Ternary complex formation of pVHL, elongin B and
elongin C visualized in living cells by a fluorescence
resonance energy transfer–fluorescence lifetime
imaging microscopy technique
Koshi Kinoshita
1,
*, Kenji Goryo
1,
*, Mamiko Takada
2
, Yosuke Tomokuni
1
, Teijiro Aso
3
,
Heiwa Okuda
4
, Taro Shuin
4
, Hiroshi Fukumura
2
and Kazuhiro Sogawa
1
1 Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Aoba-ku Sendai, Japan
2 Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku Sendai, Japan
3 Department of Functional Genomics, Kochi Medical School, Kohasu, Okoh-cho, Nankoku Kochi, Japan
4 Department of Urology, Kochi Medical School, Kohasu, Okoh-cho, Nankoku Kochi, Japan
The von Hippel–Lindau (VHL) gene is located on the
short arm of chromosome 3 and its deletions or muta-
tions are associated with VHL disease [1,2]. Affected
individuals develop a variety of tumors, including

elongin C in CHO-K1 cells. FRET signals were examined by measuring a
change in the fluorescence lifetime of donors and by monitoring a corre-
sponding fluorescence rise of acceptors. Clear FRET signals between elon-
gin B and elongin C were observed in all combinations, except for the
combination of elongin B-cerulean and citrine-elongin C. Although similar
experiments to examine interaction between pVHL30 and elongin C linked
to cerulean or citrine were performed, FRET signals were rarely observed
among all the combinations. However, the signal was greatly increased by
coexpression of elongin B. These results, together with results of coimmuno-
precipitation experiment using pVHL, elongin C and elongin B, suggest that
a conformational change of elongin C and ⁄ or pVHL was induced by binding
of elongin B. The conformational change of elongin C was investigated by
measuring changes in the intramolecular FRET signal of elongin C linked to
cerulean and citrine at its N- and C-terminus, respectively. A strong FRET
signal was observed in the absence of elongin B, and this signal was modestly
increased by coexpression of elongin B, demonstrating that a conformation
change of elongin C was induced by the binding of elongin B.
Abbreviations
FLIM, fluorescence lifetime imaging microscopy; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; VHL, von
Hippel–Lindau.
FEBS Journal 274 (2007) 5567–5575 ª 2007 The Authors Journal compilation ª 2007 FEBS 5567
second methionine [5,6]. Both pVHL proteins are asso-
ciated with two ubiquitous proteins, elongin B and
elongin C, to form a ternary complex (hereafter
referred to as the VBC complex), and its formation is
required for tumor suppressor functions.
Elongin B and elongin C were initially found
together with elongin A in the elongin (SIII) complex
that increases the efficiency of elongation by RNA
polymerase II [7,8]. Biochemical analysis of the com-

radation domain of the factors are recognized by the
pVHL in the E3 ligase and subsequent ubiquitination
of the factors results in degradation by proteasomes.
Lowered oxygen levels in hypoxia down-regulate prolyl
hydroxylation and increase stabilization of the factors.
Degradation of the factors in normoxia and their sta-
bilization in hypoxia comprise the pivotal mechanism
for cellular hypoxic responses such as the promotion
of glycolysis and vascularization [17,18].
Fluorescence lifetime imaging microscopy (FLIM) is
a recently developed technique that can be applied to
measure fluorescence lifetimes of fluorescent proteins
such as green fluorescent protein (GFP) in living cells.
When combined with fluorescence resonance energy
transfer (FRET), this measurement presents unambigu-
ous evidence for spatial and temporal interactions
between proteins and conformational changes of pro-
teins occurring in living cells. The occurrence of FRET
can be accurately and finely determined by measuring
the reduced fluorescence lifetime of donor proteins in
the presence of acceptors. Because fluorescence lifetime
is, in principle, unaffected by changes in probe concen-
tration or excitation intensity, FRET–FLIM has
advantages over intensity-based FRET techniques. In
particular, FRET–FLIM has advantages in intermole-
cular FRET measurement in which expression levels of
the two fluorescent proteins cannot be easily controlled
in individual cells [19–21].
In the present study, we monitored the fluorescence
rise of acceptor fluorescent proteins as distinctive evi-

