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No mitochondrial uncoupling artefact is caused by expression of uncoupling protein 1 in a mammalian cell culture: A new system to study mitochondrial carrier proteins
Author(s) -
Jastroch Martin,
Hirschberg Verena,
Klingenspor Martin
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.1044.3
Subject(s) - hek 293 cells , mitochondrion , microbiology and biotechnology , thermogenin , uncoupling protein , biology , brown adipose tissue , biochemistry , cell culture , yeast , cell , adipose tissue , genetics , gene
Functional and comparative studies of uncoupling proteins in their native environment are hampered by different mitochondrial, cellular and genetic backgrounds. Artificial systems such as yeast ectopically expressing UCPs or liposomes were consulted to address crucial mechanistic questions but these systems also produced inconsistencies with results from native mitochondria. We here introduce a novel mammalian cell system (HEK293) to study UCP1 function. We derived stably transfected HEK293 cell lines containing mouse UCP1 at concentrations that are comparable to brown adipose tissue mitochondria. UCP1 displays native behavior as it can be activated with fatty acids (palmitate) and inhibited with purine nucleotides (GDP). Importantly, UCP1 in HEK cell mitochondria is fully inhabitable in contrast to yeast mitochondria; and does not contribute to basal proton conductance. Furthermore, the catalytic centre activity of HEK cell UCP1 is comparable to native UCP1 activity in brown adipose tissue. Using this UCP1 test system, we are currently investigating modulators of UCP function. We emphasize that the mammalian HEK293 cell system is not only suitable for mechanistic studies on UCPs, but also for comparative functional studies between UCP orthologues and paralogues as it provides a standardized mitochondrial, cellular and genetic background.