Electron Cryomicroscopy Structure of a Membrane-anchored Mitochondrial AAA Protease

Sukyeong Lee,Steffen Augustin,Takashi Tatsuta,Florian Gerdes,Thomas Langer,Francis T.F Tsai

doi:10.1074/jbc.m110.158741

Abstract

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

Highlights

Energy-dependent proteases form oligomeric ring complexes and harbor conserved ATPase domains of the AAAϩ family [1]
Yeast possess two FtsH-related AAA proteases, an i-AAA protease and an m-AAA protease present in the mitochondrial inner membrane. i-AAA proteases expose their catalytic side toward the intermembrane space, whereas AAA proteases are active on the matrix side and are composed of homologous Yta10 and Yta12 subunits [10]
These chimeras were co-expressed with their wild-type counterpart in ⌬yta10⌬yta12 cells and analyzed for their ability to suppress the respiratory deficiency of cells lacking m-AAA proteases (Fig. 1, B and C)

Summary

Introduction

Energy-dependent proteases form oligomeric ring complexes and harbor conserved ATPase domains of the AAAϩ family [1]. Atomic Structure Fit—To generate an atomic model for the m-AAA protease, we manually fitted the x-ray structure of the Thermatoga maritima apo-FtsHCyt hexamer (PDB code 3KDS [7]) into our cryo-EM density.

Results

Conclusion