Description: This cell line can be used to determine the function of a protein by rapidly inactivating the protein of interest by rapamycin induced rerouting of the protein to the mitochondria. The HeLa-Mitotrap-Ap1g1-FKBP is a stably transfected HeLa cell line expressing two proteins containing rapamycin binding activity. To generate this cell line, the HeLa-Mitotrap cell line, ECACC 15042201, expressing a single construct expressing a mitochondrial trapping protein containing; an FRB domain, (the term FRB refers to protein containing a FKBP12 and Rapamycin Binding (FRB) domains within the mTOR (mammalian Target of Rapamycin) protein, has been transfected with a second construct expressing siRNA-resistant subunits, FKBP-tagged subunits of the adaptor protein (AP1) complexes of clatharin-coated vesicles (CCVs) together with an FKBP and rapamycin-binding domain with a mitochondrial targeting signal. An FKBP domain, FKBP is a cellular chaperone protein and prolyl isomerase which is a target for rapamycin binding and will dimerize with FRB-containing protein. Upon treatment with rapamycin a target protein containing an FKBP domain, e.g, AP1, will dimerize with FRB domains and sequester the resulting protein complexes to the mitochondria. Treatment of cell lines containing both FRB and FKBP rapamycin binding proteins can sequester target proteins to the mitochondria within minutes of treatment rendering the cells ready for immediate assay.The a-FKBP construct was based on a pIRESneo2 plasmid encoding an siRNA-resistant form of a, which includes a brain-specific insert that acts as an epitope tag (Motley et al.,2006, PMID: 17035630). The FKBP coding sequence was amplified by PCR and inserted into the linker region of a;. The open reading frame was sequenced, the plasmid was transfected into HeLaM cells, and clonal cell lines were isolated as previously described (Motley et al., 2006).

Also Known As:

Species: Human

Tissue: Cervix

Growth Properties: Adherent

Morphology: Epithelial polygonal

Growth Medium: Dulbecco's Modified Eagle'?s Medium high glucose (DMEM) + 2mM Glutamine + 10% Foetal Bovine Serum (FBS)

Subculturing Procedure: Trypsinise cultures at 80% confluence with 0.05% Trypsin/EDTA. Seed flasks at 2x10e4 cells/ cm2. Cultures must be incubated in a humidified 10% CO2/90% air incubator at 37°C.

Release Conditions: Yes. Commercial organisations and for-profit users see Deposit Release Form for Commercial Organisations.

Depositor: Margaret S Robinson, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK

Originator: Yes

References: Robinson MS, Sahlender DA, Foster SD. Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria. Dev Cell. 2010 Feb 16;18(2):324-31. doi: 10.1016/j.devcel.2009.12.015. PMCID: PMC2845799, PMID: 20159602

Hirst J, Borner GH, Antrobus R, Peden AA, Hodson NA, Sahlender DA, Robinson MS. Distinct and overlapping roles for AP-1 and GGAs revealed by the "knocksideways" system. Curr Biol. 2012 Sep 25;22(18):1711-6. doi: 10.1016/j.cub.2012.07.012. Epub 2012 Aug 16. PMCID: PMC3485558

Hyperlink to ECACC Cell Line Data Sheet: 15042203