Ubiquitination of soluble and membrane-bound tyrosine hydroxylase
and degradation of the soluble form
Anne P. Døskeland and Torgeir Flatmark
Department of Biochemistry and Molecular Biology, University of Bergen, Norway
Tyrosine hydroxylase (TH) demonstrates by two-dimen-
sional electrophoresis a microheterogeneity both as a soluble
recombinant human TH (hTH1) and as a membrane-bound
bovine TH (bTH
mem
). Part of the h eterogeneity is likely due
to deamidation of l abile asparagine residues. Wild-type
(wt)-hTH1 was found to be a substrate for the ubiquitin ( Ub)
conjugating enzyme system in a reconstituted in vitro system.
When wt-hTH1 was expressed in a coupled transcription-
translation TnT
R
-T7 reticulolysate system
35
S-labelled
polypeptides of the expected molecular mass of native
enzyme as well as both higher and lower molecular mass
forms were observed. The amount of high-molecular-mass
forms increased by time and was enhanced in the presence of
Ub and clasto-lactacystin b-lactone. In pulse-chase experi-
ments the amount of full-length hTH1 decreased by first-
order kinetics with a half-time of 7.4 h and 2.1 h in the
absence and presence of an ATP-regenerating system,
respectively. The ATP-dependent degradation was inhibited
by clasto-lacta cystin b-lactone. Our findings support t he
conclusion that hTH1 is ubiquitinated and at least p artially
degraded by the proteasomes in the reticulocyte lysate

mem
), associated
with the catecholamine secretory granules [7–9]. The
molecular and cellular mechanisms involved in the
degradation of t his k ey enzyme of neurotransmitter
biosynthesis is, however, not yet known. The half-life
of rat TH in PC-12 cells, in a subclone of PC-12 cells
and in chromaffin cells has been reported to be 17 h [10],
30 h [11] and 29 ± 3 h [12], respectively, and the
possibility that PEST motifs could be involved in its
turnover has been suggested [13]. The possibility that the
ubiquitin-proteasome pathway could play a role in its
degradation is considered in the present study as the
structurally closely related recombinant h uman phenylal-
anine h ydroxylase (PAH, EC 1.14.16.1) [ 14,15] h as been
shown to be a substrate for the ubiquitin (Ub)-conju-
gating enzyme s ystem of r at liver [16].
MATERIALS AND METHODS
Materials
Mouse monoclonal anti-Ub Ig which recognizes free and
conjugated Ub was obtained from Z ymed laboratories, Inc.
(San Francisco, CA, USA). Polyclonal antibodies directed
against recombinant hTH1 e xpressed in E. coli were
prepared in rabbit and partially purified by ammonium
sulfate precipitation. Peroxidase-conjugated antibodies
[goat anti-(mouse IgG) Ig and goat anti-(rabbit IgG) Ig]
were from Biorad. Rabbit anti-(mouse IgG) Ig was from
Trichem Aps, Denmark. Mouse monoclonal anti-(26S
proteasome) IgG (directed against p27 subunit of 20S
cylinder particles) was from American Research Products

23 January 2002)
Eur. J. Biochem. 269, 1561–1569 (2002) Ó FEBS 2002
Purification of recombinant hTH1 expressed in
E. coli
Isoform 1 of recombinant human TH (hTH 1) expressed i n
Escherich ia coli was p urified by affinity chromatography on
heparin–Sepharose as described previously [17]. The con-
centration of the hydroxylase was expressed in terms of
enzyme subunits of 62 kDa [18].
Ubiquitination of wt-hTH1 in a reconstituted
in vitro
system
Ubiquitination of wt-hTH1 was assayed at 3 7 °Cina
reconstituted in vitro system with [
125
I]ubiquitin and the
isolated Ub-conjugating enzymes [i.e. a fraction containing
the Ub-activating (E1), Ub-carrier (E2s) and Ub-protein
ligase (E3)] as described f or ubiquitination of phenylalanine
hydroxylase [16]. Following preincubation of the
Ub-conjugating enzymes (7.6 lgproteinper55lL assay),
with 1.5 l
M
Ubal, ubiquitination was performed with
 18 l
M
[
125
I]Ub by the standard assay procedure in the
absence and presence of 8 l

