Chaperoning Preassembly Modules for Mitochondrial Ribosome Assembly

Henry, Michael F. Abstract The goal of the proposed research is to define the mechanisms of ribosome assembly within mitochondria. While remarkable progress has recently been made towards understanding the structure of mitoribosomes, the unique pathways and factors that facilitate their biogenesis remains largely unknown.

Dysfunctional mitoribosome assembly abolishes the synthesis of several essential components of the respiratory chain, which can compromise cellular energy production and generate reactive oxygen species that promote degenerative disease, aging, and cancer. Thus, a clearer understanding of how these highly complex macromolecular structures are assembled is necessary to better understand mitochondrial

disease. To gain insight into this process, this proposal will examine how an evolutionarily conserved yeast protein called Mam33 effectively chaperones a subset of newly imported mitoribosomal proteins and facilitates their incorporation into the assembling large subunit. This mitoribosomal chaperone contrasts

with those in bacterial and eukaryotic assembly pathways because it binds multiple mitochondrial ribosomal proteins (MRPs), rather than a single ribosomal protein. This and emerging data suggest that mitochondria might minimize the complexity of ribosome biogenesis by forming small RNA-free preassembly blocs complexed with Mam33, rather than the individual addition of MRPs observed for

bacterial and eukaryotic cytosolic ribosomes. The first aim will determine the composition and binding characteristics of Mam33-MRP preassembly complexes. These experiments will determine 1) the number and composition of the preassembly complexes, 2) their binding domains and 3) whether they can form

without Mam33. This information will advance our understanding of Mam33 function and mitochondrial ribosome assembly chaperones in general. The second aim will assess the binding properties of Mam33- MRP preassembly complexes. These experiments will 1) establish whether Mam33 targets the N-terminal regions of its mtLSU client proteins, 2) delineate the docking sites for its 5 known mtLSU cargo proteins, 3)

identify potential new interactants and 4) examine Mam33-client protein incorporation at a specific mtLSU assembly step. Since Mam33 is conserved in eukaryotic organisms, information gained in yeast will be applicable to human disorders. In human patients, bi-allelic mutations in its ortholog p32/HABP1/gC1qR

cause severe multisystemic defects in mitochondrial energy metabolism, which directly result from oxidative phosphorylation deficiencies and mitochondrial instability. Furthermore, p32 overexpression has been detected in nearly all tissue specific forms of cancer and associated with poor prognosis. For

these reasons, understanding the physiological role of this protein in the mitochondrion is timely.