; SCHWARTZ et al. 1975;


and SOFER 1979; PAPEL et al. 1979; WINBERC,

THATCHER and MCKINLEY-MCKEE 1983), each dimer

can exist in two additional states (SCHWARTZ, O’DON-

NELL and SOFER 1979), the singly and doubly bound adduct isozymes. Each differs from the parent dimeric

form by having a lower isoelectric point (PI) and a

lower specific activity. T h e average decrement in PI is 0.64 & 0.13 unit (HOLLOCHER and PLACE 1987).

Hence, a heterozygous individual has the potential of

exhibiting nine electrophoretic forms, although only seven and sometimes five isozymes are realized be- cause of overlap with other bands and a low abun- dance of the doubly bound adduct isozyme

(SCHWARTZ et al. 1975).3 T h e banding protein is

simplified further when inactive subunits are involved since the adduct isozymes are only formed by active

enzymes (HOLLOCHER and PLACE 1987). A three-

banded pattern is seen when all three proteins (the two homodimers and the heterodimer) are inactive and each have different charges.

A nitrocellulose transfer of an isoelectric focusing polyacrylamide gel in which protein extracts from heterozygous individuals are separated is shown in

The Drosophila ADH is also prone to spontaneous deamidation and, thus, to give rise to further intermediate zones of activity (WINDBERG, THATCHER and MCKINLEY-MCKEE 1983). Deamidation of one amino acid in a protein lowers the PI by 0.1-0.2 pH unit (DICE and GOLDBERG 1975). We do not observe these products in fresh extracts but only on stored samples.

Figure 1 . Protein is visualized using a peroxidase

based antibody stain specific for Drosophila ADH. In

the AdhF X Adh”’ extract one observes seven bands

(ADHn5, a temperature sensitive allele, is active at the

temperature these gels were run). T h e protein with

the highest PI (i.e. closest to cathode) is the ADHF

homodimer. T h e heterodimer (ADHF/ADHn5) is

found next, followed by the singly-bound adduct form

of the ADHF homodimer. T h e faint band found next in the sequence is the homodimer of ADHn5. [Even at the permissive temperature the steady state level of

ADHn5 is only 5% of the steady-state level of ADHF

(PELLICCIA and SOFER 1982).] These assignments were

determined by running extracts of homozygous flies involved in the cross in adjacent wells during the initial screening. In all cases, the heterodimer appeared as a band of intermediate charge between the two homo- dimers.

T o estimate the distribution of protein (homodi- mers versus heterodimer) on such transfers, we at- tempted to quantitate the peroxidase deposited stain o n the nitrocellulose and calculate a ratio of relative peak areas. Ratios were obtained by integrating the areas under the peaks traced by a gel scanner. Three-

fold dilutions of each sample were measured to ensure

a linear proportional response (KLEBE 1975) which

for the peroxidase-deposited stain corresponded to a

range of 10-100 ng. We also attempted to obtain

relative enzymatic activities by a similar quantitation

of the deposited formazan stain on the isoelectric focusing gel. T h e linear range of formazan deposition

corresponded to 0.0-0.12 international enzyme unit.

We view both of these estimates as being semiquan- titative for several reasons. First, because of the ad- duct-bound isozymes, many bands represent more than one electromorph. Moreover, the amount of adduct-bound isozyme varied with each variant and

preparation examined. Second, it was very difficult to

obtain day to day consistent deposition of stain. Day

to day variations in staining intensity for replicates are

typically 25% or greater, while replicate samples on

the same transfer have variations of less than 20%.

We have adopted a relative ratio approach to over- come this difficulty. Each gel and nitrocellulose trans- fer acts as its own control and experiment. A further

difficulty involves the low in vivo stability of the mu-

tants; we are unsure how much degradation o r alter- ation occurred during the preparative stages of the work. Despite these caveats, however, we believe the data are internally consistent and contain useful infor-

mation which are summarized in Tables 1 and 2.

