Catalytic core of a membrane-associated eukaryotic polyphosphate polymerase.

Abstract

Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and its synthesis has only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains. Mutational analysis of the transmembrane domain indicates that VTC may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.

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CatalyticCoreofaMembrane-Associated
Eukaryotic Polyphosphate Polymerase
Michael Hothorn,1,2Heinz Neumann,3* Esther D. Lenherr,1Mark Wehner,4Vladimir Rybin,1
Paul O. Hassa,1Andreas Uttenweiler,3† Monique Reinhardt,3Andrea Schmidt,3Jeanette Seiler,1
Andreas G. Ladurner,1Christian Herrmann,4Klaus Scheffzek,1‡ Andreas Mayer3,5
Polyphosphate(polyP)occursubiquitouslyincells,butitsfunctionsarepoorlyunderstoodanditssynthesishas
only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate
polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom
crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP
winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the
enzyme functions by metal-assisted cleavage of the ATP g-phosphate, which is then in-line transferred to an
acceptorphosphatetoformpolyPchains.MutationalanalysisofthetransmembranedomainindicatesthatVTC
may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-
synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.
I
responses (1). In eukaryotes, polyP also acts in
phosphate transport between mycorrhizal fungi
and symbiotic plants (2), in osmoregulation (3),
and in bone calcification (4). Large quantities of
plankton-generatedpolyPformmarinesediments
(5). PolyP kinase generates bacterial polyP (6),
but in eukaryotes this enzyme has only been
reported in slime mold (7). Genetic screens in
yeast yielded ≥250 alternative candidates whose
deletion decreases cellular polyP (8, 9), but the
identity of the polyP-synthesizing enzyme has
remained elusive. Among the candidates is the
yeast vacuolar transporter chaperone (VTC) com-
plex, a membrane protein assembly whose dele-
tion reduces polyP accumulation (8) but also
affects membrane transport and vesicular traffic
(10–12). We have used x-ray crystallography to
identify a polyP polymerase in VTC.
Four Saccharomyces cerevisiae Vtc proteins
form hetero-oligomeric complexes (8, 10, 12).
Vtc1p is a small transmembrane protein. Vtc2p,
3p, and 4p contain the transmembrane domain
and additional large and sequence-related cyto-
plasmic segments (13). Within these segments,
we proteolytically defined 35-kD-sized domains.
We determined the 2.1 Å crystal structure of the
fragment Vtc2p183-553and the 2.6 Å structure of
Vtc4p* (Vtc4p189-480) in the presence of Li2SO4
or adenosine triphosphate (ATP)–MnCl2, respec-
norganic polyphosphate (polyP) occurs in all
life forms. In prokaryotes, polyP is a store of
phosphate and energy and augments stress
tively (see supporting online material) (tables S1
and S2). The structures are similar (root mean
squaredeviation~1.7Åbetween241correspond-
ing Caatoms) and structurally related to the RNA
triphosphataseCet1p(14).LikeCet1p,theycom-
prise a tunnel-shaped domain formed by antipar-
allel b strands (fig. S1A) with the tunnel walls
linedbyconservedbasicresidues.Vtc2p183-553and
Cet1pbothhaveasulfatecoordinatedinthetunnel
center (fig. S1B). In the case of Cet1p, the sulfate
mimics the g-phosphate of nucleoside triphos-
phates (14), which Cet1p hydrolyzes in the pres-
enceofMn2+(15).TheVtc4p*structurecontained
a long chain of electron density winding through
the tunnel domain (Fig. 1A), which suggests that
this module had generated phosphate polymers
from ATP during dialysis or crystallization. Dif-
ference density in our structure accounts for 29
phosphate units that are bound by two neighbor-
ing molecules in the asymmetric unit (fig. S2A).
Consistently, Vtc4p*-synthesized polyP can be de-
tected in solution (Fig.1B). Vtc4p* generates polyP
from ATP in a phosphotransfer reaction releas-
ing adenosine diphosphate (ADP) (Fig. 1C) (16).
