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 Bio
Trang 1Ubiquitination 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 (bTHmem) Part of the heterogeneity is likely due
to deamidation of labile 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 TnTR-T7 reticulolysate system 35S-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-lactacystin b-lactone Our findings support the conclusion that hTH1 is ubiquitinated and at least partially degraded by the proteasomes in the reticulocyte lysate system Finally, it is shown that the integral TH of the bovine adrenal chromaffin granule membrane (bTHmem) is ubiquitinated, most likely monoubiquitinated Additional Ub-conjugates of this membrane, detected by Western blot analysis, have not yet been identified
Keywords: tyrosine hydroxylase; ubiquitin; proteasome; chromaffin granule membrane; neuroendocrine cells
Tyrosine hydroxylase (TH, EC 1.14.16.2) catalyzes the
conversion of L-tyrosine to L-dihydroxyphenylalanine
(L-DOPA), the rate-limiting step in the biosynthesis of
dopamine and noradrenaline/adrenaline [1] The cellular
activity of TH is regulated by several alternative
mech-anisms in response to, e.g neuronal and hormonal
stimuli of neuroendocrine target cells Both long-term
transcriptional and short-term post-transcriptional
mech-anisms (notably phosphorylation) are involved in its
regulation [2,3] Beside its localization in the brain [2,4],
TH is present in high amount in the adrenal chromaffin
cells mainly as a soluble cytosolic form (THsol) [5,6] and
partly as a membrane-bound form (THmem), associated
with the catecholamine secretory granules [7–9] The
molecular and cellular mechanisms involved in the
degradation of this key 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 human phenylal-anine hydroxylase (PAH, EC 1.14.16.1) [14,15] has been shown to be a substrate for the ubiquitin (Ub)-conju-gating enzyme system of rat liver [16]
M A T E R I A L S A N D M E T H O D S
Materials Mouse monoclonal anti-Ub Ig which recognizes free and conjugated Ub was obtained from Zymed laboratories, Inc (San Francisco, CA, USA) Polyclonal antibodies directed against recombinant hTH1 expressed 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 (Belmont, MA, USA) Protein A–Sepharose CL-4B was from Amersham Pharmacia Biotech (Oslo, Norway) Ub C-terminal hydrolase, isopeptidase T was from Affiniti Research Products Ltd (UK), Ub aldehyde (Ubal) and clasto-lactacystin b-lactone were from Boston Biochem Inc (Cambridge, MA, USA) Yeast hexokinase was from Roche Molecular Biochemicals (Mannheim, Germany) [125I]Protein A and [35S]methionine (code AG 1094) were from Amersham (Buckinghamshire, UK) The TnTR-T7 reticulocyte lysate system was from Promega (Madison, USA)
Correspondence to T Flatmark, Department of Biochemistry and
Molecular Biology, University of Bergen, A˚rstadveien 19, N-5009
Bergen, Norway Fax: + 47 55 586400, Tel.: + 47 55 586428,
E-mail: torgeir.flatmark@pki.uib.no
Abbreviations: TH, tyrosine hydroxylase; hTH, human TH; bTH,
bovine TH; PAH, phenylalanine hydroxylase; Ub, ubiquitin;
L -DOPA, L -dihydroxyphenylalanine.
