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-
DHFRÂMTX 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).
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/3971 |
| Date | 16 August 2006 |
| Creators | Cargill, Holly Beth |
| Contributors | Johnson, Arthur E. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Thesis, text |
| Format | 3805470 bytes, electronic, application/pdf, born digital |
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