Our results define Vtc4p* as a polyP-synthesizing
1European Molecular Biology Laboratory, Structural and Com-
putational Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg,
Germany.2PlantBiologyLaboratory,SalkInstituteforBiological
Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037,
USA.3Département de Biochimie, Universitéde Lausanne,
1066 Epalinges, Switzerland.4Physikalische Chemie 1, Ruhr-
Universität Bochum, 44780 Bochum, Germany.5Department
of Biochemistry and Molecular Biology, Baylor College of
Medicine, One Baylor Plaza, Houston, TX 77030, USA.
*Present address: Medical Research Council Laboratory of
Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
†Present address: Novartis Pharma Schweiz AG, Monbijoustrasse
118, 3007 Bern, Switzerland.
‡To whom correspondence should be addressed: E-mail:
scheffzek@embl.de
A
-10+10
B
C
A259nm [arbitrary units]
σ 65 marker
Vtc4p* + ATP
Vtc4p* + AppNHp
Vtc4p* alone
N
C
0
AMPADP ATP cAMP
retention time [min]
A259nm [arbitrary units]
0
1
234
56
7
01234567
0
10
20
30
40
0
10
20
30
40
Fig. 1. Vtc4p* is a polyP polymerase. (A) Ribbon diagram of Vtc4p* with the phosphate polymer (in bonds
representation) and an omit electron difference density map contoured at 4.5 s included. The helical plug is
shown in magenta. Mapping of the electrostatic potential [in kBT/e; as output by program GRASP (24)] on
the surface of Vtc4p* highlights the basic tunnel center and the negatively charged polymer (B) Urea
polyacrylamide gel electrophoresis of polyP generated by Vtc4p* from ATP and in the presence of Mn2+ions.
(C) High-performance liquid chromatography (HPLC) analysis of nucleotide products in the polymerization
reaction. (Top) Elution profile showing the retention times for different nucleotides. (Bottom) HPLC analysis
after incubating 30 mM Vtc4p* with 200 mM ATP for 0 (black line), 15 (blue), 45 (green), and 90 min (red).
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K200
R266
R264
S457
K458
Y359
E426
B
0.00.51.01.52.0
-14
-12
-10
-8
-6
-4
-2
0
-1.6
-1.4
-1.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
020 406080 100 120140
Time (min)
Molar Ratio
kcal/mole of injectant
µcal/sec
ligand
Kd
ATP
0.37 ± 0.06
GTP
1.10 ± 0.28
CTP
1.92 ± 0.20
dATP
0.28 ± 0.03
ADP
8.62 ± 1.65
polyP12-13
8.26 ± 0.53
AppNHp
0.33 ± 0.04
GppNHp
0.53 ± 0.08
Vtc4p*
Kd
(µM)
(µM)
wild-type
0.37 ± 0.06
0.30 ± 0.06
K200A
R264A
0.93 ± 0.13
R266A
1.58 ± 0.36
R264A/R266A
7.87 ± 0.55
Y359F
0.32 ± 0.03
S457A
1.80 ± 0.90
K458L1.74 ± 0.31
A
Vtc4p* vs. ATP
Kd= 0.37±0.06 µM
∆H= -13.5 kcal mol-1
Fig. 2. Unusual modes of nucleotide and metal cofactor binding in
Vtc4p*. (A) Close-up view of Vtc4p* tunnel center bound to AppNHp (in
bonds representation) and Mn2+(gray sphere). The sugar and base
portions were modeled stereochemically. The pentavalent coordination of
Mn2+is distorted square-based pyramidal. A phased anomalous differ-
ence map calculated from data collected at the Mn K edge and contoured
at 15 s is shown in green. (B) Isothermal titration calorimetry of wild-type
Vtc4p* versus ATP, together with table summaries for the binding constants
for different ligands and Vtc4p* mutants (versus ATP). Shown are experi-
mental values T fitting errors.
CD
A
K458
Y241
S457
K281
R264
B
R264
R266
Y359
K458
E426
K200
K458
R266
R264
Y241
S457
K200
K458
R361
K200
ATP [µM]
100
150
200
0102030 40
specific activity [10–3 min–1]
0
Vtc4p*
20
40
60
80
K200A
Y241F
R264AR266A
R264/266A
K281A
Y359F
E426A
S457A
K458L
EFG
polyP content [% of full−length Vtc4p WT]
K281
H283
K256
R253
K455
n=3
n=3
0
∆vct4
20
40
60
80
100
K200A
Y241F
R264AR266A
K281A
Y359F
E426A
S457A
K458L
time [min]
10 mM EDTA
1 mM Mn2+
1 mM Mn2+ + 1 mM PPi
Fig. 3. Structural views of the Vtc4p* polymerase cycle. (A) Structure of
Vtc4p* bound to orthophosphate. Coordinating residues are shown, along
with the catalytic Lys458(marked with an arrow) to facilitate comparison.
