Chaperoning Preassembly Modules for Mitochondrial Ribosome Assembly

"Chaperoning Preassembly Modules for Mitochondrial Ribosome Assembly," aims to define the mechanisms that govern ribosome assembly within mitochondria by characterizing an evolutionarily conserved chaperone, Mam33. Although recent structural studies have illuminated mitoribosome architecture, the pathways and factors that facilitate their biogenesis remain poorly understood. Defects in mitoribosome assembly prevent synthesis of key respiratory chain components, impairing cellular energy production and increasing reactive oxygen species that contribute to degenerative disease, aging, and cancer. This proposal addresses a critical gap: how mitochondria assemble highly complex ribonucleoprotein machines with a distinct set of chaperones and pathways compared to bacterial and cytosolic eukaryotic ribosomes.

The central hypothesis is that Mam33 functions as a multivalent chaperone that forms small, RNA-free preassembly modules containing multiple mitochondrial ribosomal proteins (MRPs). Unlike bacterial or cytosolic ribosome assembly, which often proceeds by the stepwise addition of individual ribosomal proteins, mitochondria may reduce complexity by preassembling clusters of MRPs coordinated by Mam33. The project is organized into two specific aims. Aim 1 will determine the composition and binding characteristics of Mam33-MRP preassembly complexes, quantifying the number and identity of MRPs within each complex, mapping Mam33 binding domains, and testing whether preassembly complexes can form independently of Mam33. These experiments will elucidate how Mam33 organizes client proteins prior to their incorporation into the large mitoribosomal subunit (mtLSU).

Aim 2 will assess the detailed binding properties of Mam33-MRP preassembly complexes. Investigations will establish whether Mam33 preferentially targets N-terminal regions of its mtLSU clients, delineate docking sites for known cargo proteins, seek novel interactors, and examine how Mam33-client complexes are incorporated during a specific mtLSU assembly step. Together, the two aims will reveal mechanistic principles of Mam33-mediated chaperoning and clarify whether mitochondrial ribosome biogenesis uses modular preassembly as a general strategy.

Because Mam33 is conserved across eukaryotes, insights from yeast are expected to inform understanding of the human ortholog p32/HABP1/gC1qR. Biallelic mutations in p32 cause severe multisystemic defects in mitochondrial energy metabolism due to oxidative phosphorylation failure and mitochondrial instability, and p32 overexpression is observed in many cancers with poor prognosis. Thus, defining Mam33/p32 function has disease relevance for mitochondrial disorders and oncology. By elucidating how Mam33 organizes MRPs into preassembly modules and how these modules integrate into the growing mitoribosome, this research will advance fundamental knowledge of mitochondrial biogenesis and provide a framework to interpret disease-linked perturbations in mitoribosome assembly.