1–4 lL[
35
S]methionine and approximately 1 lg of plasmid
DNA were routinely used in the 50 lL assay. Reactions
were incubated at 30 °C for the time periods indicated in the
figure legen ds. From the reaction mixture 5 lL a liquots
were quenched at given time points and subjected to SDS/
PAGE after heating to 56 °C for 15 min in the classical
Laemmli s ample buffer as treatment of proteins at high
temperature (95 °C)hasbeenshowntoresultinthe
formation of aggregates especially for samples containing
membrane proteins [19] and observed in the present study.
The stability of hTH1 was studied in a reaction mixture
containing in a final volume of 50 lL: 15 m
M
Hepes
(pH 7 .5), 5 m
M
MgCl
2
,0.25m
M
dithiothreitol, 1 m
M
methionine and 25 lL of f reshly thawed rabbit reticulocyte
lysate. T he reaction was performed at 37 °C i n the pr esence
of added 0.5 m
M
ATP, 10 m
M

35
S]hTH1
and its derivatives, was estimated r elative t o t he position of
the standard proteins.
Preparation of chromaffin granule membranes
Chromaffin granules from the bovine adrenal medulla
were isolated by a discontinuous sucrose density-gradient,
lysed (hypotonic) and centrifuged in a final discontinuous
density-gradient to yield chromaffin granule ghosts essen-
tially free from mitochondrial and microsomal contamin-
ation [20].
Polyacrylamide gel electrophoresis
Protein samples for e lectrophoresis, either from ubiquiti-
nation assay or from isolated chromaffin granule ghosts,
were precipitated with ice-cold acetone (sample/acet-
one ¼ 1 : 3 by vol.) and kept on ice for 30 min After
centrifugation (12 000 g for 15 min), the pe llets were
dissolved in sample buffer and subjected to one-dimen-
sional or two-dimensional gel electrophoresis. SDS/PAGE
was performed according to the Laemmli p roce dure [21] in
10% (w/v) gel. One volume of the samples was routinely
mixed with 1 vol. of Laemmli sample buffer a nd incubated
for 15 min at 56 °C. Two-dimensional electrophoresis was
performed as described previously [16]. Acetone precipita-
ted proteins were dissolved in a medium containing 9.5
M
urea, 2% (w/v) Chaps, 1.6% (w/v) Bio-Lyte p H 5–7, 0.4%
Bio-Lyte pH 3–10 and 100 m
M
dithiothreitol and kept at

body.
Isotopic detection and quantitation using [
125
I]protein A
was preferentially used to ensure specificity of the TH and
Ub immunoreactivity. Thus, the transferred proteins were
probed with rabbit anti-TH Ig at dilution 1 : 1000 or in
paralell with anti-Ub serum at the recommanded working
concentration o f 2 lgÆmL
)1
and w ith rabbit anti-(mouse
IgG) Ig as the secondary antibody. Nitrocellulose mem-
branes were then incubated with [
125
I]protein A at the
concentration o f 0.2 lCiÆmL
)1
in phosphate buffered s aline
containing 2.5% (w/v) dried non fat milk and 0.1% (v/v)
Tween-20, and in order to vizualize
125
I-labeled proteins,
they were counted in a b scanner ( Packard Instant Imager,
Packard Inc., Canberra, Australia) or exposed to X-ray film
for autoradiography.
For Western blot analysis of chromaffin granule mem-
brane proteins on one-dimensional gel, 280 lgofproteins
were applied in a large well. After electrophoresis and
blotting, the membrane was divided in two identical parts
and probed against anti-TH Ig or anti-Ub Ig, respectively,

