The mitochondria of cultured mammalian cells: II. Expression and visualization of exogenous proteins in fixed and live cells.
Mitochondria are almost ubiquitous organelles in Eukaryota. They are highly dynamic and often complex structures in the cell. The mammalian mitochondrial proteome is predicted to comprise as many as 2000-2500 different proteins. Determination of the subcellular localization of any newly identified protein is one of the first steps toward unraveling its biological function. For most mitochondrial proteins, this can now be done relatively easily by cloning a complementary deoxyribonucleic acid of interest in frame with an additional sequence for a fluorescent or nonfluorescent protein tag. Transfection and subsequent visualization, either by direct fluorescence microscopy or by indirect immunofluorescence microscopy, will give the first clue to mitochondrial localization. In combination with a fluorescent "marker" dye, the mitochondrial localization can be confirmed. This chapter describes some of the methods used in determining mitochondrial protein localization, which can also be used to study dynamics of mitochondria or individual mitochondrial proteins or protein complexes.
Goffart
Steffi
S
FinMIT Centre of Excellence, Institute of Medical Technology and Tampere University Hospital, University of Tampere, Finland.
Martinsson
Peter
P
Malka
Florence
F
Rojo
Manuel
M
Spelbrink
Johannes N
JN
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002470:Cell Survival; D004396:Coloring Agents; D004272:DNA, Mitochondrial; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D016707:Tissue Fixation / Q000379:methods*; D014162:Transfection; D001616:beta-Galactosidase
D004396:Coloring Agents; D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase