Table Of ContentEJB Reviews 1989
European Joumal of
Biochemistry
1989
Contributing Authors:
J.-P. Battioni, Paris W. Hummel, Jiilich
P. Battioni, Paris M. C. Kennedy, Milwaukee
H. Beinert, Milwaukee M.-R. Kula, Jiilich
T. Boehm, Cambridge D. Mansuy, Paris
M. Chabre, Grenoble V. Massey, Ann Arbor
P. R. Cook, Oxford T. H. Rabbitts, Cambridge
P. Deterre, Grenoble R. Timpi, Martinsried
S. Ghisla, Konstanz B. Westermark, Uppsala
L. A. Grivell, Amsterdam R. J. P. Williams, Oxford
C.-H. Heldin, Uppsala
Springer-Verlag Berlin
Heidelberg GmbH
Professor Dr. P. Christen
Biochemisches Institut der Universităt Ziirich
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CH-8057 Ziirich
Professor Dr. E. Hofmann
Institut fiir Biochemie der Karl-Marx-Universităt Leipzig
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DDR-7010 Leipzig
ISBN 978-3-642-75191-2 ISBN 978-3-642-75189-9 (eBook)
DOI 10.1007/978-3-642-75189-9
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Originally pub1ished by Federation of European Biochemica1 Societies 1989
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Preface
In the mid-1980s the European Journal of Biochemistry set out to publish
review articles. The enterprise proved successful with many high-level reviews
written by well-known scientists appearing in the Journal. The reviews are
intended to represent emerging and rapidly growing fields of research in
fundamental as well as in applied areas of biochemistry, such as medicine,
biotechnology, agriculture and nutrition. Novel methodological and techno
logical approaches which stimulate biochemical research are also included.
The authors of the reviews are explicitly asked to be critical, selective, evalua
tive and interdisciplinarily oriented. The reviews should encourage young
scientists toward independent and creative thinking, and inform active inve
stigators about the state of the art in a given field.
The good reception of the reviews by the readers of the European Journal
of Biochemistry has induced the Editorial Board and Springer-Verlag to
publish them annually in a separate booklet, 'EJB Reviews', in order to
further their dissemination among biochemists and scientists in related bio
logical and medical disciplines.
P. Christen E. Hofmann
Chairman of the Reviews Editor
Editorial Board
Contents
Molecular mechanism of visual transduction
M. Chabre and P. Deterre, 179/2 (Feb. I) ................ 255 - 266
Structure and biological activity of basement
membrane proteins
R. Timpl, 180/3 (April I) . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 487 - 502
Mechanisms of flavoprotein-catalyzed reactions
S. Ghisla and V. Massey, 181/1 (April II). . . . . . . . . . . . . . . .. 1 - 17
Nucleo-mitochondrial interactions in yeast
mitochondrial biogenesis
L. A. Grivell, 182/3 (July I) . . . . . . . . . . . . . . . . . . . . . . . . . .. 477 -493
NMR studies of mobility within protein structure
R. J. P. Williams, 183/3 (Aug. II) ....................... 479-497
Dehydrogenases for the synthesis of chiral compounds
W. Hummel and M.-R. Kula, 184/1 (Sept. I) . . . . . . . . . . . . .. 1 - 13
Chemical model systems for drug-metabolizing
cytochrome-P-450-dependent monooxygenases
D. Mansuy, P. Battioni and J.-P. Battioni, 184/2 (Sept. II) .... 267-285
Growth factors as transforming proteins
C.-H. Heldin and B. Westermark, 184/3 (Oct. I). . . . . . . . . . .. 487 -496
A chromosomal basis of lymphoid malignancy in man
T. Boehm and T. H. Rabbitts, 185/1 (Oct. II) .............. 1 - 17
The nucleoskeleton and the topology of transcription
P. R. Cook, 185/3 (Oct. II) ............................ 487-501
Engineering of protein bound iron-sulfur clusters
H. Beinert and M. C. Kennedy, 186/1-2 (Dec. I) .......... . 5- 15
Subject index ..................................... .
