The presequence pathway is essential for mitochondrial biogenesis. The TIM23 complex mediates the translocation of precursor proteins with N-terminal targeting signals into the matrix or sorted to the inner mitochondrial membrane (IMM). The core of the TIM23 complex (translocase) interacts with the presequence translocase-associated motor complex (PAM), which drives the import process into the matrix and precursor unfolding with the mitochondrial Hsp70’s (mtHsp70) ATPase activity. How these factors facilitate mtHsp70-driven import of the precursor is still poorly understood. Here, we will elucidate the molecular and functional architecture of the PAM complex. This characterization will explain the assembly, dynamic, and mechanistic events relevant to PAM activity. The quantitative assessment of subunit-subunit interactions will provide the kinetic and thermodynamic parameters that are typically unreachable in native systems. At the same time, an array of in vivo strategies will shed light on the influence of subunits on PAM assembly and precursor import.