Vol. 179, No. 3, 1991 September 30, 1991

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1225-l 231

SIGNAL TRANSDUCTION IN COfWNUS CONGREGATUS: EVIDENCE FOR THE INVOLVEMENT OF G PROTEINS IN BLUE LIGHT PHOTOMORPHOGENESIS Katherine

R. Kozak and Ian K. Ross

Department of Biological Sciences University of California, Santa Barbara Santa Barbara, CA 93106 Received

August

5,

1991

SUMMARY: This paper reports the presence of several G proteins and light-sensitive GTP-binding proteins in the fungus Coprinus congregatus, a filamentous eukaryote. (Mono)ADP-ribosylation experiments with crude membranes in the presence of the (poly)ADP-ribosyltransferase inhibitor, &amino-benzamide, resulted in the detection of a cholera toxin substrate of 52kDa and two pertussis toxin substrates, 33 and 39kDa. Two-dimensional polyacrylamide gel analysis of GTP-binding proteins exposed in vivo to [35S]-labeled guanosine S-[r-thio]-triphosphate in the presence or absence of light demonstrated light enhanced analog binding. These results support the concept of the 9 1991AcademicPESS, Ir involvement of G proteins in phototransduction in C. congregafus.

Among the various families of guanine nucleotide binding proteins now known to exist, the heterotrimeric GTP-binding proteins (G proteins) are mainly involved in transducing external signals to the inside of cells (1). These G proteins can be distinguished from each other and other GTP-binding proteins on the basis of their molecular weight and their susceptibility to modification by certain bacterial toxins (i.e., pertussis and cholera toxin). These toxins catalyze the slow hydrolysis of [32P]-labeled NAD+ to [32P]ADP-ribose and nicotinamide and the transfer of individual, labeled ADPribose moieties to specific G-protein o subunits (2), thus they are mono(ADP)ribosyltransferases (3). It is the a subunit of a G protein which interacts with both a gr dimer, and a specific effector molecule (which functions to pass the signal on), as well as regulating the duration of effector action via its internal GTP hydrolysis activity (4). Abbreviations: ADP, adenine diphosphate; cc, California clone; GTP, uanosine triphosphate; [35S]GT~S, guanosine 5’-[r-thio]-triphosphate, [35S]-; [ $2 P]NAD+, nicotinamide adenine dinucleotide, di(triethylammonium) salt, [adenylate-32P]; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tris, tris(hydroxymethyl)aminomethane; YpSs, Yeast, powdered, Soluble starch.

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0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. AN rights of reproduction in any form reserved.

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In the mushroom-forming fungus C. congregatus, a light signal, along with nutrient depletion, initiates the differentiation of the filamentous hyphal tips that develop into multi-tissued fruiting bodies capable of producing millions of meiotically derived spores. Two blue light signals are required for photodifferentiation: a short (1 5 min) light signal followed by a second, longer (minimum 30 min) light signal, 3 hours after the first (5). Evidence supporting G protein mediated light-induced signal transduction in fungi are as follows: there are G proteins present in the plasma membranes of the fungus Metarhizium anisopliae (6); adenylate cyclase, known to be regulated by G proteins in higher eukaryotes, is also activated by blue light in Phycomyces (7); GTPbinding proteins are thought to be involved in the light signal transduction in Neurospora cfassa (6); studies with C. macrorhizus mutants have demonstrated that increases in cyclic adenine monophosphate levels correlate with the ability to fruit (9,lO); this correlation is also found in Schizophy//um commune (11) and other basidiomycetes (12); and transducin, the signal-transducing G protein of the retinal cyclic guanosine monophosphate phosphodiesterase system, is well known for its action in mediating the light signal in animal vision (13). In view of these facts, experiments were undertaken to determine the existence of G proteins in C. congregatus via ADP-ribosylation experiments and from two-dimensional gel analysis, to determine if light effects the GTP-binding of any GTP-binding proteins. MATERIALS

AND METHODS

Crude Membrane Isolation: Coprinus congregatus was isolated by Ross in 1971 (14), and stock cultures of single-spore-derived homokaryons maintained at 4-C in test tubes on Difco Emerson’s YpSs agar. The dikaryon used in our studies was obtained by crossing the California homokaryon cc9 with the homokaryon cc16 (15). Crude membrane isolation procedures were modified from Hasunuma (6), and done under red safelight conditions. Hyphal tip regions of cc9x16 dikaryons grown on YpSs overlaid with cellophane were harvested from 140, 6-day old cultures grown in the dark. The hyphal material was ground in liquid nitrogen and homogenized with a Polytron in 40ml buffer (25mM Tris-HCI, pH 7.5, 0.02mM MgCl2, 0.25mM ethylenediaminetetraacetic acid, and 1 mM phenylmethylsulfonyl fluoride), and centrifuged at 16,OOOxg, 20 min in a Sorval GSA swinging bucket rotor at 4’C. The supernatant was stored in aliquots at -60°C as the crude membrane preparation. The protein concentration of the crude membrane preparation was determined by the method described by Lowry et aI.( Inhibitor Treatment: 150~1 of the crude membrane preparation incubated for 30 min at 37’C with 10mM 3-amino-benzamide endogenous poly(ADP)ribosyltransferases.