Eur. J. Biochem. 179,255-266 (1989)
© FEBS 1989
Review
Molecular mechanism of visual transduction
Marc CHABRE and Philippe DETERRE
Laborato~re de Biophysique Moleculaire et Cellulaire (Unite Associee 520 au CNRS) Departement Recherche Fondamentale
Centre d'Etudes Nucleaires, Grenoble ' ,
(Received September 30,1988) - EJB 881153
INTRODUCTION AND OVERVIEW into the disc membrane lipids, it was often speculated that the
liberated retinal might playa direct role in the disc membrane,
Visual transduction covers the sequence of photochemical,
but its dissociation from bleached rhodopsin is much too
biochemical and electrophysiological events through which
slow to be significant for the visual transduction process.
the absorption of a photon in a pigment molecule in a
photoreceptor cell generates an electrical cellular response Ph.~toexcited rh~d?psin itself has to be the trigger. The pre
vatlmg hypothests m the seventies was that the soluble trans
that will be detectable at the level of the synaptic connection.
mitters were calcium ions released from the disc, either directly
Our understanding of visual transduction has considerably
progressed recently and much of the new developments have t~rough photoexcited rhodopsin acting as a channel, or in
dtrectly through another channel controlled by photoexcited
been in the field of biochemistry and enzymology (for others
rhodopsin [4]. It had also been recognized that illumination
recents review, see [1-3]).
interferes in the rods with the metabolism of cyclic nucleotides,
The best studied system is the rod cell of the vertebrate
and more precisely cGMP; this was generally considered as a
retina, which provides unrivalled advantages for biochemical
secondary process, too slow for a role in transduction and
studies, due to the exceptional segregation and concentration
more likely to be involved in adaptation.
of the molecular machinery of visual transduction in its outer
Biochemical studies concentrated then on rhodopsin. In
segment. The initial molecular event, the photoisomerization
the late seventies, reasonable agreements were reached on its
of retina in rhodopsin, and the physiological cellular response,
molecular mass, around 40 kDa, from the fact that it crosses
a hyperpolarization resulting from the closure of a cationic
the disc membrane and the predominance of rx helices in the
conductance of the cellular membrane, were both well
transmembrane region. This was confirmed by the determi
characterized by the late sixties. When less than a hundred
nation of the sequence in which could easily be recognized
photons are absorbed per rod flash, that is in the non-saturat
seven hydrophobic stretches corresponding to seven trans
ing range, the hyperpolarization rises in about 0.2 s in mam
membrane helices. The search for a possible effect of photo
malian rods. After reaching its maximum, it decays nearly as
excited rhodopsin on calcium release from the disc however
fast so that the response is terminated within 0.5 s. In the rod
remained very disappointing, while by contrast evidence had
outer segment the disc membrane which contains rhodopsin
accumulated on the interaction of photoexcited rhodopsin
is not connected to the cellular membrane which contains the
with peripheral GTP- and ATP-dependent enzymes. Three
cationic channels whose conductance is modulated by light.
of these enzymes: a GTP-binding protein, (transducin), the
This morphology imposed the concept of a soluble transmit
ter, released or modulated at the level of the disc membrane rhodopsin kinase and arrestin (also called S antigen or 48K
and acting at the level of the cellular membrane. protein), were originally isolated through their specific inter
Early studies had concentrated on the photochemistry of action with photoexcited rhodopsin [5 -7]. Transducin is a
retinal, the molecule acted upon by light, which induces its member of the large class ofG proteins that mediate the action
isomerization from the ll-cis to the all-trans conformation. of a membrane receptor on internal secondary effectors; it
As this transconformation is followed by the release of the was demonstrated [8] that, in the rod, transducin is responsible
all-trans retinal from its membrane-bound protein cofactor for the amplified activation of the cGMP phosphodiesterase
[9]. It then became clear that the main, and probably only,
role of photoexcited rhodopsin is to trigger an amplifying
cascade which activates with high gain and fast kinetics the
This review is dedicated to the memory of our friend Hermann
hydrolysis of cGMP. But the mechanism through which
Kuhn, a pioneer of the biochemistry of visual transduction.
cGMP could modulate the cationic permeability of the cell
Correspondence to M. Chabre, Laboratoire de Biophysique
Moleculaire et Cellulaire, CENG Bolte Postale 85X, F-38041 Gre membrane remained unknown and was assumed to be com
noble, France plex: according to a classical scheme one expected that cyclic
Abbreviations. ROS, rod outer segment; R *, photoexcited nucleotides activated cGMP-dependent kinases, hence the
rhodopsin; T.GDP, T.GTP, T.empty, a-subunit oftransducin bear necessity of ATP, and a possible link with a calcium release at
ing a GDP, a GTP, or with an empty site; Tp, Ty, fJ and y subunit of a later stage. Fesenko etal. in 1985 [10] checked an improbably
transducin; PD E, cGMP-specific phosphodiesterase; PDE.p, catalytic 'simplistic' alternative: they demonstrated that cGMP alone
subunits ofPDE; I, inhibitory subunit ofPDE; GDP[fJS], guanosine
acts directly on the cationic conductance of the cell mem
5' -[fJ-thio ]diphosphate; GTP[yS], guanosine 5' -[y-thio ]triphosphate;
brane! A cGMP-binding polypeptide that reconstitutes
GuoPP [NH]P, guanosine 5' -[fJ,y-imino ]triphosphate.