ide, 20 lgÆmL
)1
leupeptin, 0.5 mg ÆmL
)1
soybean trypsin
inhibitor, 14 lgÆmL
)1
pepstatin, 1 m
M
benzamidine) were
added, followed by addition of 120 lL immunoadsorbent
Protein A–Sepharose with bound IgG. The immunoad-
sorbent was Protein A–Sepharose ( 10 mg of dry beads
suspended and washed twice in 50 m
M
potassium phos-
phate, pH 8.0) to which were coupled 5 lLanti-THIgGby
incubating for 1 h on a rotating wheel at 4 °C. For
immunoisolation the beads were mixed with samples of
the membrane proteins and rocked in Eppendorf tubes f or
2hat4°C. The p rotein A–Sepharose with bound IgG–TH
was pelleted b y centrifugation a t 12 000 g for 15 s, washed
nine times with phosphate buffer containing 0.2% (w/v)
Triton X-100 a nd finally twice with the same buffer without
Triton X-100. The pellet was kept at )20 °C until used, then
heated (56 °C, 10 min) in sample buffer (40 lL added).
Immunoreactive material resolved by SDS/PAGE was
thereafter immunoblotted with either anti-Ub Ig or anti-
TH Ig, and the immunoreactivities compared.
RESULTS

I]Ub in the
absence of hydroxylase (A), the presence of 8 l
M
hPAH (B) and
(CandD)of8l
M
hTH1. After 90 m i n, the reaction was quenched by
the addition of acetone and precipitated proteins analysed on two-
dimensional electrophoresis [12.5% (w/v) ( gel)] in ( A–C); 10% (w /v)
gel in (D). Inset in B and C: Coomassie Brilliant Blue stained proteins
from the reaction mixture containing PAH (B) and TH (C). The
multiple molecular forms of hTH1 have a molecular mass for the
subunit of  62 kDa; the doublet with a more acidic p I a nd a
molecular mass of  100 kDa r epresents presumably the E1 enzyme
[16,49]. The main forms of hTH1 are also indicated by arrows in Panel
D. The [
125
I]Ub-labelled conjugates of hTH1 are v isualized as diagonal
spots of radioactivity in the autoradiogram (Panel C and D). The
polyUb chains derived from
125
I-labelled Ub with 8.5 kDa a nd neutral
pI are observed as vertical spots (A–C). (D) An expanded view of the
area of interest (i.e. a bove  60 kDa) and co rrespon ds to the pattern of
superimposed profils of stained g el and t he respective autoradio gram
obtained after short exposure time. The main Coomassie Blue stained
spots indicated by arrows correspond to hTH1 and E1. The auto-
radiographic pattern of [
125
I]Ub-labelled conjugates corresponds

periodic pattern co rresponding to poly Ub chains [16].
Finally, some insignificant amounts of poly/multi-ubiquiti-
nated proteins, representin g ubiquitination o f not yet
identified liver proteins, present in the E3 preparation [16],
were also o bserved ( Fig. 1A).
Microheterogeneity of wt-hTH1 and bTH as observed
by two-dimensional electrophoresis
The recombinant wt-hTH1 expressed in E. coli revealed a
microheterogeneity on two-dimensional electrophoresis
(Fig. 2A) with 5–6 components of  62 kDa, differing
in pI by  0.1 pH unit. A similar type o f microhetero-
geneity was observed (Fig. 2 B) when the enzyme was
expressed (1 h at 37 °C) in an in vitro transcription-
translation system as a protein of either  62 kDa or
 60 kDa subunits, where the difference in molecular
mass is explained by a second initiation site in this
expression system [22].
When the membrane form of bovine TH (bTH
mem
),
extracted from i solated adrenal chromaffin g ranule ghosts,
was subjected to two-dimensional electrophoresis, the
Western blot a nalysis revealed a broad distribution pattern
in terms of pI (Fig. 2C) with the apparent molecular mass of
 60 kDa, a value typical of the subunit of bTH in
chromaffin cells [7,9]. The streaky pattern of bTH
mem
(Fig. 2C) is characteristic of proteins with a tendency to
aggregate/precipitate around the pI [23]. In addition, two
post-translational modifications of bTH may also contri-

