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1. A role for ubiquitinylation and the cytosolic
proteasome in turnover of mitochondrial
uncoupling protein 1 (UCP1)
Kieran J. Clarke Xingyun Wang

2. Background
1.Uncoupling protein 1 (UCP1) is a 32 kDa tripartite mitochondrial carrier protein of 306 amino acids.
2.Adenine nucleotide translocator (ANT), also known as the ADP/ATP translocator, exports ATP from the
mitochondrial matrix and imports ADP into the matrix. ANT is the most plentiful protein in the inner
mitochondrial membrane. 298 amino acids.
UCP1 ANT (ADP/ATP

3. （Calpains/Caspases）
泛素-蛋白酶体途径
溶酶体途径
线粒体蛋白酶等其他途径
胞液蛋白酶水解途径
Background

4. The proteasome represents a cancer drug target, and its inhibition results in the accumulation
of tumor-suppressive proteins
UBIQUITIN-PROTEASOME PATHWAY
BACKGROUNDS

5. Background
In regard to protein turnover, UCP1 degradation in BAT has only ever been described in the context of lysosomal
degradation.
Recently identified serine phosphorylation of UCP1 in BAT mitochondria, the proportion of which is augmented following
cold acclimation. Phosphorylation has been identified as a flag for ubiquitinylation .
To investigate whether UCP1 can be ubiquitinylated and whether the cytoplasmic proteasome plays a role in turnover of
UCP1.

6. Results UCP1 has the potential to be ubiquitinylated
BAT mitochondria were incubated with purified ubiquitin in a ubiquitin conjugating system.
BAT (500 μg/ml)
mitochondria were
incubated at 37 °C in a
non-ionic medium (250
mM sucrose 5 mM
Tris–HCl, pH 7.4, 1 mM
EGTA) containing an ATP
regenerating system
(1mM ATP,
10mM phosphocreatine
and 20 U/ml creatine
kinase) and a ubiquitin
conjugating system
(100 μg ubiquitin, 1.4 μg
fraction 1 and 1.4 μg
fraction 2 from
Calbiochem

7. Results
Immunoblots illustrating the immunoprecipitation of UCP1 using an anti-ubiquitin antibody and the
immunoprecipitation of ubiquitinated proteins using an anti-UCP1 antibody.

8. Results
Immunoblots illustrating the immunoprecipitation of UCP1 using an anti-ubiquitin antibody and the
immunoprecipitation of ubiquitinated proteins using an anti-UCP1 antibody.

9. Results
Immunoblots illustrating the immunoprecipitation of UCP1 using an anti-ubiquitin antibody and the
immunoprecipitation of ubiquitinated proteins using an anti-UCP1 antibody.
磷酸化丙酮酸脱氢酶

10. Results
Brown adipocytes were isolated and differentiated from C57BL6 mice and treated
with the proteasome inhibitor MG132 (+) or the vehicle DMSO (.) for 12 h.
UCP1 protein levels in mature brown adipocytes are sensitive to
proteasome inhibition

11. Results
Succinic acid 琥珀酸
BAT mitochondria were isolated as described and an in vitro
degradation assay for UCP1.
For each assay BAT mitochondria (500 μg/mL) were incubated with an
ATP regenerating system, consisting of 1 mM ATP, 10 mM phosphocreatine,
20 U/mL creatine kinase, 2 mM MgCl2 and 0.1% (w/v) defatted bovine
serum albumin. Where indicated 20 mM succinate, 20 μM FCCP, ubiquitin
mix (35 μg ubiquitin, 1.4 μg fraction 1, 1.4 μg fraction 2 from Calbiochem
cat. #662096) and 5 μg 26S proteasome fraction were added to the assay.

12. Results

13. Results
cycloheximide：环己亚胺

14. Results

15. UCP1 protein levels are stable in isolated BAT and thymus mitochondria incubated at 37 °C in the absence of a
proteasome conjugating system.

16. BAT mitochondria isolated from cold acclimated rats contain greater amounts of UCP1 with a
molecular weight >32 kDa compared to BAT mitochondria isolated from rats housed at room
temperature.

18. Results

20. 结论
• 1.UCP1通过UPS降解
• 2UCP1在WB实验中的条带位置，曝光强度等。