Enzymes. cGMP-specific phosphodiesterase (EC 3.1.4.35); cGMP-dependent conductance in artificial membranes has
guanylate cyclase (EC 4.6.1.2). been isolated [11], so that the molecular path is now complete,
256
,
cGMP -Chapen
CGMP ....... cGMP .... Ch
clOsed
T!.POE (
A
"II 5'GMP
T*/ 'POE
1 photon _lR* _102-3T* _102-3 POE* _105cGMP hydrolyzed
Fig. 1. The basic scheme of the photo transduction cascade
from the light-absorbing chromophore to the response-gener analogy may even start at the ligand-receptor interaction site:
ating channel. one pictures now the catecholamine ligand binding in the
But if the simple scheme of Fig. 1 accounts well for the adrenergic receptor in a site analogous to that of retinal in
kinetics of the rising phase of a response to a short flash, it rhodopsin.
does not account for the fast decay of this response, nor for The retinal system is however unique in many respects, for
the slower adaptation processes that lower the sensitivity after example by the very high segregation and concentration of
prolonged illumination. The light-triggered cascade that acti the components of the transduction chain, the large excess
vates the degradation pathway of the cGMP pool seems insen number of receptor molecules with respect to the other
sitive to calcium. By contrast the synthesis of cGMP, which components, transducin and cGMP phosphodiesterase, and
is required for the regeneration of dark-adapted levels, is the unusual solubility of these membrane-associated proteins.
ensured by a guanylate cyclase that is not directly acted upon This may be related to the extreme sensitivity of the rods,
by light but is sensitive to calcium: it is inhibited by implying an enormous amplification, and their short response
micromolecular concentrations of calcium [12, 13], probably time (compared to other G-protein-coupled receptors)
through the action of an inhibitory calcium-binding protein implying fast kinetics.
[14]. There at last the elusive calcium ion comes into the story,
but in a totally different way and with an opposite effect
to that originally postulated: intracellular calcium decreases
INTRACELLULAR COMPARTMENTATION
after illumination, as a secondary effect of the closure of the
IN ROD OUTER SEGMENTS:
cGMP-sensitive cationic channels [15]. In their open state the
SOLUBLE, MEMBRANE-ASSOCIATED
channels also have a low permeability for calcium. In the dark,
AND INTRINSIC PROTEIN COMPONENTS
the calcium homeostasis was maintained by the counteracting
effect ofNa + lea 2 + exchangers present in the outer membrane The compact stack of discs fills most of the volume of the
of the rod outer segment. After illuminaton the closure of outer segment an divides the cytoplasm into interdiscallayers
the cGMP-dependent cationic channels blocks the influx of about 15 nm thick, communicating only around the disc edge
calcium. But the exchangers still extrude calcium, probably with the continuous extradiscal layer underneath the outer
at an even faster rate, since the cell hyperpolarizes and the cell membrane. The intradiscal compartment probably has
intracellular sodium level bocomes lower. The ensuing de an 'extracellular' ionic composition but no well documented
crease in intracellular calcium induces the activation of the exchange with the cytoplasm, at least on the time course of
guanylate cyclase, hence an increase of cGMP synthesis. This the response to a flash. The rhodopsin molecules are intrinsic
counteracts the effect of the earlier activation of the cGMP components of the disc membrane and the two major proteins
phosphodiesterase, tends to restore the dark level of cGMP of the cGMP cascade, transducin and cGMP phosphodiester
and to close the light-sensitive channels. ase are only peripherally attached to the disc membrane, from
This feedback reaction scheme of the cyclase is very recent which they can easily be dissociated by lowering the ionic
and still partially tentative [16]. By contrast, the light-triggered strength of the medium. In its activated form, the GTP-bind
cascade that controls cGMP hydrolysis has been thoroughly ing subunit of transducin is even physiologically soluble, but
analyzed: the proteins involved have been isolated, cloned, it will very soon interact with a disc-membrane-bound
most of them are sequenced, and their interactions have been phosphodiesterase. The two regulatory components of the