(B) Structure of the AppNHp-Mn2+complex. The Mn2+ion is shown as a
gray sphere; the position of the orthophosphate has been inferred from
(A). (C) Rotated view of Vtc4p* bound to PPi. One PPi occupies the
nucleotide binding site mimicking ADP; a second molecule occupies the
acceptor pocket. (D) Close-up view of the polyP exit tunnel. (E) ATP
turnover analyzed by HPLC at 30 mM Vtc4p* and 1 mM Mn2+(filled
circles), 1 mM additional PPi(open circles), or 10 mM EDTA (squares). (F)
ATP turnover rates for various Vtc4p* mutants at 30 mM. (G) Cellular
polyP content for Vtc4p point mutants expressed under the control of
their native promoter in yeast BY4742 Dvtc4. Error bars represent the
standard deviation derived from three independent measurements.
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enzyme, an activity that rationalizes the loss of
vacuolarpolyPinyeastvtc4deletionstrains(8)and
the concomitant increase in cellular ATP (9).
We located the substrate binding site in the
structureofVtc4p*crystallizedinthepresenceof
the substrate analog adenosine-5′-[(b, g)-imido]
triphosphate (AppNHp) and MnCl2. Difference
electron density accounts for the Mn2+-bound
triphosphate moiety of AppNHp, which is coor-
dinated by seven conserved basic residues and a
tyrosine in the tunnel center (Fig. 2A, and fig.
S3A).Thenucleosidemoietyappearsdisordered.
We can define the directionality of the nucleotide
by the topology of the tunnel domain that is
closed by a helical segment on one end (Fig. 1A)
(17). To validate the unusual substrate binding
mode, we quantified interaction of Vtc4p* with
different nucleotides by isothermal titration calo-
rimetry. Vtc4p* tightly interacts with ATP, as
well as with other nucleoside triphosphates
[dissociation constant (Kd) ranging from 0.3 to
2mM],witha1:1stoichiometryandhighbinding
enthalpy (Fig. 2B). Vtc4p* does not discriminate
ATP and its 2′-deoxy form, but the absence of a
g-phosphate substantially weakens the interac-
tion (Fig. 2B). Mutation of Arg264or Arg266,
which contact the ATP a- and b-phosphates, to
alanine decreases nucleotide binding (by a factor
of2.5to4)(Fig.2B)andVtc4pactivity(Fig.3,F
and G). Their simultaneous mutation substantial-
ly impairs binding (by a factor of 20) (Fig. 2B)
and inactivates Vtc4p* (Fig. 3F). Consistent with
these observations, our structure reveals extensive
protein contactsonly with the triphosphate moiety
of the nucleotide substrate (Fig. 2A).
ATP turnover is impaired in the presence of
EDTA (Fig. 3E), which indicates that Vtc4p*
activity requires bivalent cations. There is mod-
erate metal ion specificity with Mn2+> Zn2+>
Co2+> Mg2+> Fe2+> Ni2+(fig. S4). The
nucleotidea-,b-andg-phosphatescoordinatethe
Mn2+,whichinturnmakesabidentateinteraction
withGlu426(Fig.2A).Mutation ofGlu426reduces
ATP turnover and polyP synthesis (Fig. 3, F and
G). Mn2+coordination in Vtc4p* differs from the
previously reported octahedral binding geometry
inCet1pandothertunnelenzymes(14,18)(fig.S4).
We next addressed the catalytic mechanism
employed by Vtc4p. A structure of Vtc4p* crys-
tallized from Na+/K+phosphate reveals an ortho-
phosphate(Pi)boundclosetothenucleotidebinding
site (Fig. 3A). Superposition with the AppNHp-
bound structure shows the phosphate in this “ac-
ceptor pocket” in a binding geometry compatible
with anin-line transfer of the ATPg-phosphatein
a nucleophilic displacement reaction (Fig. 3B).
