Year: 2006
Type: dissertation
Abstract: Most proteins residing in the mitochondrial matrix are synthesized in the cytosol\nand post-translationally imported into the mitochondrial matrix. The matrix-targeted\npreproteins traverse the outer mitochondrial membrane (OM) via the Translocase of the\nOuter Membrane (TOM) complex, and the inner mitochondrial membrane (IM) via the\nTranslocase of the Inner Membrane 23 (TIM23) complex. A novel system was set up to\nexamine the import of matrix-targeted preproteins into mitochondria using fluorescence\nspectroscopy. The fluorescent probe 6-(7-nitrobenz-2-oxa-1,3-diazol-4-\nyl)aminohexanoic acid (NBD) was site-specifically incorporated into different positions\nalong the model matrix protein Su9-DHFR. The fluorescent-labeled polypeptides were\neither fully imported into isolated mitochondria or were arrested along the translocation\npathway by the binding of methotrexate (MTX) to the DHFR moiety, creating NBDSu9-\nDHFR•MTX import intermediates. The NBD-Su9-DHFR polypeptides were able\nto be fully imported into the mitochondrial matrix in the absence of MTX, and were\ninaccessible to externally-added iodide ion quenchers. Treatment of the mitochondria\nwith the pore-forming antibiotic alamethicin allowed the iodide ion quenchers access to\nthe matrix through pores in the inner membrane (IM). After Alamethicin treatment the fully-imported NBD-Su9-DHFR polypeptides were accessible to the externally-added\niodide ions. The extent of collisional quenching of the NBD fluorophores by the iodide\nions was measured as the Stern-Volmer quenching constant, Ksv. Ksv values were\nobtained for the NBD-Su9-DHFR polypeptides in the presence of MTX (import\nintermediates) or in the absence of MTX (fully-imported). The Ksv values for NBD-Su9-\nDHFR import intermediates were similar, despite the location of the NBD probe along\nthe translocation pathway. These Ksv values were similar to those obtained for the fullyimported\nNBD-Su9-DHFR polypeptides (-MTX). The locations of the varying probe\npositions along the import pathway were addressed using chemical crosslinking of Su9-\nDHFR Cys mutants. The use of fluorescence spectroscopy, in association with chemical\ncrosslinking, to analyze the mitochondrial protein import pathways will prove a useful\ntool to probe the environment of the nascent chain as it is crossing the import pathway\n(the TOM, TIM23 complexes).
Author: Holly Beth Cargill
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2276565158