(Received 1 November 2001, revised 2 January 2002, accepted
23 January 2002)
Trang 2Purification of recombinant hTH1 expressed inE coli
Isoform 1 of recombinant human TH (hTH1) expressed in
Escherichia coliwas purified 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 reconstitutedin vitro
system
Ubiquitination of wt-hTH1 was assayed at 37°C in a
reconstituted in vitro system with [125I]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 for ubiquitination of phenylalanine
hydroxylase [16] Following preincubation of the
Ub-conjugating enzymes (7.6 lg protein per 55 lL assay),
with 1.5 lM Ubal, ubiquitination was performed with
18 lM [125I]Ub by the standard assay procedure in the
absence and presence of 8 lM hydroxylase After 90 min,
the reaction was quenched by the addition of acetone, and
the precipitated proteins were analysed on two-dimensional
electrophoresis (for details, see below) After
electrophor-esis, the gels were stained with Coomassie Blue R250 and
dried in vacuo at 70°C between two sheets of cellophane
To determine the distribution of 125I-radioactivity, gels
were then exposed to Hyperfilm TM-b-max for
autoradi-ography The apparent molecular mass of the 125
I-containing bands in each lane, representing [125I]Ub, free
poly Ub chains and [125I]Ub-conjugates, respectively, was
estimated by comparison with the position of the standard
proteins
Expression and degradation of hTH1 in a coupled
transcription-translation reticulocyte lysate system
The hTH1 was expressed in a coupled in vitro
transcription-translation system using the pET3a-hTH1 vector [18] and
the TnT-T7 reticulocyte lysate system in the presence of
[35S]methionine essentially as described by the supplier
1–4 lL [35S]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 legends From the reaction mixture 5 lL aliquots
were quenched at given time points and subjected to SDS/
PAGE after heating to 56°C for 15 min in the classical
Laemmli sample buffer as treatment of proteins at high
temperature (95°C) has been shown to result in the
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 mM Hepes
(pH 7.5), 5 mM MgCl2, 0.25 mM dithiothreitol, 1 mM
methionine and 25 lL of freshly thawed rabbit reticulocyte
lysate The reaction was performed at 37°C in the presence
of added 0.5 mM ATP, 10 mM phosphocreatine and
0.2 mgÆmL)1 creatine phosphokinase (Sigma), or in an
ATP-depleted lysate obtained by adding 2-deoxy-D-glucose
(20 mM) and hexokinase (230 UÆmL)1) The mixture was
preincubated for 10 min and incubation started by the
addition of the last component, i.e [35S]methionine-labelled
hTH1 (6.5% of the final volume) freshly obtained by the
coupled in vitro transcription-translation system To mon-itor hTH1 degradation, aliquots (6 lL) were, at selected time points, added to 10 lL of reducing SDS/PAGE sample buffer containing 2 mercaptoethanol (5%), incubated
15 min at 56°C, and applied to 10% SDS/PAGE gels The distribution of radioactivity in each sample lane of one-dimensional gel or in two-one-dimensional gel was first deter-mined in unstained gels by a b-scanner (Packard Instant Imager, Packard Inc., Canberra, Australia) and then exposed to Biomax MR (Kodak) or Hyperfilm TM-b-max for autoradiography The apparent molecular mass of the
35S-containing bands in each lane, representing [35S]hTH1 and its derivatives, was estimated relative to the 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 electrophoresis, 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 pellets were dissolved in sample buffer and subjected to one-dimen-sional or two-dimenone-dimen-sional gel electrophoresis SDS/PAGE was performed according to the Laemmli procedure [21] in 10% (w/v) gel One volume of the samples was routinely mixed with 1 vol of Laemmli sample buffer and 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 9.5M urea, 2% (w/v) Chaps, 1.6% (w/v) Bio-Lyte pH 5–7, 0.4% Bio-Lyte pH 3–10 and 100 mMdithiothreitol and kept at )20 °C until used After 1 h pre-electrophoresis at 200 V, the proteins were loaded at the basic end of the isoelec-trofocusing gel, and electrophoresis was performed at
400 v for 16 h and at 1000 v for an additional hour The second dimension was run according to Laemmli using 10% (w/v) acrylamide slab gels (1 mm) The prestained protein standards (Biorad) used were phosphorylase b (101 kDa), BSA (79 kDa), ovalbumin (50.1 kDa), car-bonic anhydrase (34.7 kDa), soybean trypsin inhibitor (28.4 kDa) and lysozyme (20.8 kDa) The gels were stained with Coomassie Brilliant Blue, dried in vacuo at 70°C between two sheets of cellophane and analysed for radioactive proteins