(4.5mg/ml protein) was (17) (Sigma) to inhibit

ADP-ribosvlation: ADP-ribosylation was carried out as described by Sternweis (la), with some modifications. Toxins (List Biological Laboratories) were activated in activation buffer (50mM Tris-HCI, pH 7.5, 50mM dithiothreitol, 2.5 ccg/ml bovine serum albumin) for 30 min, 37°C. The ADP-ribosylation reactions (total volume 80~1) contained 2.5 mM MgC12, 50mM Tris-HCI, pH 7.5, 10mM GTP (Sigma), 7 tig 1226

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preactivated cholera toxin or 0.7 pg preactivated pertussis toxin, 7.5 &i of 13*P]NAD’ (NEN research products, 800 Ci/mmol) and 18Opg of crude membrane preparation (2.25mg/ml) with or without 3-amino-benzamide pretreatment. After incubating the reactions for 1 hr at 35” C, the proteins were precipitated with trichloroacetic acid, centrifuged at 16,OOOxg for 5 min and washed three times with ethyl ether. The precipitated [32P]-labeled proteins were dissolved in 50~1 of SDS sample buffer, heated 2 min at 100 “C and electrophoresed on 3%-27% gradient SDS polyacrylamide gels according to Laemmli (19). Unincorporated r*P]NAD was removed by discarding the lower region of the gels before fixing in two large volumes of a 9% acetic acid, 45% methanol solution. Dried gels were then exposed to film. Gels: Cc9x16 dikaryons growing in the dark on cellophane/YPSS plates were pulsed with 12ml of [35S]GTP 7 S (4.2&i/ml) in 80mM Tris-HCI, pH 7.5, 0.4M NaCI, 20mM MgC12 in the presence or absence of light for 12 hr. The cellophane bound fungus was rinsed once in quenching buffer (40mM Tris-HCI, pH 7.5, 0.2% Lubrol PX [Sigma], 0.2M NaCl, 25mM MgCl2, 0.2M GTP), twice in cold wash buffer (20mM Tris-HCI, pH 7.2, 0.1 M NaCI, 25mM MgC12) and once in water. Hyphal tips were harvested and ground with a mini-pestle in a microfuge tube. The ground material was centrifuged at 12,OOOxg, 5 min, and the protein concentration of the supernatant was assayed. Samples from light and dark exposed cultures were resolved on a nondenaturing isoelectric focusing gel with a pH range of 3.5-9.5 (LKB) run on a flatbed unit at 30 Watts, constant power for 30 min. The strips were equilibrated in 30ml nondenaturing sample buffer (30mM Tris-HCI, pH 6.8, 60% v/v glycerin, 10% w/v bromophenol blue), 30 min. Gel strips were loaded on non-denaturing 10% PAGE gels. Both light and dark sample gels were run simultaneously at constant current, 40 mA for 2 hr. The gels were fixed overnight in a 9% acetic acid, 45% methanol solution. Fluorography of the gels was completed according to E.M. Corp. Resolution instructions, and then dried and exposed to Kodak X-OMAT AR film, 8 days. Two-dimensional

RESULTS AND DISCUSSION

The autoradiogram reactions

results from the polyacrylamide

show that several ADP-ribosyl-substrates

congregatus membrane

crude membrane benzamide

preparations

preparations

that cholera

toxin ADP-ribosylates cyclase

molecular

weight

messenger

RNA transcribed

cholera toxin-sensitivity

45,000

cholera

congregatus

52kDa G-protein

o subunit

toxin substrate

or 52,000

from a single gene. Therefore,

toxin substrate

implies

Q subunit.

has the same molecular

It is known

has an apparent splicing

of the

both the shared specific

weight of 52kDa

a possible

(lane

that stimulates

due to alternative

and the identical molecular

congregatus

toxin substrate

33 and 39kDa (lane 15), identified. G,, the G-protein

the

inhibitor 3-amino-

are a distinct 52kDa cholera toxin substrate

(20), and that this cholera of either

in the crude C.

(fig. 1, lanes 3 and 10). Only after pretreating

toxin substrates,

adenylate

are present

with the poly(ADP)ribosyltransferase

prior to ADP-ribosylation,

8) and two pertussis

gel of the ADP-ribosylation

stimulatory

of G, and the C. role for the C.

Also, the C. congregatus 39kDa pertussis weight and the same toxin sensitivity 1227

as Go

Vol. 179, No. 3, 1991

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&gJ. Autoradiogram of SDS-PAGE resolved ADP-ribosylated proteins from Coprinus congregatus membranes.Lane (1) High molecularweight markers (kDa), reactions containing (2) CM+CT, (3) CM+CT+GTP, (4) CT+GTP (no membrane), (5) CM+GTP (no toxin), (8) 3AB pretreated CM+CT+GTP, (9) CM+PT, (10) CM+PT+GTP, (11) PT+GTP (no membrane), (12) CM+GTP (no toxin), (15) 3AB pretreated CM+PT+GTP. Cholera toxin (CT), pertussistoxin (PT), crude membranepreparation (CM), guanosine 5’-triphosphate (GTP), 3-amino-benzamide(3AB).