with ratio coefficients of 37.9% and 62.1%, respec-
tively (Table 1). The decay curve of elongin B-cerulean
was similarly analyzed as shown in Fig. 1E,F, and the
lifetimes, 1.38 ns and 3.41 ns, were almost identical to
FRET imaging of the VBC complex K. Kinoshita et al.
5568 FEBS Journal 274 (2007) 5567–5575 ª 2007 The Authors Journal compilation ª 2007 FEBS
those of cerulean-elongin B (Table 1). The v
2
values of
the fit were between 1.0 and 1.3 and between 1.0 and
1.2, respectively, indicating that the overall model fit-
ting was statistically significant. The decays were also
analyzed according to a three-exponential model as
reported by Millington et al. [22], resulting in only a
modest improvement of fit as judged from v
2
values;
the values were reduced by approximately 4% or less
by the three-exponential fitting.
Next, we coexpressed acceptor fluorescent proteins
together with donor fluorescent proteins in the follow-
ing four combinations: cerulean-elongin B and citrine-
elongin C; cerulean-elongin B and elongin C-citrine;
elongin B-cerulean and elongin C-citrine; and elon-
gin B-cerulean and citrine-elongin C. Transfected cells
with coexpression of moderate amounts of two fluo-
rescent proteins, cerulean-elongin B and citrine-
elongin C, were randomly chosen for measuring
fluorescence decay of the two proteins. As shown in
Fig. 1C, decay of fluorescence of cerulean-elongin B in

0.01
0
1
0.1
Intensity/a.u.
2
4
6
810
12
14
Time/ns
Cit
0.01
0
1
0.1
Intensity/a.u.
2
4
6
810
12
14
Time/ns
0.01
0
1
0.1
Intensity/a.u.

12
14
Time/ns
Ceru
0.01
0
1
0.1
Intensity/a.u.
2
4
6
810
12
14
Time/ns
0.01
0
1
0.1
Intensity/a.u.
2
4
6
810
12
14
Time/ns
Ceru-EloB
Ceru-EloB

3.5
4.0
(ns)
Ceru-EloB
Ceru-EloB + Cit-EloC
Ceru-EloB Cit-EloCEloB-Ceru EloC-Cit
mock
Ceru-EloB
EloB-Ceru
Cit-EloC
EloC-Cit
A
C
D
E
F
B
Fig. 1. FLIM analysis of interaction between elongin B and elon-
gin C in CHO-K1 cells. (A) Cellular localization of elongin B linked to
cerulean and elongin C linked to citrine. Chimeric proteins, ceru-
lean-elongin B (Ceru-EloB), elongin B-cerulean (EloB-Ceru), citrine-
elongin C (Cit-EloC) and elongin C-citrine (EloC-Cit) were transiently
expressed in CHO-K1 cells by DNA transfection using the lipofec-
tion method. Forty hours after transfection, fluorescence of ceru-
lean and citrine moieties of the chimeric proteins was observed
with an Olympus BX50 fluorescent microscope with a filter set
(Olympus U-MCFPHQ and U-MYFPHQ). Scale bar ¼ 20 lm. A typi-
cal result of immunoblot analysis of whole cell extracts of cells
expressing cerulean-linked elongin B or citrine-linked elongin C was
shown using anti-GFP serum, as shown below. Lane 1, mock;