PAGE revealed two major bands, corresponding to
subunits of  62 kDa and  60 kDa, respectively. In
Fig. 2. Microheterogeneity of recombinant human tyrosine hydroxylase
(hTH1) an d the me mbrane-bound form o f the bov ine enzyme (bTH
mem
)
as revealed by 2D-electrophoresis. (A) R ecombinant hTH1 (40 lg)
expressed in E. coli and visualized by Coomassie B rilliant Blue stain-
ing. ( B) [
35
S]Methionine-labelled hTH expressed in the in vitro
transcription-translation system (10 lL assay) and dete cted b y a uto-
radiography. (C) bTH
mem
of the bovine adrenal chromaffin granula
membrane. (part a) two-dimensional profil of Coomassie Brilliant
Blue stained membrane proteins (500 lg). ChgA (chromogranin A)
and DBH (dopamine b-hydroxylase) represent the major spots as
described previously [50,51]. The position of the multiple molecular
forms of bTH are indicated by bracket as confirmed by immuno-
blotting using ECL detection with 20 s (part b) a nd 5 min (part c)
exposures.
1564 A. P. Døskeland and T. Flatmark (Eur. J. Biochem. 269) Ó FEBS 2002
addition, on prolonged exposure (> 60 min),
35
S-labelled
proteins of molecular mass around 80 kDa were observed,
concomittantly to the formation of the main product. Less
defined
35

somal activity [ 32]. In the presence of M gATP a s ignificant
decrease in the a mount of wt-hTH1 was obs erved (Fig. 4A,
lower i nset) while the hTH1 degradation was relatively
moderate on depletion of MgATP (Fig. 4A, upper inset).
The h alf-life of hTH1 disappearance was estimated to be of
7.4 h when the lysate was depleted for MgATP vs. 2.1 h in
the presence of an ATP regenerating sys tem. Based on three
independent experiments the MgATP-dependent proteoly-
sis gave a half-life of  4.3 h (Fig. 4B). Furthermore, when
clasto-lactacystin b-lactone (2 l
M
) a nd anti-(26S protea-
some) IgG (2 lLper50lL assay) were added to the
degradation assay in the presence of an excess of MgATP,
the MgATP-dependent degradation was reduced by
 60%. This finding further supports the conclusion that
proteasomes in the reticulocyte lysate are involved in t he
degradation of TH.
Fig. 4. MgATP-dependent degradation of hTH1. Semilogarithmic plot
of the degradation of [
35
S]methionine labelled full-length ( 62 kDa )
and truncated form (  60 kDa) of hTH1 p rotei n synthesize d by the
coupled in vitro transcription-translation (reticulocyte lysate) system.
After synthesis for 1.5 h at 30 °C, 1 m
M
cold me thionine was added
and incubated at 37 °C in the presence of ex cess (j, h)ordepletionof
MgATP (m) (fo r details, see Materials a nd method s). Aliquots w ere
removed at t imed inter vals, and labelled hTH1 (  62 plus  60 kDa

150minincubationtime,and(D)theprofileobtainedafteranaddi-
tional 3 h incubation in the presence of a regenerating system for ATP
(degradation assay). The main products in (B) t o (D) co rrespond to
proteins  62 and  60 kDa (i.e. full-length and truncated form of
hTH1) and minor proteolytic products of  34 and 28–30 kDa.
(C) T he area containing [
35
S]methionine-labelled proteins which are
considered to represent U b-conjugates of hTH1 are indicated by
bracket. O, origin and F, dye fr ont. Th e value 1 o n the ordinate c or-
responds to about 214 cpm in (A) and (B), to 76 c.p.m. in (C) and (D).
Ó FEBS 2002 Ubiquitination of tyrosine hydroxylase (Eur. J. Biochem. 269) 1565
Immunodetection of Ub-TH conjugates in bovine
adrenal chromaffin granule ghosts
TH is also a t arget protein for ubiquitination in vivo,as
shown by SDS/PAGE and Western blot analysis of proteins
extracted from highly purified bovine chromaffin granule
ghosts. Immunoblots with anti-Ub IgG revealed several
Ub-conjugates of molecular mass ‡ 55 kDa, with the
highest intensity near the top of the gel (Fig. 5A, lane 2).
One o f t he bands revealed a mobility c orresponding to that
of TH immunoreactivity ( 60 kDa), with a trace amount
of reactivity a t the top of the gel (Fig. 5A, la ne 1 ). O n t wo-
dimensional electrophoresis, t he U b-conjugates were found
to be distributed over a l arge pI interval, m ostly d etected as
a smear, especially dense in t he high-molecular-mass region.
Most interestingly, a strong immunoreactivity was observed
as a s eries of distinct s pots of 5 .0 < pI < 5.8 in t he 63 kDa
region (Fig. 5B, panel 2) w hich revealed cross-r eactivity
with anti-TH Ig (Fig. 5B, panel 1). The same pattern was