Western blot analysis Proteins from chromaffin granule membranes separated by SDS/PAGE [10% (w/v) gel] were blotted electrophoretically for 3 h at 300 mA on a nitrocellulose membrane (0.45 lm pore diameter, BA 85 from Schleicher & Schuell, Dassel, Germany) in a buffer containing 48 mM Trizma base,
39 m glycine (pH 9.2) and 20% (v/v) methanol
Trang 3Western blot analysis of chromaffin granule membrane
proteins was performed using the enhanced
chemilumines-cence detection method with polyclonal rabbit anti-hTH1 Ig
or anti-Ub Ig as primary antibody and anti-rabbit or
mouse horseradish peroxidase-labelled secondary
anti-body
Isotopic detection and quantitation using [125I]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 of 2 lgÆmL)1 and with rabbit anti-(mouse
IgG) Ig as the secondary antibody Nitrocellulose
mem-branes were then incubated with [125I]protein A at the
concentration of 0.2 lCiÆmL)1in phosphate buffered saline
containing 2.5% (w/v) dried non fat milk and 0.1% (v/v)
Tween-20, and in order to vizualize125I-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 lg of proteins
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,
as illustrated below For analysis of proteins by Western
blot on two-dimensional gel, the membrane blot was, after
immunodetection with one antibody, for example anti-TH
IgG, stripped and probed with anti-Ub IgG and vice versa
Stripping of bound antibodies was performed by
incuba-ting the membrane in a buffer containing 63 mMTris/HCl
pH 6.7, 100 mM2-mercaptoethanol and 2% (w/v) SDS at
50°C for 30 min with occasional agitation and finally
extensive washing in a large volume of Tris/NaCl/Pi/
Tween
Immunoisolation
Chromaffin granule membrane proteins (200 lg) were
solubilized in 1% (w/v) SDS and incubated at room
temperature for 5 min 10 vol (920 lL) of buffer (50 mM
potassium phosphate pH 7.0 containing 190 mM NaCl,
6 mMEDTA, 2.5% Triton X-100 and a cocktail of protease
inhibitors including 0.2 mM phenylmethanesulfonyl
fluor-ide, 20 lgÆmL)1 leupeptin, 0.5 mgÆmL)1 soybean trypsin
inhibitor, 14 lgÆmL)1pepstatin, 1 mM 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 mM potassium
phos-phate, pH 8.0) to which were coupled 5 lL anti-TH IgG by
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 for
2 h at 4°C The protein A–Sepharose with bound IgG–TH
was pelleted by centrifugation at 12 000 g for 15 s, washed
nine times with phosphate buffer containing 0.2% (w/v)
Triton X-100 and 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 Ub Ig or
anti-TH Ig, and the immunoreactivities compared
R E S U L T S
Ubiquitination of recombinant wt-hTH1
by a reconstituted ubiquitin conjugating enzyme system
As expected from our previous studies [16] on the ubiqui-tination of recombinant wild-type human phenylalanine hydroxylase (wt-hPAH), it is seen from Fig 1 that recom-binant wt-hTH1 is also a substrate for the reconstituted Ub-conjugating enzyme system of rat liver After incubation
of the hydroxylase with125I-labelled Ub and a mixture of the purified preparations of the E1, E2 and E3 enzymes, the 2D-electrophoresis revealed the formation of 125I-labelled
Fig 1 Mono- and multi/poly ubiquitination of recombinant hTH1 by a reconstituted ubiquitin conjugating enzyme system Ubiquitination of recombinant hTH1 was performed in a reconstituted in vitro system with the Ub conjugating enzymes E1, E2 and E3 isolated by affinity chromatography from rat liver and [ 125 I]Ub [16] hPAH with subunit molecular mass of 51 kDa was used as a positive reference protein for ubiquitination The reaction mixture (55 lL) contained 7.6 lg of proteins (E1, E2 and E3 proteins), 1.5 l M Ubal, 18 l M [ 125 I]Ub in the absence of hydroxylase (A), the presence of 8 l M hPAH (B) and (C and D) of 8 l M hTH1 After 90 min, 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 pI and a molecular mass of 100 kDa represents presumably the E1 enzyme [16,49] The main forms of hTH1 are also indicated by arrows in Panel
D The [125I]Ub-labelled conjugates of hTH1 are visualized 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 and neutral
pI are observed as vertical spots (A–C) (D) An expanded view of the area of interest (i.e above 60 kDa) and corresponds to the pattern of superimposed profils of stained gel and the respective autoradiogram 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 mainly to the poly/multi Ub-TH conjugates vizualized as a ladder of
at least eight distinct Ub-TH conjgates with a microheterogeneity corresponding to the enzyme as isolated.
