Tauchmannova 2015 Abstract MiP2015
|Assembly of subunit Fo-a into mammalian ATP synthase.|
The biogenesis of mammalian ATP synthase is a complex process believed to proceed via several modules. In the later steps, the membranous subcomplex is formed and the final phase is represented by incorporation of the two mtDNA-encoded subunits Fo-a and A6L (Atp6 and Atp8). However, little is known about the position of two newly described Fo accessory subunits DAPIT (also termed Usmg5) and MLQ (also known as c14orf2) in the assembly scheme and about their role in regulation of ATP synthase biogenesis. We have utilised several model systems, namely rho0 cells lacking mtDNA and thus both subunits o-a and A6L, cells harbouring 9205delTA microdeletion, which results in the absence of the subunit o-a, HEK293 cells with knockdown of DAPIT protein and HEK293 cells with knockout of MLQ protein and followed the assembly state of ATP synthase among them. Contrary to previously reported data, we observed normal levels of assembled ATP synthase in DAPIT knockdown and MLQ knockout cells. Our results indicate, that lack of DAPIT protein leads to the assembly of more labile, but complete and functional enzyme. Absence of either Fo-a alone or Fo-a and A6L results in normal levels of structurally altered, labile, and ~60 kDa smaller enzyme complex which also lacks DAPIT and MLQ. This complex retains the ATP hydrolytic activity but is unable to synthesize ATP. Cells with the MLQ knockout presented with the phenotype similar to the lack of Fo-a: normal content of smaller and labile complex. In the absence of MLQ, ATP synthase did not contain subunit Fo-a and the total Fo-a content was also decreased, presumably due the degradation of unassembled subunit. This complex also retained ATP hydrolytic activity, while its phosphorylating capacity was affected. Based on our data, we conclude that MLQ and Fo-a closely associate and their incorporation into the enzyme complex depends on each another. On the contrary, DAPIT protein seems to be incorporated at the very last step and its presence stabilises the holoenzyme.
Labels: MiParea: mt-Biogenesis;mt-density, mt-Membrane, mtDNA;mt-genetics, Genetic knockout;overexpression
Organism: Human Tissue;cell: Other cell lines, HEK
Event: B1, Oral MiP2015
Inst Physiology Czech Acad Sc, Prague, Czech Republic. - [email protected]
This project is supported by the Czech Science Foundation grants 14-36804G and P303/12/1363.