If we assume random assortment of subunits and,

thus, compliance to the expected 1 :2: 1 homodi-

mer:heterodimer:homodimer ratios, we find in the

majority of cases the data fit the expected distribution




- - ~ -.- .. . .~

' AdhD/Adhnl2'' Adh?Adhn3 ' I AdhF/Adhn5'


'Adhn5/Adhn7 "Adhn5/Adhn71"Adhn5/Adhn1~

FIGURE 1 .-Nitrocellulose transfers of isoelectric focusing polyacrylamide gels. Alcohol dehydrogenases in extracts of heterozygotes of Adh-negatives with Adh-positive electrophoretic variants were separated on a pH 3.5-10.0 gradient. Protein blots were probed for ADH cross-reacting material using the procedure described by TOWBIN, STAEHELIN and COR" (1979) modified as described in MATERIALS AND

METHODS. Threefold serial dilutions (increasing dilution from left to right) of each extract were loaded. T h e open triangles mark the location of one homodimer while the tilled triangles mark the location of the other homodimer. Except for the sample from the Adh' X Adh"' cross,

the heterodimer is the only band found between the two homodimers.


Relative protein m m and enzyme activity d i ~ t ~ i b ~ t i ~ ~ in Adh heterozygotes d t h g from mosses between active variants and null activity variants

ADH protein ratio ADH activity ratio Heterodimer

CrosS homodimer:heterodimer:homodimer homodimer:heterodimer:homodimer specific activity SE^ Adh' X Adh"'

Adh' X Adh"'

Adh' X Adh" Adh' X Adh"' Adh" X Adh"' Adh" X Adh" Adh" X Adhw7 Adh" X Adh* Adh" X Adh"' AdhD X Adh"" Adh" X Adh"" Adh' X Adh"' Adh' X Adh"'

Adh' X Adh* Adh' X Adhn7 Adh' X Adhd Adh' X Adh"' Adh' X Adh"" Adh' X Adh"" Adh"' X Adh"' Adh" X Adh"' Adh"' X Adh*

Adh"' X Adhn7 Adh"' X Adhd Adh"' X Adh" Adh"'X Adh""

Adh"' X Adh""

1.0 0.12 f 0.04 0.14 f 0.04

1.0 0.80 f 0.07 0.30 f 0.10

1.0 1.57 f 0.37 0.63 f 0.12

1.0 2.11 fO.ll 1.29f0.06

1.0 1.57 0.50

1.0 0.32 0.62

1 .O 1.02 f 0.20 0.80 f 0.07

1.0 1.24 0.65

1.0 0.23 f 0.08 0.56 f 0.09

(ADH"" homodimer and heterodimer overlap)

1.0 1.59 f 0.45 0.83 f 0.27

1.0 1.66 f 0.30 1.23 f 0.13

1.0 1.54 f 0.04' 0.98 f 0.10

1.0 1.20 f 0.05 1.10 f 0.26'

1.0 2.76 f 0.16' 1.05 f 0.1 1

1.0 1.78 f 0.14 0.89 f 0.04

1 .O 0.30 f 0.02 0.65 f 0.02'

1.0 2.60 f 0.2Y 2.22

1.0 1.32 1.49'

1.0 1.68 f 0.36 0.61 f 0.09

1 .O 0.15 f 0.002 0.86 f 0.06

1.0 1.37 1.58

1 .O 2.28 f 0.34 3.13 f 0.78

1.0 2.40 f 0.64 2.92 f 0.18

1.0 C0.05 1.85

(ADH"" homodimer and heterodimer overlap)

1.0 1.93 f 0.10 3.83 f 0.94





1 .o 1 .o 1 .o 1 .o

1 .o 1 .o 1 .o 1 .o 1 .o



1 .o 1 .o 1 .o

1 .o

1 .o 1 .o 1 .o 1 .o

1 .o

1 .o 1 .o

(heterodimer unstable, activity < 10% of ADH?

0.21 f 0.18 0.14 f 0.08 0.27 f 0.08

[0.47 f 0.1 11"

0.90 f 0.32 0.57 f 0.24

1.15 f 0.18 0.54 f 0.09

0.72 f 0.08 0.45 f 0.08

0.13 0.41 f 0.15

0.51 f 0.13 0.50 f 0.16

0.22 f 0.05 0.96 f 0.39

1.54 f 0.17

0.59 f 0.14 0.37 f 0.14

1.09 f 0.30 0.65 f 0.21

0.87 f 0.06' 0.48 f 0.10 0.56 f 0.04

[0.49 f 0.1 1


0.53 f 0.10 0.64 f 0.12

3.09 f 0.5T 1.12 f 0.21'

0.52 f 0.1 1

0.93 f 0.19

0.24 0.80 f 0.33

2.82 f 0.79' 1.08 f 0.33'