Trang 4Ub-protein conjugates derived from hTH1 in addition to
poly Ub-chains Mono-ubiquitinated and
poly/multi-ubiq-uitinated species were visualized as a diagonal pattern of
high MrÔladderÕ of radioactive spots (Fig 1C) which reflects
the heterogeneity of the Ub-protein conjugates of wt-hTH1
in terms of size and pI Multiple molecular forms of
62 kDa and different pI values around 5.5 (Fig 1C inset)
were observed for the nonubiquitinated enzyme A ladder of
at least eight Ub-TH conjugates could be identified in the
molecular mass range higher than 66 kDa (Fig 1, panel
D), which also revealed a microheterogeneity corresponding
to the enzyme as isolated (arrow in Fig 1D) The
background in the control was negligible in this relevant
area as earlier reported for this reconstituted in vitro assay
[16] Thus, the diagonal spots observed on the
autoradio-gram (Fig 1D) correspond to mono- and multi/poly Ub
adducts, respectively, for the wt-hTH1 with 70 kDa,
78 kDa, etc (molecular mass increasing by multiple of
8 kDa) and increasing pI In addition, a series of
predominant spots with the same neutral pI as free Ub
and increasing Mr, observed in the absence (Fig 1A) or
presence of hydroxylase (Fig 1B,C), was distributed in a
periodic pattern corresponding to poly Ub chains [16]
Finally, some insignificant amounts of
poly/multi-ubiquiti-nated proteins, representing ubiquitination of not yet
identified liver proteins, present in the E3 preparation [16],
were also observed (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 of
microhetero-geneity was observed (Fig 2B) 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 (bTHmem),
extracted from isolated adrenal chromaffin granule ghosts,
was subjected to two-dimensional electrophoresis, the
Western blot analysis 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 bTHmem
(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-bute to this pronounced microheterogeneity Thus, the
enzyme has four possible phosphorylation sites at Ser
residues in the regulatory domain, and each
phosphoryla-tion lowers the pI by 0.1 U [24,25] Deamidation of labile
amide groups has a similar effect on pI as shown for the
structurally related phenylalanine hydroxylase [26] and
most likely explains the microheterogeneity of the
nonphos-phorylated recombinant wt-hTH1 (Fig 2A) Thus, hTH1
contains three aspargine residues of which Asn414
(posi-tioned in a short loop between two b strands) [14] is
predicted to be the most labile one on the basis of its nearest
neighbour amino acids (QNG), with a half-life of 1.5 days
days [27] in Tris buffer, pH 7.0 Thus, similarly to hPAH [26,28], hTH1 also occurs in multiple molecular forms which could be explained by a progressive deamidation of labile Asn residue(s)
Ubiquitination and degradation of hTH1
in the reticulocyte lysate system wt-hTH1 was expressed in the coupled in vitro transcrip-tion-translation (TnTR) system and the net accumulation
of [35S]hTH1 was followed as a function of time (Fig 3A,B) The typical profile of [35S]hTH1 on SDS_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) and the membrane-bound form of the bovine enzyme (bTH mem )
as revealed by 2D-electrophoresis (A) Recombinant hTH1 (40 lg) expressed in E coli and visualized by Coomassie Brilliant Blue stain-ing (B) [35S]Methionine-labelled hTH expressed in the in vitro transcription-translation system (10 lL assay) and detected by auto-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) and 5 min (part c) exposures.