human enzyme and in vivo as a membrane-bound form of
the enz yme i n t he c atecholamine storage/secretory gra nules
of the a drenal medulla, w hich may h ave important
functional implications in the central nervous system.
Ubiquitination of the soluble recombinant hTH1
The finding that recombinant hTH1 is a substrate for the
in vitro reconstituted Ub conjugating enzyme system of rat
liver (Fig. 2) was indeed expected as the structurally
homologous enzyme phenylalanine hydroxylase and its
catalytic core enzyme [14] have already been found to be
ubiquitinated [16]. Similarly, the in vivo turnover of the
structurally homologou s enzyme tryptophan h ydroxylase
[14,15] is reported to be mediated by the Ub-proteasome
pathway [36]. Furthermore, ou r previous studies on two
mutant forms of hTH1, associated with the clinical and
metabolic phenotype of
L
-DOPA responsive dystonia and
infantile p arkinsonism, h ave revealed a reduced cellular
stability c ompared t o the wild-type form when expressed in
human embryonic kidney (A293) cells [37] supporting the
in vivo relevance of the observed Ub-conjugates of hTH1
formed in vitro. Thus, elimination of proteins by the
Ub-proteasome pathway is co nsidered to be most active
towards misfolded/misassembled and abnormal mutant
proteins [38].
Energy-dependent degradation of recombinant hTH1
in the
in vitro
reticulolysate system

contain m ultiple proteolytic systems including the MgATP-
ubiquitin-proteasome-dependent pathway, calpains and
MgATP-independent proteases, but they contain no lyso-
somes [31]. Due to the lack of lysosomal activities, the
system has one limitation as compared to regular eukaryotic
cells.
In order to clearly distinguish proteasomal activity from
other proteolytic activities, an established effective concen-
tration (2 l
M
) in vitro of the selective and potent protea-
somal inhibitor clasto-lactacystin b-lactone was used in the
present study. The inhibitor is a lactacystin analog with a
potency of  10 times that of lactacystin and which beside
epoxomycin [39] is the most potent and specific proteasome
inhibitor [29,30,40]. In this coupled transcription-translation
system a time-dependent formation of [
35
S]methionine-
labelled hTH1 w as observed, followed by i ts degradation to
molecular species of  34 and 28–30 kDa, which is related
to the 34-kDa core fragment of hTH1 observed on limited
tryptic proteolysis [4 1]. Its degradation was found to be
partly MgATP-dependent which was inhibited to about
60% by anti-(26S proteasome) IgG plus clasto-lactacystin-
b-lactone.
The o verall half-life of [
35
S]methionine-labelled h TH1
was estimated to 2.1 h in the p resence of an ATP-

possible at this point to answer the question of whether
bTH
mem
is inserted into the membrane before or after its
ubiquitination. The finding that bTH
mem
is ph osphorylated
by cAMP-dependent protein kinase on Ser40 in the
regulatory domain [9] may support an ubiquitination of
the e nzyme by the cytosolic Ub-conjugating enzyme system
after its membrane insertion.
In contrast to the multi/poly Ub conjugates observed for
the soluble recombinant hTH1 the membrane-bound form
of bTH is mono-ubiquitinated, which m ay be related to t he
function of the u biquitin C-terminal h ydrolase (UCH-L1 o r
PGP9.5) which is widely and often highly expressed in
neuroendocrine cells [34,35], including the rat chromaffin
cells [45]. From a functional point of view, the membrane
localization may protect the catalytically active enzyme
from degradation by t he cytosolic proteases. Thus, ubiqui-
tination may play a role in the degradation of both
membrane-bound and soluble TH. However, the accurate
role of the ubiquitination remains t he subject of further
investigation and the reason why TH is detected mainly as
mono-ubiquitinated form is still unclear.
Ubiquitination of proteins in the chromaffin granule
membrane
Previous studies on subcellular fractions of rat brain
homogenates have revealed that the synaptic membrane
fraction contains multiple Ub-immunoreactive bands, i.e.

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