0.77 f 0.01 0.58 f 0.17

0.53 f 0.1 1 0.31 f 0.09

2.60 f 0.47

0.39 f 0.07

0.67 f 0.08 0.25 f 0.18

0.56 0.23 f 0.09

4.89 f 0.46

0.73 f 0.19 2.39 f 0.54 0.37 f 0.10

(heterodimer activity < 5% of Adh")

(inactive heterodimer)

(inactive heterodimer)

r0.62 f 0.21p

' T h e standard error of this estimate is: 4 = A/E[a2/A2


b 2 / p ] " where A is the measured enzymatic activity, E the protein concentrations


Relative specific activity of null variant homodimer.

' Fstimates include adduct-bound isozyme.



Relative protein mass and enzyme activity distributions in Adh heterozygotes resulting from crosses between null variants

Cross homodimer:heterdimer:homdimer ADH protein ratio Heterdimer activity

Adh"' X Adhn3 1 .o 0.81 1.32


Adh"' X A d V 7 1 .o 1.58


0.21 1.84 Jr 0.21


Adh"' X Adh"" 1 .o 3.05 3.08 1.52 f 0.19'(0.50



Adh"' X Adhn7 1 .o 1.63


0.05 1.89




Adh"' X Adh"' 1 .o 1.76 1.84


Adh"' X Adhn9 1 .o 1.64 1.46


Adh"' X Adh"" 1 .o 3.21 3.56


Adh"' X Adh"I2 1 .o 2.99 f 0.32 3.95 f 0.80


Adhn6 X Adh"" 1 .o 1.73 f 0.19 1.29 f 0.003


A d V 6 X Adhn7 1 .o 1.26 1.16


A d V 7 X Adhug 1 .o 1.43 1.53


Adhn7 X Adh"" (bands overlap)


Adhn7 X Adh"12 1 .o 1.20 f 0.10 1.38 f 0.21


Adh" X Adh"" 1 .o 1.42 2 0.38 1.09 f 0.14


Adha9 X Adh"" 1 .o 0.80 1.08


Adh"I2 X Adh"" 1 .o 1.18 f 0.01 1.11 f 0.14


We have not attempted to quantitate single activity bands. Peaks above background (approximately 5% wild type or 0.01 enzyme units) are registered as plus (+). In the crosses Adh"' X Adh"", Adhn6 X Adh"" and Adhng X Adh"" quantitation was not possible because the homodimers have identical pLs. We have no quantitative data for crosses Adh"' X Adh* and Adhn7 X Adh". However, both produce an inactive heterodimer.


There is restoration of ADH"' homodimer activity in this cross. The activity of the heterodimer is normalized to that of the ADH"' homodimer.

' Relative specific activity of heterodimer.

one should not expect compliance for proteins of

dissimilar in vivo stability. It has been shown that each

of the null variants when homozygous has a lower in

vivo steady-state level of ADH protein (PELLICCIA and SOFER 1982). Furthermore, this lower level of ADH has been shown to reside in a higher degradative rate

and not differences in rates of synthesis (PELLICCIA

and SOFER 1982). Hence, we view the protein distri-

butions in Table 1 and 2 as reflecting the steady-state

stability of each protomer of ADH in a heterozygous

individual. Deviation from the expected ratio is most apparent when variants with protein species of strik-

ingly different in vivo steady state levels, as determined

by PELLICCIA and SOFER (1982), are crossed. In these instances the heterodimer stability is usually not inter- mediate and resembles one or the other of the paren-

tal types. In only one case, AdhS X Adh"', do we find

the heterodimer protein level to be higher than ex- pected; even in this case the value for the heterodimer is suspect because of the single adduct of ADH'.