A chimeric fluorescent protein, pVHL30-cerulean, was
expressed in CHO-K1 cells by DNA transfection. As
shown in Fig. 2A, it was distributed throughout the
cells with stronger expression in the cytoplasm. By
western blotting analysis, it was found that a small
amount of pVHL19-cerulean was also expressed. Life-
times were determined on the FLIM microscope as
shown in Table 2. We constructed a plasmid only for
expression of pVHL19-cerulean, introduced it into the
Table 1. Fluorescence decay data for cerulean-linked elongin B and citrine-linked elongin C expressed in living CHO-K1 cells. Data are
derived from whole cell regions of interest and are expressed as mean ± SD. a
1
and a
2
are the exponential coefficients (%) for the s
1
and s
2
decay times, respectively. n, number of cells examined.
Combination of protein a
1
(%) s
1
(ns) a
2
(%) s
2
(ns) n v
2
Cerulean-elongin B 37.9 1.32 ± 0.06 62.1 3.54 ± 0.08 10 1.0–1.3

Time/ns
0.01
1
0.1
Intensity/a.u.
02468101214
Time/ns
HA-pVHL
FLAG-EloC
Myc-EloB
-
-
-
-
+
-
+
+
-
-
+
+
+
+
+
WB : anti-FLAG
WB : anti-Myc
WB : anti-FLAG
WB : anti-HA
pVHL

ric protein, pVHL-cerulean (pVHL-Ceru), was transiently expressed
in CHO-K1 cells by DNA transfection using the lipofection method.
Forty hours after transfection, fluorescence of cerulean moiety of
the chimeric proteins was observed with an Olympus BX50 fluores-
cent microscope with a filter set (Olympus U-MCFPHQ). Scale
bar ¼ 20 lm. A typical result of western blotting for expressed pro-
teins of pVHL-cerulean is shown on the right. CHO-K1 cells were
transfected with plasmids encoding (B) pVHL-cerulean and citrine-
elongin C and (C) pVHL-cerulean and citrine-elongin C coexpressed
with elongin B. The fluorescence decay curve of cerulean (shown
in blue) and citrine (shown in green) represents an average of fluo-
rescence decay data obtained from cells observed. For comparison,
the decay curve of pVHL-cerulean without acceptor protein (shown
in black) or the decay curve of citrine-linked elongin C without
donor protein (shown in black) are also shown. (D) Coimmunopre-
cipitation analysis of pVHL, elongin B and elongin C. HA-pVHL,
myc-elongin B and Flag-elongin C were expressed in CHO-K1 cells.
Whole cell extracts were treated with anti-Flag serum. Co-precipi-
tated proteins were visualized with anti-HA, anti-Flag or anti-myc
sera after electrophoresis and subsequent electroblotting to a nitro-
cellulose membrane; 5% input is shown.
FRET imaging of the VBC complex K. Kinoshita et al.
5570 FEBS Journal 274 (2007) 5567–5575 ª 2007 The Authors Journal compilation ª 2007 FEBS
cells and measured lifetimes of expressed pVHL19-
cerulean. Almost identical lifetimes to those of
pVHL30-cerulean were obtained (data not shown).
When an acceptor chimeric protein, citrine-elongin C
was coexpressed with pVHL30-cerulean, the lifetimes
of cerulean moiety showed only a minimal decrease
(Fig. 2B and Table 2). We expressed donor and accep-

Increased FRET signals between pVHL-cerulean and
citrine-elongin C by coexpression of elongin B suggest
that a conformation change of elongin C induced by
binding of elongin B may occur and that this confor-
mational change of elongin C leads to stabilization of
elongin C and pVHL. To visualize the conformational
change in living cells, intramolecular FRET measure-
ment using a chimeric protein of cerulean-elongin C-
citrine was carried out in the presence or absence of
elongin B. Without the coexistence of elongin B, a con-
siderable decrease in donor fluorescence lifetime was
observed (Fig. 3B and Table 3) compared to that of
cerulean-elongin C-citrine(Y66A) in that fluorophore
formation in the citrine moiety was abolished by the
mutation of Tyr66 to Ala (Fig. 3A). A decrease in the
lifetimes was further augmented by the coexpression of
elongin B as shown in Fig. 3D and Table 3. This
decrease was modest but reproducible in three indepen-
dent experiments. Coimmunoprecipitation experiments
indicated that the presence of fluorescent proteins at
N- and C-terminal ends of elongin C did not affect the
binding of elongin B to elongin C moiety (Fig. 3C).
Discussion
We used cerulean as the FRET donor because the flu-
orescence lifetime of this protein is reported to be the
best fit by a single exponential [23], which greatly sim-
plifies quantitative analysis of FRET data compared to
donors with a double exponential decay. However, our
results clearly demonstrated that the decay curve of
cerulean is the best fit by a double exponential such as

