Linked Life Data

19733184

Source:http://linkedlifedata.com/resource/pubmed/id/19733184

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2009-11-13
pubmed:abstractText
Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. Using fragments of different fluorescent proteins, we investigated the temporal resolution and the quantitative accuracy of BiFC analysis. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of yellow fluorescent protein fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 min after the addition of rapamycin and a 10-fold increase in the mean fluorescence intensity in 8 h. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and yellow fluorescent protein produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment with the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized before the addition of rapamycin formed BiFC complexes with the same efficiency as did newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggests that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular adaptation to protein folding stress. In summary, BiFC analysis enables detection of protein interactions within minutes after complex formation in living cells, but does not allow detection of complex dissociation. Conditional BiFC complex formation depends on the folding efficiencies of fluorescent protein fragments and can be affected by the cellular protein folding environment.
pubmed:grant
pubmed:commentsCorrections
http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-10318894, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-11753368, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-11904411, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-11983170, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-12415118, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-12438689, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-12692560, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-15121849, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-15282548, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-15509571, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-15631464, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-15796538, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-16103094, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-16426976, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-16454041, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-16461889, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-16489332, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-16614209, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-16625152, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-16862132, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-17090218, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-17099704, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-17512539, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-17785458, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-17959720, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-19007309, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-19771334, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-2123553, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-7092917, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-7518356, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-7792603, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-7809080, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-8008069, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-8448132, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-9184161, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-9223271, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-9237989, http://linkedlifedata.com/resource/pubmed/commentcorrection/19733184-9651680
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
1089-8638
pubmed:author
pubmed:issnType
Electronic
pubmed:day
4
pubmed:volume
394
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
391-409
pubmed:dateRevised
2010-12-3
pubmed:meshHeading
More...