If we compare the relative protein ratios of the homodimers in each heterozygous cross with the ex-

pected relative steady state levels of these proteins

(PELLICCIA and SOFER 1982) in homozygotes, a rea- sonably close concordance is found. T h e rank order

of the steady state levels found by PELLICCIA and

























We find, for example, in crosses involving ADHF a

rank order of:








with relative levels of 1.00, 0.6, 0.30 and 0.14 as

compared to 1.00, 0.54, 0.05 and 0.15 for ADHF,

ADH"', ADH"' and ADH"', respectively (PELLICCIA

and SOFER 1982). We attributed the higher ADH"'

value in our study to a lower temperature of fly

rearing. If we examine crosses involving ADH"' we

find a rank order in steady state levels of: ADH""








ADH"'> ADH"'





which again is consistent with PELLICCIA and SOFER

(1982). If we combine all our data and establish a consensus rank order we find:



ADH"', ADHn7




ADH"', ADH"'






ADH"', ADH""

which, except for two discrepancies, agrees with PEL-

LICCIA and SOFER (1982). In all our crosses we find ADH"" to have higher steady state levels (approxi-

mately wild type) while ADH"' appears to have lower

steady state levels (-30%). T h e reason for this dis-

crepancy is not known.




Relative protein mass and enzyme activity distributions in AdhF/AdhD heterozygotes

Apoenzyme Single adduct


Protein 1


1.49 & 0.06 1.26 & 0.15 0.82 1.12 1.16

Activity 1


1.06 & 0.05 0.68 f 0.27 0.29 & 0.15 0.41 & 0.15 0.32 & 0.2

70% of the cases) that the heterodimer has half the relative specific activity of the active homodimer. We would classify these heterozygotes as demonstrating

neutral complementation. Only in four cases (AdhD X

Adh"'; AdhS X Adhn7; AdhS X Adh""; Adhn5 X Adhn3)

d o we have sufficient precision in our data to argue that the heterodimer's specific activity is equivalent o r greater than the active homodimer. Although for

both Adhn5 X Adhn3 and AdhD X Adh"' very little stable

heterodimer is formed. Moreover, in the crosses in-

volving Adh", the data is suspect because the hetero-

dimer co-migrates with the single adduct for ADH'.

In crosses between AdhF, AdhS and AdhD, only with

the AdhF X AdhD cross were we able to resolve all the

enzymatic forms of ADH (Table 3). T h e double ad-

duct isozymes were too low in abundance to quanti-

tate. T h e lower specific activity of the ADHD homo-

dimer, as well as that for the adduct bound isozymes

is consistent with previous work (VIGUE and SOFER

1974; SCHWARTZ, O'DONNELL and SOFER 1979). In a

AdhS X AdhF cross the protein distribution was

1.0:1.94 k 0.46:2.14 & 0.60 (normalized to the ADH'

homodimer). T h e ADHF homodimer value is suspect

because of comigration with the ADH' single adduct

isozyme. In a AdhS X AdhD fly, the heterodimer value

(1.0:2.18:2.10, again normalized to the ADH' homo-

dimer) is questionable because of comigration with the ADHS single adduct isozyme. Again the higher

steady state level of ADHF and ADHD relative to

ADHS is consistent with previous work (VIGUE and SOFER 1974; ANDERSON and MCDONALD 1983).

We have compiled a protein complementation pro-

file resulting from all pairwise crosses of 12 ADH

inactive protein variants and from crosses between the

inactive variants and ADH"', ADH', ADHF and

ADHD in Figure 2. T h e F1 progeny were scored on

the basis of whether they possessed ADH heterodi-

mers, and if so, whether these heterodimeric proteins were active o r inactive. Reciprocal crosses gave an identical complementation pattern.

Four of the mutant proteins, ADH"',, ADHn4,

ADH"" and ADH"13, d o not form heterodimers with

any of the other subunits tested. ADHn4 and ADH"",

shown to be CRM(-) (PELLICCIA and SOFER 1982),

produce no heterodimeric proteins with any of the

other subunits. Although ADH"' and ADH"" possess

immunodetectable polypeptides of the same length as wildtype, they cannot be stabilized under native con-

ditions (HOLLOCHER and PLACE 1987) and do not

produce any heterodimeric proteins with any of the other subunits. All of the other ADH mutant proteins were shown to exist naturally as homodimers under

n l n2 n3 n4 n5 n6 n7 n8 n9 n10 n l l n12 1113 S F D

Inactive heterodimer

Active heterodimer

(neubal complementation for null x Active heterodimer

(positive complementation)

Coold n d be distinguished

n l









n 10

n 11

n 12

n 13

FIGURE 2. Complementation profile of each Adh


native conditions (HOLLOCHER and PLACE 1987) and display distinct complementation patterns.

ADH"' in combination with ADHn5, ADHn7, ADH"', ADH"" and ADH"" shows heterodimeric activity. In the cross Adh"' X Adh"", in addition to the heterodimeric protein being active, residual ADH"' homodimer enzyme activity is observed (Table


ADH"' dimerizes readily with ADHS and ADHD.