Consistently, we found that ATP turnover can be
stimulated by “priming” the reaction with either
orthophosphate (by a factor of 3 at 10 mM Pi) or
pyrophosphate (by a factor of 90 at 1 mM PPi)
(Fig. 3E and fig. S5). ATP cleavage is supported
by Mn2+that is positioned over the terminal scis-
sile phosphoanhydride (Fig. 2A). Mutation of
Lys458originating from the “helical plug” and di-
rectlyfacingtheg-phosphateimpairsATPturnover
(Fig.3,BandF),whichsuggestsaroleincatalysis.
To further validate the key position of the ac-
ceptorphosphate,wesolvedthestructureofVtc4p*
cocrystallized with PPi. The structure reveals dif-
ferencedensitypeaksinthesubstratebindingpock-
et and in the acceptor pocket. It may thus mimic
Vtc4p* after the first round of ATP cleavage
(Fig. 3C). Lys200contacts the g-phosphate of
AppNHp (Fig.3B) andthe transferred phosphate
in the acceptor pocket (Fig. 3C). Mutation of this
residue to alanine reduces polyP formation and
substantially enhances ATP turnover (Fig. 3, F
and G), seemingly uncoupling these steps.
Thesubstrate-boundstructuressuggestthatthe
polyP complex (Fig. 1A) represents a product-
bound state in the polymerization process where
polyPoccupiestheentiretunneldomain,including
the substrate binding and acceptor pockets (fig.
S2B). This structure defines a path along which
polyP may be transported. Several conserved basic
residues align like bristles, guiding the polymer
away from the active site (Fig. 3D). Notably, the
tunneldomaininVtc4p*presentsitselfmoreclosed
in the nucleotide-bound state, when compared with
thePi-boundstructure(fig.S2C).Wespeculate that
substrate binding and polyP transport in Vtc4p*
maybedrivenbysimilarconformationalchanges.
We next investigated Vtc4p-catalyzed polyP
formationinvivo.Yeastvtc4deletionstrainslack
the entire vacuolar polyP pool (8) (Fig. 3G).
Point mutations in the full-length Vtc4 protein
that correspond to those found essential for
Vtc4p* function in vitro (R264A, R266A,
K281A, E426A, and K458L) (Fig. 3F) cannot
complementthis deficiency(Fig.3G).Inmedia
with limiting phosphate concentration, where the
internal polyP store is used, these point mutants
also arrested growth clearly faster than wild-type
cells (fig. S3B). Thus, VTC accounts for most of
the polyP synthesis in yeast, and the catalytic ac-
tivity resides in the tunnel domain of Vtc4p.
Infungi,polyPaccumulatesinthevacuoleand
in the extracellular space (19). Correspondingly,
we detected two VTC subcomplexes composed
of Vtc1/2/4p or of Vtc1/3/4p (fig. S6). Vtc2p and
3p are catalytically impaired and appear to be
accessory subunits in VTC (fig. S7). We studied
green fluorescent protein (GFP)–tagged Vtc2p
and 3p in yeast cells by microscopy. GFP-Vtc3p
predominantly stains vacuoles on Pi-rich media
as well as under low Piconditions (Fig. 4A). In
contrast,GFP-Vtc2pisonlypartiallyonvacuoles
but mainly around the nucleus and at the cell
periphery along the plasma membrane, where it
is highly enriched in patches. Upon transfer of
cellstolow-phosphate medium,these patches dis-
appear and GFP-Vtc2p concentrates on vacuoles
(Fig. 4A), perhaps to maximize synthesis of the
vacuolar polyP store that buffers fluctuations in
exogenous phosphate (20). We assume that the
peripheral VTC pool may generate extracellular
polyP(19).Atbothmembranes,thecatalytictunnel
domain in VTC faces the cytoplasm (13), and thus
the polymer must pass the membrane. Notably, all
Vtcs share related transmembrane domains that
contain conserved basic residues. Vtc1 point mu-
tations targeting these residues drastically reduce
cellular polyP levels (Fig. 4B and fig. S8), while
leavingVTCcomplexesstableandcorrectlysorted
(fig. S6). We thus speculate that Vtc trans-
membrane domains participate in the transport of
polyP across the membrane. Phosphate polymer-
ization and membrane translocation would thus be
orchestrated functions of VTC. Purification and
GFP-
Vtc2p
GFP-
Vtc3p
-Pi
+Pi
A
∆vct1
K24EK24S
R31E
R83E
R88−90E
R98E
polyP content [% of Vtc1 WT]
0
20
40
60
80
100
B
n=3
Fig. 4. Phosphate-dependentsub-
cellular distribution of Vtc2p or 3p
subcomplexes. (A) GFP-Vtc2p or
GFP-Vtc3p integrated into strain
BY4742 was expressed under the
control of an ADH promoter. Logarithmically growing cells were transferred for 2 hours to phosphate-
depleted yeast extract, peptone, and dextrose medium (YPD) replenished with 0 (-Pi) or 5 mM (+Pi)
Na-phosphate pH 5.5 and analyzed by spinning disc microscopy. Nucleus (N), vacuole (V), and
peripheral (P) concentrations are labeled. (B) Point mutations in the Vtc1p transmembrane domain
impact cellular polyP levels. Vtc1 mutants were expressed under the control of the vtc1 promoter
from pRS304 plasmids integrated into the TRP1 locus of BY4727 vtc1::HIS.
