Abstract
Disturbances in the assembly of mitochondrial complex I (CI) are a frequent cause of mitochondrial disorders. Several lines of evidence hint at a semi-sequential assembly pathway, in which the 45 individual subunits that form the holoenzyme are pieced together by means of smaller intermediates. To understand this process, it is necessary to explain the exact order, the rate-limiting steps, and the dynamics of subunit incorporation. In this chapter, we describe an approach to regulate the expression levels of an AcGFP(1)-tagged subunit (NDUFS3) in mammalian cells by means of a tetracycline-inducible promoter. This strategy allows the study of the dynamics of CI assembly intermediates in living cells on native gels. After establishing that the AcGFP(1) tag does not interfere with the activity and assembly of the enzyme, we show how this system can be used to trace the labeled subunit in an induction pulse-chase experiment or to study its accumulation in specific assembly intermediates after inhibition of mitochondrial translation.