Trang 5addition, on prolonged exposure (> 60 min),35S-labelled
proteins of molecular mass around 80 kDa were observed,
concomittantly to the formation of the main product Less
defined 35S-labelled proteins were also observed in the
high-molecular-mass region (Fig 3C), in amount enhanced
by the presence of Ub (20 lM) and an inhibitor of
proteasome proteolytic activity [29,30], clasto-lactacystin
b-lactone (2m M) dissolved in dimethylsulfoxide (1%) (data
not shown), and were identified as post-transcriptionally
modified TH such as Ub-conjugates Furthermore, in the
hTH1-expression sytem,35S-labelled peptides of 34 and
28–30 kDa were observed in increasing amount
concom-itantly to the formation and subsequent decrease of
[35S]TH at longer incubation time points Thus,
degrada-tion of wt-hTH1 by components in the rabbit reticulocyte
lysate influencing its proteolysis were further studied
(Fig 3C,D)
MgATP-dependent degradation of wt-hTH1
[35S]Methionine-labelled wt-TH1 was incubated with a
reticulocyte lysate as the degradation machinery (Fig 4)
Reticulocytes do not contain any lysosomes [31] and any
MgATP-dependent degradation correlates with
proteo-somal activity [32] In the presence of MgATP a significant
decrease in the amount of wt-hTH1 was observed (Fig 4A,
lower inset) while the hTH1 degradation was relatively moderate on depletion of MgATP (Fig 4A, upper inset) The half-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 system 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 lM) and anti-(26S protea-some) IgG (2 lL per 50 lL 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 the degradation of TH
Fig 4 MgATP-dependent degradation of hTH1 Semilogarithmic plot
of the degradation of [35S]methionine labelled full-length ( 62 kDa) and truncated form ( 60 kDa) of hTH1 protein synthesized by the coupled in vitro transcription-translation (reticulocyte lysate) system After synthesis for 1.5 h at 30 °C, 1 m M cold methionine was added and incubated at 37 °C in the presence of excess (j, h) or depletion of MgATP (m) (for details, see Materials and methods) Aliquots were removed at timed intervals, and labelled hTH1 ( 62 plus 60 kDa forms) was quantitated by INSTANT IMAGER or subjected to autoradiography after SDS _ PAGE (A) The autoradiograms shown as inset represent experiments with excess of MgATP (lower inset) vs depletion of MgATP (upper inset) The half-lives for TH were esti-mated to 7.4 h in the assay depleted in MgATP (m), and to 2.1 h with excess of MgATP (j, h) (B) The MgATP-dependent degradation (total minus MgATP-independent) gave a half life of 4.3 h (mean of three experiments shown by separated symbols) The curves were drawn by linear regression analysis [A, r ¼ 0.809 for curve (m) and
r ¼ 0.961 for curve (j, h); B, r ¼ 0.872 for the curve].
Fig 3 In vitro ubiquitination and degradation of [35S]methionine
labelled hTH1 in the reticulocyte lysate system hTH1 was expressed in
a coupled in vitro transcription-translation (reticulocyte lysate) system
as described in Materials and methods From the 50 lL reaction
mixture 5 lL aliquots were quenched at given time points subjected to
SDS_PAGE and image analysis (A) Zero control; (B) the pattern of
[35S]methionine-labelled proteins after 30 min incubation; (C)
repre-sents the image analysis profile of [ 35 S]methionine-labelled hTH1 at
150 min incubation time, and (D) the profile obtained after an
addi-tional 3 h incubation in the presence of a regenerating system for ATP
(degradation assay) The main products in (B) to (D) correspond 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) The area containing [ 35 S]methionine-labelled proteins which are
considered to represent Ub-conjugates of hTH1 are indicated by
bracket O, origin and F, dye front The value 1 on the ordinate
cor-responds to about 214 cpm in (A) and (B), to 76 c.p.m in (C) and (D).