Dimerization with ADHF could not be determined

because of similarity in charge.

ADH"' and ADH"' subunits dimerize readily with the other subunits yet these heterodimers remain inactive o r are only marginally active (0.5% wild-type activity) as with Adh"' X Adhn7 and Adh"' X Adh"".

However exceptions to this statement are the Adhn5 X

Adh"' and A d h D X Adh"' crosses, where the heterodi- mers have specific activities greater than expected. Although higher in specific activity, the heterodimers

formed in these cases are not stable. ADH"' has a

deactivating effect on the ADHD and ADHF subunits,

making the heterodimer inactive. ADH"' dimerizes

with ADH'. Whether dimerization of ADH"' with

ADHS and ADH"' with ADHF occurs cannot be de- termined because of identical PIS on these pairs of proteins.

Although ADHn5 is active in the homodimer state

and dimerizes readily with the other subunits, for the most part it is neutral with respect to complementa-

tion, i.e., the heterodimers formed under these con-

ditions showed no more activity than expected if the ADHn5 subunit is the only active subunit.

ADH"' forms heterodimers readily with ADH"', ADHn5, ADHn7, ADH"", and ADH"", yet the result- ing heterodimers are only active in crosses with ADH"" ADH"". ADH"' forms active heterodimers

with ADHF, ADHD and ADHS.

ADHn7 and ADHnB dimerize readily with the other subunits and both show heterodimeric enzyme activity

in crosses involving ADH"', ADHn5 and ADH"", yet

d o not restore activity when in combination with each

other. ADHn7 in combination with ADHn3 and

ADH"'* shows marginal heterodimeric activity. ADH"' forms a heterodimer with ADHS, yet is deac-

tivating in combination with ADHD subunit. Hetero-

dimer formation between ADHF and ADH"' could

not be scored because of their similarity in charge. ADHn7, on the other hand, forms active heterodimers

with ADHF and ADHD, and forms a heterodimer with

greater specific activity than expected with ADH'. T h e most successful complementing subunit is ADH"" , which forms active heterodimers with


T h e specific activity of the ADH in the Adh"" X Adhn6

cross has been shown to be 13% that of the wild-type

b ADHF cn vg (PELLICCIA and COOPER 1984). ADH""

also forms active heterodimers with ADHF, ADHS and

ADHD. In the Adh"" X AdhS cross, the heterodimer has a greater specific activity than expected.

complements ADH"', ADHn5, ADH"' and

ADH"" to a great extent. To a lesser extent, ADH""

complements ADHn7 and ADH"'. In the cross Adh""

X Adhn5, the ADH"" homodimer shows residual ac- tivity in addition to the heterodimeric protein. ADH"" readily dimerizes with ADHS and ADHD.

Dimerization of ADH"" with ADHF could not be

determined because of their similarity in charge.


T h e results of this study corroborate the previous

findings of GRELL, JACOBSON and MURPHY (1 968) and

SCHWARTZ and SOFER (1976b). In addition, by prob-

ing for ADH cross-reacting material on protein blots,

we were able to distinguish between the following four explanations when no heterodimeric activity was detected on the gel: (1) one of the mutant subunits

existed in drastically reduced levels, diminishing the

amount of detectable heterodimer formed (e.g., AdhF

X Adh"');

( 2 )

the subunits failed to dimerize (e.g., Adhn5

X Adhng); (3) the subunits formed a heterodimer that

remained inactive (e.g., Adhn7 X Adh""); or (4) the

subunits formed a heterodimer that resulted in the

loss of activity in the previously active subunit (e.g.,

AdhD X Adh"'). These distinctions could not be made if activity is used as the only criterion for scoring F1 progeny.

In the AdhS X Adh"" and the AdhS X Adhn7 heter-

odimers there is an indication of either hyperactiva-

tion of the ADHS subunit or repair of the ADH""

and ADHn7 subunits. Both ADH"" and ADHn7 have

similar charge substitutions. It has been shown that the glycine to aspartate substitution (Gly14 to Asp1 4)

found in ADH"" results in an inability to bind coen-


and RETZIOS 1980). ADHS, in contrast to ADHF and

ADHD, is characterized by having a lysine at position

192 (RETZIOS and THATCHER 1979; FLETCHER et al.

