Expression of algal nuclear ATP synthase subunit 6 in human cells results in protein targeting to mitochondria but no assembly into ATP synthase

Artificial transfer of mitochondrial genes to the nucleus has implications for the understanding of mitochondrial function, evolution, and human health. Therefore, we created nuclear compatible versions of human subunit a (A6) of ATP synthase, linked to a mitochondrial targeting signal. Expression and targeting of human nuclear subunit a were compared to subunit a of Chlamydomonas reinhardtii, which naturally occurs in the nucleus. Algal subunit a was targeted to mitochondria more efficiently than human nuclear subunit a variants. However, there was no evidence of improved mitochondrial function in cultured cells; on the contrary, long-term expression of algal subunit a was associated with poor survival and intolerance of growth conditions that demand heavy reliance on oxidative phosphorylation. Analysis of enriched mitochondrial membrane fractions on native gels revealed a high-molecular-weight complex containing FLAG-tagged subunit a; however, this complex did not colocalize with ATP synthase. Thus, there was no evidence of assembly of algal subunit a into holoenzyme, nor did human nuclear subunit a colocalize with ATP synthase holoenzyme. In conclusion, obstacles remain to functional expression of mitochondrial genes transferred to the nucleus.