(18), the most abundant G protein retinal rod light receptor-coupled

present in bovine brain, and as transducin, the G-protein Q subunit (13), implying that similar G

proteins may exist in C. congregatus. The crude membrane preparations have retained the soluble cofactors (e.g. ADP-ribosylation factor) required for our assays, but have also retained endogenous poly(ADP)ribosyltransferases.

As a result of these contaminating transferases, toxin-

independent incorporation of ADP-ribose leads to the nonspecific radiolabeling of nonG proteins. This is observed when a sample is incubated without toxin, but with the toxin activation buffer (fig. 1, lane 5 and 12). To deal with this problem, thymidine can be included

in the reaction

poly(ADP)ribosyltransferases

mixture

to diminish

protocols

activity

of the

often present in membrane preparations, but even then,

some ADP-ribose can become incorporated ADP-ribosylation

the catalytic

into non-G proteins (21). Several [32P]-

suggest adding cold ADP-ribose

in order to reduce

background labeling in crude membrane samples, but this can alter the amounts of various forms of a subunits and may not allow small quantities of a subunits to be detected (2). Alternatively, treatment of C. congregatus crude membrane preparations with 3-amino-benzamide efficiently reduced the background created by endogenous poly(ADP)ribosyltransferases,

with minimal effects on mono(ADP)ribosyltransferases

(in fig. 1, compare lane 3, untreated, with lane 8, inhibitor treated) (22). ADP-ribosyl-G-protein

a subunits in crude cell preparations may be resolved by

the O’Farrell procedure of denaturing SDS-PAGE (23), but throughout our procedure we have used non-denaturing conditions to prevent the disruption of the structurally 1228

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AND

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IEF

B

A

Fio. 2. Two-dimensional non-denaturing gels of GTP-binding proteins from Coprinus congregatus pulsed with [35S]GTP6 in (A) the absence of light, and in (B) in the presence of light. Equaf protein concentrations were loaded on both first dimensions. The GTP-binding proteins ran with pls of approximately 5.

supported

radioligand(GTP)-binding

separated

GTP-binding

observed mycelia

proteins

were exposed

dark grown

to [35S]GTPrS

were

applied

after the second

from GTP-binding

proteins

in image

points

extracted

of the light-sensitive

different

subtypes

of Q subunits

endogenous

proteolysis

GTP-binding

supports

congregatus

spots

and equal

from the light induced The GTP-binding

or the proteins

The image derived

mycleia

proteins

from dark-grown

mycelia.

in light-induced

mycelia

extracted proteins

of GTP to the proteins.

or the result

prior to electrophoretic

proteins)

significantly

could

represent

products

either

caused

This observed

of a light

role in mediating

was

The similar isoelectric

of degradation analysis.

existence

G protein and its possible

and

from light-induced

(GTP-binding

the possible

of light,

by autoradiography.

of GTP-binding

the binding

gels

(Fig. 2). C. congregatus

or absence

preparations

dimension

intensity

implies that light enhanced

activity

to the first dimension.

than the image of GTP-binding

The difference

We have successfully two-dimensional

in the presence

of crude membrane

mycelia

revealed

darker

on non-denaturing

the effect of light on their GTP binding

protein concentrations

were

of the G proteins.

by

light-induced

receptor-coupled

signal transduction

C.

for fruiting

body formation. In this paper, we have shown G proteins

do exist in crude membrane

We have also shown GTP-binding are strong be involved

that several

proteins evidence

fractions

from the analysis

cholera

of the basidiomycete

of our two-dimensional

may exist in the hyphal tip regions that G proteins

and pertussis

are present

in its blue-light-signal-induced 1229

C. congregatus.

gels that light sensitive

of the fungus.

in C. congregatus

differentiation.

toxin sensitive

These results

and that they may

In addition,

preliminary

Vol.

179. No. 3, 1991

studies

BIOCHEMICAL

of PCR amplified

sequences subunits

using primers

have revealed the presence (data not shown). Furthermore,

non-hydrolyzable G proteins

sequences

AND BIOPHYSICAL

from known G-protein

in earlier studies using aluminum

receptor activation

in the dark responded

derived

of at least two different putative

analog which mimics the r-phosphate

by bypassing

RESEARCH COMMUNICATIONS

G-protein

a

fluoride,

a

of GTP and directly activates

(24)) we found that cultures treated with

aluminum

fluoride

stimulus.

Further analysis of these G proteins should provide a better understanding

of signal transduction

as though

they had received

the first light

in fungi and may shed some light on fungal photodifferentiation.

ACKNOWLEDGMENTS

This work was supported in part by a grant from BP Venture Research to I.K.R. and in part from a Stiftung Fur Medizinische Forschung to K.R.K. Special thanks to Lyndon Foster for his support.

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