studied in reconstitution experiments. The elucidation of the cascade, rhodopsin kinase and arrestin, are soluble but have
amplification and regulation mechanisms of this cascade is a to bind and interact with rhodopsin [19]. The cascade triggered
major biochemical achievement of the last few years and by one photoexcited rhodopsin and controlling the hydrolysis
serves as a model for the very general problem of coupling by of cGMP develops therefore entirely whithin one interdiscal
G proteins of membrane transducers to intracellular effectors compartment, on a time course too short to allow any signifi
[17, 18]. Indeed the overall visual transduction mechanism has cant exchange of disc-membrane-associated proteins between
become a model not only for other sensory processes, as in different interdiscal compartments. The small messenger and
olfactory and gustatory transduction, but even for a large metabolite molecules (ATP, GTP, cGMP and calcium) ex
class of a priori unrelated types of hormonal or neuronal change rapidly between the interdiscal compartments and the
signal transduction. The analogy is striking at the receptor continuous cytoplasmic layer that surrounds the stack of dis
level: rhodopsin is the prototype of a large class of 'seven cs. The guanylate cyclase is not associated with the discs but
transmembrane IX helical' transducers, that includes the vari with the axoneme of the ciliary structure of the outer segment
ous adrenergic and muscarinic receptors and the receptor to [12]; the cGMP-sensitive cationic channel is an intrinsic
substance K; many others are coming into the picture. The component of the outer cell membrane. The pool of cG MP is
257
eG GTP
RhK Na __
~
Z T
ILl
:E
CI
ILl Ca
en
Ir disc
ILl Na
~
::::l
0 photon
..
0
0 c
co
Ir 1..;
GTP E
.. E
.. iii
E c
c .G.i
0 )(
><
co
CILIUM
Fig. 2. Intracellular compartments of the rod outer segment and the main molecular effectors. Rh, rhodopsin; RhK, rhodopsin kinase; A,
arrestin; GC, guanylate cyclase. PDE and GC reciprocally control the cGMP pool, while the Ca2+ content is balanced between entry by the
channel and extrusion by the Na + ICa2+ exchanger. The negative control of GC by Ca2+ ensures the correlation between Ca2+ and cGMP
concentration
Table 1. The main proteins involved in the photo transduction cascade
Protein Relation to membrane Molecular mass Equivalent Number of Reference
concentration molecules im- for sequence
in cytoplasm plicated in one
photon event
kDa IlM
Rhodopsin intrinsic 39 1 [2,21,22)
Transducin (IX + fJ + y) peripheral or soluble 80 500 102 -103 [3,36)
(39 + 37 + 6)
Phosphodiesterase (IX + fJ + I + I) peripheral 200 150 102 -103 [3, 85, 86)
(88 + 84 + 13 + 13)
Rhodopsine kinase soluble 65 5
Arrestin soluble 48 500 [75 -77)
Guanylate cyclase attached to cytoskeleton ? ? ?
cGMP-activated channel intrinsic 66 ? 102 -103
Na + ICa2+ exchanger intrinsic ? ? ?
therefore controlled negatively by the cascade in the in helical segments, adding up to half of the total length of the
terdiscal space, positively by the cyclase in the outer cytoplas polypeptide chain (Fig. 3). The predominance of transmem
mic layer and acts on the cationic channel. The pool of calcium brane helices had long been suggested by a variety ofbiophysi
is controlled at the level of the outer membrane, by the con cal measurements: X-ray and neutron small-angle diffraction,
ductance of the cGMP channels and the activity of the Na + j circular dichroism, diamagnetic anisotropy, near infrared lin
Ca2+ exchanger, and it regulates the cyclase in the extradiscal ear dichroism [20]. Seven hydrophobic stretches correspond
compartment (Fig. 2 and Table 1). ing to the transmembranes helices, separated by regions rich
in hydrophilic and polar amino acids, appeared clearly on the
hydropathy pattern of the sequence of bovine rhodopsin [21,
RHODOPSIN: A SEVEN-HELICES RECEPTOR 22] and are a constant feature of all sequenced visual pigments,
FOR RETINAL
much in invertebrates as well as in vertebrates [2]. Retinal is
Rhodopsin is an integral membrane protein. The trans linked by a protonated-Schiff-base bond to a lysine located
membrane core is made up of a bundle of hydrophobic IX- in the middle of the last helix and lies therefore in the middle
258
si les 0 f in teroe tion phosphorylation sites
wilh orreslin ond transducin C~sSs:s:sSs::>SSS:S:S:S::;;C::=;-'
..; .