(%) s
1
(ns) a
2
(%) s
2
(ns) n v
2
VHL-cerulean 39.0 1.26 ± 0.06 61.0 3.40 ± 0.07 8 1.0–1.2
VHL-cerulean-citrine-elongin C 43.5 1.23 ± 0.13 56.5 3.38 ± 0.18 10 1.0–1.2
VHL-cerulean-citrine-elongin C-elongin B 51.4 1.05 ± 0.05 48.6 3.18 ± 0.20 10 1.0–1.2
K. Kinoshita et al. FRET imaging of the VBC complex
FEBS Journal 274 (2007) 5567–5575 ª 2007 The Authors Journal compilation ª 2007 FEBS 5571
the FLIM microscope used in the present study were
very low (approximately 15 mWÆcm
)2
) so that no pho-
todynamic reactions took place.
FRET signals between cerulean-linked elongin B
and citrine-linked elongin C can be detected in the fol-
lowing donor-acceptor combinations in decreasing
order: cerulean-elongin B and citrine-elongin C >
cerulean-elongin B and elongin C-citrine  elongin B-
cerulean and elongin C-citrine. FRET signals from the
pair of elongin B-cerulean and citrine-elongin C were
modest (Table 1). Since the rate of energy transfer
depends on the inverse sixth power of the distance
between donor and acceptor, this result matches with
the results from the X-ray crystallography of the VBC
complex [26]; the distance between the C-terminal end

0.1
Intensity/a.u.
0
2
4
6
810
12
14
Time/ns
0.01
1
0.1
Intensity/a.u.
0
2
4
6
810
12
14
Time/ns
0.01
1
0.1
Intensity/a.u.
0
2
4
6

Ceru(W66A)-EloC-Cit
Ceru-EloC-Cit(Y66A)
-
-
-
-
+
-
-
-
+
+
-
-
+
-
+
-
+
-
-
+
WB : anti-Myc
WB : anti-GFP
WB : anti-Myc
WB : anti-GFP
Elongin C
Elongin C
Elongin B
Elongin B

B
C
D
E
Fig. 3. Intramolecular FRET of elongin C conjugated with cerulean
and citrine at its N- and C-termini, respectively. (A) Cellular
images expressing cerulean-elongin C-citrine or its mutant pro-
teins. Chimeric proteins, cerulean-elongin C-citrine and its mutant
proteins, cerulean(W66A)-elongin C-citrine and cerulean-elongin C-
citrine(Y66A), were transiently expressed in CHO-K1 cells by DNA
transfection using the lipofection method. Forty hours after trans-
fection, fluorescence of cerulean and citrine moieties of the chime-
ric proteins was observed with an Olympus BX50 fluorescent
microscope with a filter set (Olympus U-MCFPHQ and U-MY-
FPHQ). Scale bars ¼ 20 lm. A typical result of western blotting for
expressed proteins is shown on the right. (B) FLIM analysis of
cerulean-elongin C-citrine in living CHO-K1 cells. CHO-K1 cells
were transfected with a plasmid encoding cerulean-elongin C-
citrine for FLIM analysis. For comparison, the decay curve of ceru-
lean-elongin C-citrine(Y66A) or cerulean(W66A)-elongin C-citrine is
shown. (C) FLIM analysis of cerulean-elongin C-citrine expressed
with elongin B. For comparison, the decay curve of cerulean-
elongin C-citrine(Y66A) or cerulean(W66A)-elongin C-citrine coex-
pressed with elongin B is shown. (D) Comparison of the decay
curves of cerulean-elongin C-citrine expressed with or without
elongin B. Two decay curves of cerulean-elongin C-citrine obtained
in the absence or presence of elongin B are shown in blue and red,
respectively. (E) Coimmunoprecipitation analysis of cerulean-elon-
gin C-citrine with elongin B. A plasmid for cerulean-elongin C-citrine
or its mutants was introduced into CHO-K1 cells with a plasmid for

FRET imaging of the VBC complex K. Kinoshita et al.
5572 FEBS Journal 274 (2007) 5567–5575 ª 2007 The Authors Journal compilation ª 2007 FEBS
elongin C is relatively long (4.7 nm) compared to dis-
tances (2–3 nm) between other combinations of termi-
nal ends of elongin B and elongin C, although the
effects caused by the binding of pVHL on the 3D struc-
ture of the elongin BC complex are not exactly known.
The present study has clarified that conformation of
pVHL and ⁄ or elongin C in the absence of elongin B
was different from that in the VBC complex and that
conformation of elongin C was changed upon binding
of elongin B. The coimmunoprecipitation experiment
(Fig. 2D) demonstrated that a remarkable stabilization
of elongin C was caused by the binding of elongin B
and, to a lesser extent, stabilization of pVHL was also
found as previously reported [27,28]. The conforma-
tional change of elongin C may be associated with the
stabilization of the proteins. To date, the role of elon-
gin B in the large E3 ubiquitin-ligase complex includ-
ing the VBC-Cul2-Rbx1 is not understood because no
direct interaction is present between elongin B and
other components except for elongin C, and the fact
that there is no obvious elongin B homologue in yeast
obscured its physiological function [29]. The present
study strongly suggests that elongin B is required to
alter the conformation of elongin C that leads to sta-
bilization of elongin C and pVHL.
In summary, we have shown that interactions
between components of the VBC complex can be visu-
alized in living cells by a FRET–FLIM technique.

CGGGATATCGGCGTAGTCGGGCACGTCGTAGGGG
TACATGGTGGT-3¢, into the XbaI site of pBOS Vector.
pBOS-Myc and pBOSFlag were constructed similarly by
using the synthesized oligonucleotides 5¢-CTAGACCA
CCATGGAGGAACAGAAGCTGATCAGTGAGGAAG
ACCTGGATATCCCGGGTTAACT-3¢ and 5¢-CTAGAG
TTAACCCGGGATATCCAGGTCTTCCTC ACTGATCA
GCTTCTGTTCCTCCATGGTGGT-3¢, and 5¢-CTAGAC
CACCATGGACTACAAAGACGATGACGATAAAGAT
ATCCCGGGTTAACT-3¢ and 5¢-CTAGAGTTAACCCGG
GATATCTTTATCGTC ATCGTCTTT GTAGTCC ATGG
TGGT-3¢, respectively. pBOS-HA-pVHL was constructed
by inserting the blunt-ended XhoI-AgeI fragment of pVHL-
cerulean into the HpaI site of pBOS-HA. PBOS-FLAG-
elongin C was constructed by inserting the blunt ended
BstBI-SmaI fragment of pCIneo-elongin C into the SmaI
site of pBOS-FLAG. PBOS-Myc-elongin B was constructed
by inserting blunt-ended XhoI-SmaI fragment of pCIneo-
elongin B into the HpaI site of pBOS-Myc. pCerulean-elon-
gin C-citrine was constructed by inserting the EcoRV-HpaI
fragment of the plasmid for elongin C-citrine into the
EcoRV-HpaI site of pcerulean-elongin C. pCerulean
(W66A)-C1 was constructed by site-directed mutagenesis,
using the primers 5¢-CGTGACCACCCTGACCGCGGG
CGTGCAGTGCTTC-3¢ and 5¢-GAAGCACTGCACGCC
CGCGGTCAGGGTGGTCACG-3¢. pCerulean(W66A)-
elongin C-citrine was constructed by inserting the
BsrGI-EcoRI fragment of pcerulean-elongin C and the
EcoRI-HpaI fragment of elongin C-citrine into the BsrGI-
HpaI site of pcerulean(W66A)-C1. pcitrine(Y66A)-N1 was

Whole cell extracts were prepared from CHO-K1 cells
transfected with plasmids encoding chimeric fluorescent
proteins by mixing 10 mm Tris ⁄ HCl buffer, pH 7.5, con-
taining 1 mm EDTA, 0.15 m NaCl, 1 mm dithiothreitol,
1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS,
10 lm MG132 and protease inhibitor cocktail (Roche). Pro-
teins were resolved by 12% SDS ⁄ PAGE, and transferred to
a nitrocellulose membrane (GE Healthcare, Piscataway, NJ,
USA). Polyclonal anti-GFP serum (Clontech, Mountain
View, CA, USA) diluted 1 : 1000 and donkey anti-rabbit
horseradish peroxidase linked IgG (GE Healthcare) diluted
1 : 10000 were used as the first and second antibodies,
respectively. The membrane was developed using the ECL
plus detection system (GE Healthcare). CHO-K1 cells were
transfected with plasmids for HA-tagged pVHL, Flag-
tagged elongin C and myc-tagged elongin B, harvested,
lysed and exposed to Flag-affinity agarose beads (Sigma)
that had been pretreated with anti-Flag serum. Proteins
bound to washed beads were eluted, boiled and separated
by 15% SDS ⁄ PAGE. After electrophoresis, the proteins
were blotted onto a nitrocellulose membrane and probed
with anti-FLAG (Sigma), anti-HA (MBL, Nagoya, Japan)
or anti-Myc (MBL) sera. Coimmunoprecipitation of elon-
gin B and cerulean-elongin C-citrine was similarly per-
formed.
Measurement of fluorescence lifetime
Techniques to measure FRET include FLIM to detect
decreases in the lifetime of donor fluorescence and fluores-
cence rise in the acceptor decay curve that are accompanied
by FRET. FLIM measurements were conducted on the live

References
1 Latif F, Tory K, Gnarra J, Yao M, Duh FM, Orcutt
ML, Stackhouse T, Kuzmin I, Modi W, Geil L et al.
(1993) Identification of the von Hippel–Lindau disease
tumor suppressor gene. Science 260, 1317–1320.
2 Chen F, Kishida T, Yao M, Hustad T, Glavac D, Dean
M, Gnarra JR, Orcutt ML, Duh FM, Glenn G et al.
(1995) Germline mutations in the von Hippel–Lindau
disease tumor suppressor gene: correlations with pheno-
type. Hum Mutat 5, 66–75.
3 Foster K, Prowse A, van den Berg A, Fleming S, Huls-
beek MM, Crossey PA, Richards FM, Cairns P, Affara
NA, Ferguson-Smith MA et al. (1994) Somatic muta-
tions of the von Hippel–Lindau disease tumour suppres-
sor gene in non-familial clear cell renal carcinoma. Hum
Mol Genet 3, 2169–2173.
4 Gnarra JR, Tory K, Weng Y, Schmidt L, Wei MH, Li
H, Latif F, Liu S, Chen F, Duh FM et al. (1994) Muta-
tions of the VHL tumour suppressor gene in renal carci-
noma. Nat Genet 7, 85–90.
5 Iliopoulos O, Ohh M & Kaelin WG Jr (1998) pVHL
19
is a biologically active product of the von Hippel–Lin-
dau gene arising from internal translation initiation.
Proc Natl Acad Sci USA 95, 11661–11666.
6 Schoenfeld A, Davidowitz EJ & Burk RD (1998) A sec-
ond major native von Hippel–Lindau gene product, ini-
tiated from an internal translation start site, functions
as a tumor suppressor. Proc Natl Acad Sci USA 95,
8817–8822.

