Defining the Substrate Spectrum of the TIM22 Complex Identifies Pyruvate Carrier Subunits as Unconventional Cargos

In mitochondria, the carrier translocase (TIM22 complex) facilitates membrane insertion of multi-spanning proteins with internal targeting signals into the inner membrane [1, 2, 3]. Tom70, a subunit of TOM complex, represents the major receptor for these precursors [2, 4, 5, 6]. After transport across the outer membrane, the hydrophobic carriers engage with the small TIM protein complex composed of Tim9 and Tim10 for transport across the intermembrane space (IMS) toward the TIM22 complex [7, 8, 9, 10, 11, 12]. Tim22 represents the pore-forming core unit of the complex [13, 14]. Only a small subset of TIM22 cargo molecules, containing four or six transmembrane spans, have been experimentally defined. Here, we used a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum. Along with carrier-like cargo proteins, we identified subunits of the mitochondrial pyruvate carrier (MPC) as unconventional TIM22 cargos. MPC proteins represent substrates with atypical topology for this transport pathway. In agreement with this, a patient affected in TIM22 function displays reduced MPC levels. Our findings broaden the repertoire of carrier pathway substrates and challenge current concepts of TIM22-mediated transport processes.