Abstract: The majority of multispanning inner mitochondrial membrane proteins utilize internal targeting signals, which direct them to the carrier translocase (TIM22 complex), for their import. MPV17 and its Saccharomyces cerevisiae orthologue Sym1 are multispanning inner membrane proteins of unknown function with an amino-terminal presequence that suggests they may be targeted to the mitochondria. Mutations affecting MPV17 are associated with mitochondrial DNA depletion syndrome (MDDS). Reconstitution of purified Sym1 into planar lipid bilayers and electrophysiological measurements have demonstrated that Sym1 forms a membrane pore. To address the biogenesis of Sym1, which oligomerizes in the inner mitochondrial membrane, we studied its import and assembly pathway. Sym1 forms a transport intermediate at the translocase of the outer membrane (TOM) complex. Surprisingly, Sym1 was not transported into mitochondria by an amino-terminal signal, and in contrast to what has been observed in carrier proteins, Sym1 transport and assembly into the inner membrane were independent of small translocase of mitochondrial inner membrane (TIM) and TIM22 complexes. Instead, Sym1 required the presequence of translocase for its biogenesis. Our analyses have revealed a novel transport mechanism for a polytopic membrane protein in which internal signals direct the precursor into the inner membrane via the TIM23 complex, indicating a presequence-independent function of this translocase.