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biochemical reconstitution of the entire VTC com-
plex will be necessary to test this hypothesis.
Ouridentificationof aeukaryotic polyP poly-
merase now allows investigating polyphosphate
metabolism in fungi such as Laccaria (21) (fig.
S9) that deliver polyP to the roots of their plant
hosts, in diatoms such as Thalassiosira (22) (fig.
S9),whosepolyP pools formmarinesediments (5),
and in parasites where VTC is essential for osmo-
regulation (23). Because the VTC complex appears
not to be conserved in animals or plants, another
classofenzymesremainstobediscoveredthatcata-
lyzes phosphate polymerization in these organisms.
References and Notes
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16. Using recombinant Vtc4p* in vitro, we did not detect
regeneration of ATP from ADP and either a polyP 3-,
~12-, or ~65-nucleotide oligomer.
17. Modeling of the nucleotide substrate in the opposite
direction leads to severe clashes of the ribose and base
portions with main chain atoms in the C-terminal helix
in Vtc4p*. The position of this helical plug appears
invariant in all crystal forms analyzed, making the helix
unlikely to move upon substrate binding. Further, our
assigned directionality of the nucleotide is consistent with
the positions of the acceptor pocket and the polyP exit
tunnel, respectively (Fig. 3, A and D, and fig. S3B).
18. N. Keppetipola, R. Jain, S. Shuman, J. Biol. Chem. 282,
11941 (2007).
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24. A. Nicholls, K. A. Sharp, B. Honig, Proteins 11, 281
(1991).
25. With the exception of the AppNHp-Mn2+and PPicomplexes
(determined at the Salk Institute), all crystallographic work
was carried out at the European Molecular Biology
Laboratory. We thank J. Noel, T. Gibson, and T. A. Steitz for
discussion and J. Chory for generous support. This work was
funded by the Peter and Traudl Engelhorn Foundation
(Penzberg, Germany) and a European Molecular Biology
Organization long-term fellowship (M.H), the Boehringer
Ingelheim Fonds (A.U.), the German Science Foundation
and the Landesstiftung Baden-Württemberg (K.S.), the
Swiss National Science Foundation (A.M, P.O.H), and the
National Science Foundation (IOS-0649389 to J. Chory). We
thank the staff at the European Synchrotron Radiation
Facility, Grenoble, France, at the Deutsches Elektronen
Synchrotron, Hamburg, Germany, and at the Advanced Light
Source, Berkeley, for technical support; and C. Müller and
S. Cusack for sharing beam time. Coordinates and structure
factors for Vtc2p183-553(PDB-ID 3g3o), Vtc4p*-polyP (3g3q),
Vtc4p*-AppNHp-Mn2+(3g3r), Vtc4p*-Pi(3g3t), and
Vtc4p*- PPi(3g3u) have been deposited with the Protein
DataBank (www.rcsb.org).
