In vivo localisation and stability of human Mcl-1 using green fluorescent protein (GFP) fusion proteins.
Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins. We have expressed full length and mutated GFP:Mcl-1 fusion proteins to define structural motifs that control protein localisation and stability. When expressed in U-937 cells, full length Mcl-1 locates primarily within mitochondria and its half-life was approximately 3 h, which was identical to the native, endogenously expressed protein. When the terminal 20 amino acids from the C-terminus of the protein were detected, the protein was diffused in the cytoplasm, but its stability was unaffected. This confirms that this region is responsible for efficient targeting to mitochondria. Surprisingly, deletion of 104 amino acids (residues 79-183) that contain putative PEST sequences and other stability regulating motifs, did not affect protein stability.
School of Biological Sciences, Life Sciences Building, University of Liverpool, L69 7ZB, Liverpool, UK.
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Akgul
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Moulding
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White
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Edwards
D020816:Amino Acid Motifs; D001692:Biological Transport; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006207:Half-Life; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013816:Thermodynamics; D014162:Transfection; D020298:U937 Cells
D008164:Luminescent Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D021382:Protein Sorting Signals; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins