Assaying protein import into mitochondria using fluorescence spectroscopy

Most proteins residing in the mitochondrial matrix are synthesized in the cytosol and post-translationally imported into the mitochondrial matrix. The matrix-targeted preproteins traverse the outer mitochondrial membrane (OM) via the Translocase of the Outer Membrane (TOM) complex, and the inner mitochondrial membrane (IM) via the Translocase of the Inner Membrane 23 (TIM23) complex. A novel system was set up to examine the import of matrix-targeted preproteins into mitochondria using fluorescence spectroscopy. The fluorescent probe 6-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminohexanoic acid (NBD) was site-specifically incorporated into different positions along the model matrix protein Su9-DHFR. The fluorescent-labeled polypeptides were either fully imported into isolated mitochondria or were arrested along the translocation pathway by the binding of methotrexate (MTX) to the DHFR moiety, creating NBDSu9- DHFR??MTX import intermediates. The NBD-Su9-DHFR polypeptides were able to be fully imported into the mitochondrial matrix in the absence of MTX, and were inaccessible to externally-added iodide ion quenchers. Treatment of the mitochondria with the pore-forming antibiotic alamethicin allowed the iodide ion quenchers access to the matrix through pores in the inner membrane (IM). After Alamethicin treatment the fully-imported NBD-Su9-DHFR polypeptides were accessible to the externally-added iodide ions. The extent of collisional quenching of the NBD fluorophores by the iodide ions was measured as the Stern-Volmer quenching constant, Ksv. Ksv values were obtained for the NBD-Su9-DHFR polypeptides in the presence of MTX (import intermediates) or in the absence of MTX (fully-imported). The Ksv values for NBD-Su9- DHFR import intermediates were similar, despite the location of the NBD probe along the translocation pathway. These Ksv values were similar to those obtained for the fullyimported NBD-Su9-DHFR polypeptides (-MTX). The locations of the varying probe positions along the import pathway were addressed using chemical crosslinking of Su9- DHFR Cys mutants. The use of fluorescence spectroscopy, in association with chemical crosslinking, to analyze the mitochondrial protein import pathways will prove a useful tool to probe the environment of the nascent chain as it is crossing the import pathway (the TOM, TIM23 complexes).