Supporting Online Material
www.sciencemag.org/cgi/content/full/324/5926/513/DC1
Materials and Methods
Figs. S1 to S9
Tables S1 and S2
References
5 November 2008; accepted 9 March 2009
10.1126/science.1168120
Homeostatic Sleep Pressure and
Responses to Sustained Attention
in the Suprachiasmatic Area
Christina Schmidt,1,2* Fabienne Collette,1,2Yves Leclercq,1Virginie Sterpenich,1
Gilles Vandewalle,1Pierre Berthomier,3Christian Berthomier,3Christophe Phillips,1
Gilberte Tinguely,1Annabelle Darsaud,1Steffen Gais,1Manuel Schabus,1Martin Desseilles,1
Thien Thanh Dang-Vu,1Eric Salmon,1Evelyne Balteau,1Christian Degueldre,1André Luxen,1
Pierre Maquet,1Christian Cajochen,4Philippe Peigneux1,5*
Throughout the day, cognitive performance is under the combined influence of circadian processes
and homeostatic sleep pressure. Some people perform best in the morning, whereas others are
more alert in the evening. These chronotypes provide a unique way to study the effects of sleep-
wake regulation on the cerebral mechanisms supporting cognition. Using functional magnetic
resonance imaging in extreme chronotypes, we found that maintaining attention in the evening
was associated with higher activity in evening than morning chronotypes in a region of the locus
coeruleus and in a suprachiasmatic area (SCA) including the circadian master clock. Activity in the
SCA decreased with increasing homeostatic sleep pressure. This result shows the direct influence
of the homeostatic and circadian interaction on the neural activity underpinning human behavior.
M
sity progressively overrides the wake-promoting
circadian signal (1–3). Morning-type individuals
wake up early and find it difficult to maintain per-
formance in the evening (4). In contrast, evening
types perform well in the evening hours (4) and
seem to tolerate elevated sleep pressure in the eve-
ningbetterthanmorningtypes.Besidesdifferences
incircadianphase(5–8),morningtypesdifferfrom
evening types by a faster build-up of homeostatic
aintaining optimal performance levels
during evening hours may become dif-
ficult because increasing sleep propen-
sleep pressure during daytime (9) and by its faster
dissipation during sleep (10, 11). Taken together,
these results support the notion that the circadian
variationinwakingperformancealsodependson
homeostatic sleep pressure (2, 12, 13). Using
functional magnetic resonance imaging (fMRI) in
extreme chronotypes, we investigated the neural
correlates of performance in the psychomotor vig-
ilance task (PVT) (14–16), a simple reaction-time
task that probes the ability to maintain sustained
attention (14), which is modulated by circadian
and sleep homeostatic regulatory processes (17).
Young, healthy participants of extreme morn-
ing (n = 16) or evening (n = 15) typology (18)
werematchedatthegrouplevelaccordingtoage,
sex, and educational level and did not differ in
their anxiety and depression levels or in sleep
qualityanddaytimesleepiness(allPvalues>0.1)
(table S1). Participants were instructed to live ac-
cording to their own preferred sleep-wake sched-
ule for at least 1 week before the study (16).
Afterwards, they reported to the sleep laboratory
for two consecutive nights, where they were
monitored by polysomnography at their preferred
bedtimes (Fig. 1). The laboratory protocol started
7 hours before their habitual sleep time, which
allowed for hourly assessments of subjective
sleepiness (19) and objective vigilance (14), as
well as hourly collections of saliva samples for
assessment of circadian melatonin phase (16).
Onaverage,bedtime,waketime,andcircadian
phase[estimatedbythemelatoninmidrangecross-
ing(MRC)time(16)]wereabout4hoursearlierin
morning than in evening types (all P values <
0.05) (table S1). These results indicate circadian
phase entrainment with a similar phase angle (i.e.,
similar distance between MRC and bedtimes, P >
0.2)(tableS1)inbothchronotypes.Oneandahalf
hours (morning session) after scheduled awaken-
1Cyclotron Research Centre, University of Liège, 4000 Liège,
Belgium.2Cognitive and Behavioral Neuroscience Centre, Uni-
versity of Liège, 4000 Liège, Belgium.3PHYSIP S.A., 75011
Paris, France.4Centre for Chronobiology, Psychiatric Hospital of
the University of Basel, CH-4025 Basel, Switzerland.5Neuropsy-
chology and Functional Neuroimaging Research Unit (UR2NF),
UniversitéLibre de Bruxelles, B-1050 Brussels, Belgium.
*To whom correspondence should be addressed. E-mail:
Christina.Schmidt@ulg.ac.beandPhilippe.Peigneux@ulb.ac.be
24 APRIL 2009VOL 324
SCIENCE
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