1978) and a twofold slower catalytic turnover than the ADHF allozyme (WINBERG, HOVIK and Mc- KINLEY-MCGEE 1985). It is conceivable that a salt bridge might form between these two residues: the

Asp14 in ADH"" and the Lys192 in ADHS, especially

if the arrangement of the dimer is antiparallel. This

may repair the defect in ADH"" by alleviating the

influence of the carboxyl group on the adenine bind- ing domain of the enzyme. An equally likely expla- nation is that the salt bridge somehow ameliorates the

effect Lys192 has on the ADHS subunit. If the salt

bridge were to exist it should be possible to crosslink the two residues. Studies are currently underway along these lines.

Four ADH mutant proteins d o not form heterodi-



Although ADHn4 and ADH"" have been classified as

CRM(-) by PELLICCIA and SOFER (1982), we still

expected to be able to detect heterodimeric proteins by the antigenic determinants on the subunits that

were able to cross-react with the anti-ADH antibody.

Because no heterodimeric proteins were detected in any crosses involving ADHn4 or ADH"", the belief that these two mutants resulted from either early translation termination o r a nonsense frameshift is

supported (HOLLOCHER and PLACE 1987). ADH"' and

ADH"I3 are CRM(+) (PELLICCIA and SOFER 1982) and

exhibit subunit polypeptides the same length as wild-


PLACE 1987). It was shown previously that these two

mutant proteins could not be stabilized under native

conditions (HOLLOCHER and PLACE 1987). T h e fact

that both of these proteins also d o not form hetero- dimers with any other ADH subunit tested supports our earlier hypothesis that they may represent muta-

tions in the dimerization process (HOLLOCHER and

PLACE 1987).

T h e relative ratios of homodimer versus heterodi- mer in these crosses indicate that the expected binom- ial ratios is not the norm. For proteins that occur at

drastically different in vivo steady state levels (PELLIC-

CIA and SOFER 1982), the amount of heterodimer may

be equal to the amount of the more abundant of the two proteins in the cross, the amount of the lower steady state level variant, o r some intermediate value. It is evident that the subunit interactions are not neutral with respect to stability and that heterodimers represent new protein species having their own char- acteristics. In fact, the heterologous interactions in the heterodimer appear to be weaker than the ho- mologous interactions in the homodimers. T h e het-

erodimer does not appear to be a state where an

unstable subunit can find relief from the degradative machinery of the cell, except, perhaps, for the case of

AdhS x Adh"' where the heterodimer exists in greater amounts than expected. No simple prediction can be made as to how two different protein subunits are going to interact in a heterodimer.

In two crosses, Adh"' X Adh"" and Adhn5 X Adh"I2,

activity for the homodimers, ADH"' and ADH"", respectively, was detected in the F1 progeny. T h e activity was most apparent and consistently reproduc- ible for ADH"" homodimer in four independent

Adhn5 X AdhnI2 crosses. There are four possible expla- nations for the apparent restoration of activity in the presumptive inactive homodimers in these two crosses. First, the homodimers may not represent proteins absolutely devoid of activity. Second, other loci may be directly involved in the production of active ADH. Third, the resulting genetic background

in the progeny fly may effect the expression of ADH.

Fourth, the association of the inactive subunits with

the other ADH subunits present in the cross may effect their level of activity.

There is a possibility that ADH"' and ADH"'* hom- odimers do not represent proteins absolutely null in activity. ADH"' is suspected of having residual activity

at permissive temperatures (VIGUE and SOFER 1976).

T h e activity levels observed for ADH"' in the Adh"'

X Adh"" cross fit that explanation. There have been no reports of ADH"'* having residual activity.

There is no molecular evidence that other loci are directly involved in the production of active ADH

enzyme. Both the ADH protein (THATCHER 1980)

and the A d h gene have been sequenced (BENYAJATI et

al. 198 l), and there is no evidence of posttranslational

modification modulated by other genetic loci.

T h e possibility of modifier genes greatly enhancing the level of ADH protein cannot be ruled out. It has

been well documented that the expression of A d h in

Drosophila can be greatly modified by loci on other

chromosomes seemingly unrelated to the Adh gene

itself (MCDONALD and AYALA 1978; MARONI 1978;

LAURIE-AHLBERG et al. 1980, 1982; MARONI et al.