:: ::
N====~==~====~
glycosylo ed siles conserved loops
Fig. 3. Structural model of bovine rod rhodopsin. Some residues conserved among known G-protein-related receptors are shown (shadowed
circles). Empty circles indicate the three negative residues specifically mutated in cone pigments. The C-terminal is anchored in the membrane
by palmitoylation of two cysteines [87)
of the membrane, nearly parallel to its surface. Glycines and in the final block of 10-20 amino acids. In photoexcited
pro lines are found in the central region of five out of the seven rhodopsin these hydroxylated residues will become phos
helices. As these are assumed to allow or induce 'kinks' in the phorylation sites for the rhodopsin kinase. Their sequence is,
helical structure, this could help provide a pocket for retinal. however, not conserved within the different visual pigments.
This seven-helices model has now been carefully checked by The site of interaction with transducin is mainly on the poly
proteolytic attack, by labelling with hydrophobic probes, and peptide loops connecting helices 5 to 6 and helices 2 to 3 [7].
is solidly established. But the packing and lateral ordering of The amino-terminal polypeptide is on the intradiscal side
the helices remains totally speculative. Rhodopsin is often (or extracellular side in cones). It varies in length and sequence
modelled by analogy with bacteriorhodopsin, the photo between visual pigments, but always carries glycosylation sites
energized proton pump of Halobacterium halobium. But for one or two oligosaccharides. Two of the polypeptide loops
although bacteriorhodopsin has seven transmembrane heli (2 - 3 and 4 - 5) are highly conserved in all visual pigments
ces, and also a retinal cofactor (hence the name) attached to and two conserved cysteine residues probably establish a di
a lysine in the last transmembrane helice, one must stress that sulfide link between these two loops. This conserved structure
its sequence has no detectable analogy with that of the visual must correspond to an important function yet to be deter
rhodopsin and its function is not that of a signal transducer. mined. It has been suggested that the loop 4 - 5 could rep
Many receptor proteins of the 'seven-transmembrane-helices' resent a calcium-binding site.
superfamily have significant sequence analogy with rhodopsin
and have analogous intracellular function; bacteriorhodopsin
INTERACTION OF RETINAL WITH RHODOPSIN
is the only one that cannot be related structurally as well
IN THE DARK AND AFTER ILLUMINATION
as functionally to rhodopsin. In the sequence of all known
rhodopsins and related seven-helices receptors few features Rhodopsin is a membrane receptor for the photoactivable
are strictly conserved: for example a sequential pair of posi ligand retinal. The protein environment of retinal in the trans
tively and negatively charged residues (Arg-Glu or Arg-Asp) membrane region fulfills multiple roles.
located at a fixed position on the cytoplasmic end of helix 2. a) In the dark, the site stabilizes the 11-cis conformation
These are not found in bacteriorhodopsin. A too-long phylo of the bound chromophore and hinders its thermal isomeriza
genetic separation does not account for this lack of detectable tion, hence protecting the system against spurious triggering.
homology: bovine rhodopsin cDNA probes have detected While the energy difference is only about 2 kJ Imol between
putative genes of analogous visual pigments in distant species the 11-cis and the all-trans conformations of free retinal, it
and one is even found, with low stringency, in Halobacterium requires more than 125 kJ/mol to isomerize a retinal bound
[23], but it is not the gene ofbacteriorhodopsin, probably that in its rhodopsin site. This estimate is based on a
of a sensory rhodopsin. The structural analogies between microcalorimetric measurement of the energy uptake upon
visual pigments and bacteriorhodopsin may therefore result illumination at liquid nitrogen temperature [24]. At this low
from converging evolution and may suggest that the 'magic temperature the chromophore can still be forced to change
number' of seven helices be required to cage a retinal oriented conformation upon absorbing the large energy (209 kJ Imol)
perpendicularly to the helices. of a visible photon but the protein movements are blocked,
The polypeptide loops connecting the helices constitute and the photoinduced process stops at the bathorhodopsin
two hydrophilic poles of about equal size. The hydrophilic stage after the isomerization of retinal: most of the energy
carboxyl-terminal polypeptide is on the cytoplamic side of the uptake must be due to the movement of the protonated Schiff
membrane. In all visual pigments, it is always rich in serine base end of the chromophore apart from a fixed negative
and threonine residues, 6 -10 of them being concentrated counterion in the protein. A candidate counterion has been