Trang 6Immunodetection of Ub-TH conjugates in bovine
adrenal chromaffin granule ghosts
TH is also a target 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 of the bands revealed a mobility corresponding to that
of TH immunoreactivity ( 60 kDa), with a trace amount
of reactivity at the top of the gel (Fig 5A, lane 1) On
two-dimensional electrophoresis, the Ub-conjugates were found
to be distributed over a large pI interval, mostly detected as
a smear, especially dense in the high-molecular-mass region
Most interestingly, a strong immunoreactivity was observed
as a series of distinct spots of 5.0 < pI < 5.8 in the 63 kDa
region (Fig 5B, panel 2) which revealed cross-reactivity
with anti-TH Ig (Fig 5B, panel 1) The same pattern was
obtained whether the membranes were first probed with
anti-Ub Ig or anti-TH Ig, and then reprobed with anti-TH
Ig and anti-Ub Ig, respectively Due to the similarity in
molecular mass between the proteins cross-reacting with
anti-TH Ig and those reacting with anti-Ub Ig, it is assumed that the Ub-conjugates correspond to monoubiquitinated forms of TH
The comigration of TH- and Ub-immunoreactivities was further studied in chromaffin granule membranes solubi-lized by the detergents SDS (1%, w/v) and Triton X-100 (2.5%, w/v) [33] bTHmem was immunoisolated, in the presence of protease inhibitors, on anti-TH IgG coupled to protein A–Sepharose beads (see Materials and methods) After resolution on one dimensional SDS/PAGE and Western blot analysis, TH of 60 kDa and some higher molecular mass forms were detected with anti-TH IgG The immunoisolates were also probed with the anti-Ub Ig, and positive immunoreactivity was then observed at 60 kDa comigrating with TH immunoreactivity (data not shown), and thus further support the results shown in Fig 5B
D I S C U S S I O N
The ubiquitination of many vital proteins plays an import-ant role in regulating their functions and turnover in eukaryotic cells including neurons and neuroendocrine cells [34,35] In the present study it is shown that a key regulatory enzyme in catecholaminergic neuroendocrine cells, i.e tyrosine hydroxylase, is a substrate for the Ub-conjugating enzyme system, both in vitro as a soluble recombinant human enzyme and in vivo as a membrane-bound form of the enzyme in the catecholamine storage/secretory granules
of the adrenal medulla, which may have 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 vitroreconstituted 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 homologous enzyme tryptophan hydroxylase [14,15] is reported to be mediated by the Ub-proteasome pathway [36] Furthermore, our previous studies on two mutant forms of hTH1, associated with the clinical and metabolic phenotype ofL-DOPA responsive dystonia and infantile parkinsonism, have revealed a reduced cellular stability compared to the wild-type form when expressed in human embryonic kidney (A293) cells [37] supporting the
in vivorelevance of the observed Ub-conjugates of hTH1 formed in vitro Thus, elimination of proteins by the Ub-proteasome pathway is considered to be most active towards misfolded/misassembled and abnormal mutant proteins [38]
Energy-dependent degradation of recombinant hTH1
in thein vitro reticulolysate system Further experimental evidence in support of degradation of hTH1 by the Ub-proteasome pathway was obtained in stability studies of recombinant hTH1 in the in vitro reticulolysate system (Fig 3) Reticulocyte lysates have been used as the degradation machinery and are especially well suitable to study ubiquitin-dependent proteasomal degradation of specific proteins [32] Indeed, reticulocytes
Fig 5 Immunodetection of bTH mem and ubiquitin-conjugates in
chromaffin granule membranes (A) Chromaffin granule ghost proteins
(130 lg) were subjected to SDS/PAGE and immunoblotted with
antibodies directed against TH (lane 1) or Ub (lane 2) and 125 I-Protein
A as described in the Materials and methods section O, origin; F,
front (B) Chromaffin granule ghost proteins (800 lg) were subjected
to two-dimensional electrophoresis and immunoblotted with
anti-bodies directed against hTH1 (panel 1) or Ub (panel 2) Ub-conjugates
were first precisely localized on two-dimensional gel electrophoresis by
immunoblotting with anti-Ub Ig The membrane was thereafter
stripped and then reprobed with anti-TH Ig As shown in panel 2,
Ub-conjugates were detected at the same position as bTH (panel 1).