1982). If ADH"" does have residual activity and is overexpressed in the F1, then homodimer activity will be detectable. However, an active ADH"" homodi-

mer is not obtained in the progeny of A d h D X Adh"12,

which involves genetic backgrounds similar to Adh"'

X Adhn12, in that Adhn5 was derived from AdhD.

T h e final possibility is that level of ADH"" activity in this cross is affected by the subunit's association with the active subunit of ADH"'. According to CRICK

and ORGEL ( I 964), a defect in the folding of a mutant

subunit can be corrected by association with a "com-

plementing'' subunit. SCHWARTZ (1973, 1975) has

determined that such an influence on the folding of a mutant polypeptide can become "fixed" and is re-

tained after dissociation and reassociation in vitro,

affecting the subsequent interaction of the mutant

polypeptide with other subunits. SCHWARTZ'S hypoth-

esis is based on observations of proteins that become

permanently inactivated (1 975), as well as proteins

that are conferred stability during the heterodimeiic phase which is maintained during the later formation of homodimers (1973). ADH"' is active, yet intrinsi-

cally unstable (VIGUE and SOFER 1974). Since an active

heterodimer is formed, association with ADH"' has a positive influence on the folding of the ADH"" sub- unit, yet the resultant heterodimer may dissociate rapidly. It is possible that an ADH"12 subunit disso- ciated from the heterodimer may then reassociate with a homologous subunit and retain the positive conformation acquired from its association with the active ADH"' subunit.

Our hypothesis is supported by the recently de- scribed phenomenon of "conformational drift" (WE-


(note that at sometime during polypeptide synthesis a subunit exists as a dissociated entity) is decreased or lost, even in situations in which a dynamic equilibrium

between the aggregate and its members is clearly

established. T h e loss in affinity is attributed to a

progressive, time-dependent disorganization of the monomers when they fail to exert influence upon each other in the dissociated state. It has been shown

with lactate dehydrogenase isozymes (KING and WE-

BER 1986a,b) that high pressures as well as cold tem-

peratures bring about dissociation, conformational drift, and reassociation into inactive tetramers. T h e reassociated inactive tetramer resembles the active form in all measurable physical properties except for being catalytically inactive. With cryoinactivation, full

activity can be restored simply by allowing the mixture

to incubate at room temperature. T h e kinetics of recovery in activity is very slow, however, when com-

pared to the rate of reassociation. Hence,

". . . that

catalysis must depend upon conformational details that are independent, to a large extent, of those that

ensure the reassociation of the isolated subunits"

(KING and WEBER 1986a).

We consider both CRICK and ORGEL'S hypothesis

and SCHWARTZ'S hypothesis for explaining interallelic

complementation as being plausible and consistent with our data. We find no reason for the two hy-

potheses to be mutually exclusive. SCHWARTZ'S hy-

pothesis and WEBER'S idea of conformational drift

extend the original idea of Crick and Orgel; which phenomenon dominates, depends on the relative in- tensity of the subunit interactions in homodimers versus heterodimers. Both explanations may be cor- rect for different sets of alleles. Our data have exam- ples of positive (Adh"" X Adh"), neutral (Adh"' X Adh""), and negative (Adh"' X AdhD) complementa- tion, as well as a possible example of conformational drift (Adh"" X Adh"').

We would like to finish our discussion by stating that, based on a variant's ability to form heterodimers (active, inactive, or not at all) and on the physical

properties of the variants as described earlier (HOL-

LOCHER and PLACE 1987), each Adh null variant ex-

amined in this study possesses a unique amino acid

substitution. N o variant has identical molecular and

interallelic complementation profiles.

We thank S. ABRAHAM, A. BEADY, A. COLDBERG and R. E.

SHERMAN for their technical assistance. This work was supported by the National Science Foundation (PCM81-10819).


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Communicating editor: W. J. EWENS SPIELMAN, H., R. P. ERICKSON and C. J. EPSTEIN, 1974

THATCHER, D. R. and A. RETZIOS, 1980


URSPRUNG, H. and J. LEONE, 1965

VIGUE, C. and W. SOFER, 1976

WEBER, G., 1986



FIGURE 0 cross-reacting material using the procedure described by Adh-negatives with Adh-positive electrophoretic variants were separated on of one homodimer while the tilled triangles mark the location of the other homodimer


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