of prolyl-4-hydroxylases that modify HIF. Science 294,
1337–1340.
14 Epstein AC, Gleadle JM, McNeill LA, Hewitson KS,
O’Rourke J, Mole DR, Mukherji M, Metzen E, Wilson
MI, Dhanda A et al. (2001) C. elegans EGL-9 and mam-
malian homologs define a family of dioxygenases that
regulate HIF by prolyl hydroxylation. Cell 107, 43–54.
15 Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh
M, Salic A, Asara JM, Lane WS & Kaelin WG Jr
(2001) HIFa targeted for VHL-mediated destruction by
proline hydroxylation: implications for O
2
sensing. Sci-
ence 292, 464–468.
16 Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert
J, Gaskell SJ, Kriegsheim AV, Hebestreit HF, Mukherji
M, Schofield CJ et al. (2001) Targeting of HIF-a to the
von Hippel–Lindau ubiquitylation complex by O
2
-regu-
lated prolyl hydroxylation. Science 292, 468–472.
17 Hirota K & Semenza GL (2005) Regulation of hypoxia-
inducible factor 1 by prolyl and asparaginyl hydroxylas-
es. Biochem Biophys Res Commun 338, 610–616.
18 Masson N & Ratcliffe PJ (2003) HIF prolyl and aspar-
aginyl hydroxylases in the biological response to intra-
cellular O(2) levels. J Cell Sci 116, 3041–3049.
19 Suhling K, French PM & Phillips D (2005) Time-
resolved fluorescence microscopy. Photochem Photobiol
Sci 4, 13–22.

27 Schoenfeld AR, Davidowitz EJ & Burk RD (2000)
Elongin BC complex prevents degradation of von Hip-
pel–Lindau tumor suppressor gene products. Proc Natl
Acad Sci USA 97, 8507–8512.
28 Kamura T, Brower CS, Conaway RC & Conaway JW
(2002) A molecular basis for stabilization of the von
Hippel–Lindau (VHL) tumor suppressor protein by
components of the VHL ubiquitin ligase. J Biol Chem
277, 30388–30393.
29 Koth C, Botuyan MV, Moreland RJ, Jansma DB,
Conaway JW, Conaway RC, Chazin WJ, Friesen JD,
Arrowsmith CH & Edwards AM (2000) Elongin from
Saccharomyces cerevisiae. J Biol Chem 275, 11174–11180.
30 Sato F, Yasumoto K, Numayama-Tsuruta K & Sogawa
K (2005) Heterodimerization with LBP-1b is necessary
for nuclear localization of LBP-1a and LBP-1c. Genes
Cells 10, 861–870.
31 Kemnitz K, Pfeifer L, Paul R & Coppey-Moisan M
(1997) Novel detectors for fluorescence lifetime imaging
on the picoscecond time scale. J Fluoresc 7, 93–98.
32 Kemnitz K, Pfeifer L & Ainbund MR (1997) Detector
for multichannel spectroscopy and fluorescence lifetime
imaging on the picosecond timescale. Nucl Instr Meth
Phys Res A 387, 86–87.
33 Beechem JM (1989) A second generation global analysis
program for the recovery of complex inhomogeneous
fluorescence decay kinetics. Chem Phys Lipids 50, 237–
251.
K. Kinoshita et al. FRET imaging of the VBC complex
FEBS Journal 274 (2007) 5567–5575 ª 2007 The Authors Journal compilation ª 2007 FEBS 5575