The immunoblotting procedure has been repeated using first anti-TH
and then anti-Ub Ig which also revealed colocalization of the two
immunoreactivities The time of exposure (membrane to film) which
was required to detect Ub-conjugates with anti-Ub Ig was usually
twofold to threefold longer than that required to detect TH
immuno-reactivity.
Trang 7contain multiple 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 lM) 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 was observed, followed by its 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 [41] 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 overall half-life of [35S]methionine-labelled hTH1
was estimated to 2.1 h in the presence of an
ATP-regenerating system and to 7.4 h when the lysate was
depleted of MgATP (Fig 4) By comparison, the half-life of
rat TH estimated 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 The shorter
half-lifes observed in the present reconstituted in vitro system
may be explained in several ways, including a stabilization
of TH in the cells as a result of its binding to membranes [9]
and to cytosolic proteins, e.g the 14.3.3 proteins [42], as
discussed below
That TH is a substrate for the Ub-conjugating enzyme
system also in vivo is further supported by our finding of
mono-ubiquitinated bTHmem in highly purified bovine
adrenal chromaffin granule ghosts (Fig 5) and for the first
time underlines a physiological role of this
post-translation-sal modification Although the TH- and Ub-
immunoreac-tivities revealed a comigration in the two selected (one- and
two-dimensional) electrophoretic systems we do not know
if all the TH subunits in the oligomer are ubiquitinated
A similar observation has been made for the ubiquitinated
form of the membrane-bound form of nitric oxide synthase
on SDS/PAGE [43] bTHmem behaves as an integral
membrane protein [7,9], but the mechanism by which it is
bound is not yet resolved Interestingly, it has been
suggested that ubiquitination might promote a structural
change (unfolding) of linked proteins [44], but it is not
possible at this point to answer the question of whether
bTHmemis inserted into the membrane before or after its
ubiquitination The finding that bTHmemis phosphorylated
by cAMP-dependent protein kinase on Ser40 in the
regulatory domain [9] may support an ubiquitination of
the enzyme 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 may be related to the
function of the ubiquitin C-terminal hydrolase (UCH-L1 or
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 the 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 the 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 Ub-conjugates of 105, 72, 60, 41 and 38 kDa, and the majority of the conjugates were found to be integral membrane proteins including some high-molecular-mass glycoproteins [46] As the synaptic membrane fraction represents a mixture of different types of membranes, the functional significance of this finding is not clear The specific localization of Ub-conjugates to secretory granules may suggest a function either in membrane fusion events or
in the turnover of the organelles Thus, it has been reported that an Ub-like conjugating enzyme system is involved in homotypic membrane fusion in Pichia pastoris [47] Alter-natively, Ub appended to membrane proteins may represent
a signal which promotes the selective sorting/sequestration and turnover of the secretory granules by autophagosomes
as already shown for the regulated turnover of other cytoplasmic organelles (reviewed in [48])
A C K N O W L E D G E M E N T S
The study was supported by grants from the Research Council of Norway, from the Novo Nordisk Foudation, The Nansen Fund, The Blix Family Fund for the Advancement of Medical Research and the Norwegian Council on Cardiovascular Diseases We greatly appreciate the expert technical assistance of Sidsel Riise for the preparation of hTH1 and that of Sissel Vik Berge for the preparation of chromaffin granule ghosts.
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