Biomedical Engineering Reference
In-Depth Information
Chapter 2
In Vitro Analysis of the Mitochondrial Preprotein Import
Machinery Using Recombinant Precursor Polypeptides
Dorothea Becker and Wolfgang Voos
Abstract
The import of proteins into mitochondria represents an essential process for the survival of eukaryotic
cells. Most mitochondrial proteins are synthesized as cytosolic precursor proteins. A complex chain of reac-
tions needs to be followed to achieve a successful transport of these precursors from the cytosol through
the double membrane system to their fi nal destination inside the mitochondria. In order to elucidate the
details of the translocation process, in vitro import assays have been developed that are based on the incu-
bation of isolated active mitochondria with natural or artifi cial precursor proteins containing the appropri-
ate targeting information. Using this basic system, most of the protein components of the import machinery
have been identifi ed and functionally characterized. However, a detailed defi nition of the molecular mech-
anisms requires more specialized assay techniques. Here we describe modifi cations of the standard in vitro
import assay technique that are based on the utilization of large amounts of recombinant preprotein con-
structs. The application of saturating amounts of substrate preproteins is a prerequisite for the determina-
tion of translocation kinetics and energy requirements of the import process. Accumulation of preproteins
as membrane-spanning translocation intermediates further provides a basis for the functional and struc-
tural characterization of the active translocation machinery.
Key words
Mitochondria,
Saccharomyces cerevisiae
, Protein import, Precursor proteins, Import kinetics,
Translocation intermediates
1
Introduction
According to proteomic studies, yeast mitochondria contain
roughly 1,000 different proteins [
1
]. Only about 1 % of these pro-
teins are encoded by the mitochondrial genome. Consequently, the
vast majority of proteins has to be imported into the organelle after
their synthesis on cytosolic ribosomes (
see
Fig.
1a
). To ensure their
proper destination, mitochondrial proteins are generated as pre-
cursor polypeptides carrying specifi c targeting signals. Most matrix-
destined precursors, as well as monotopic inner membrane proteins
possess N-terminal, typically cleavable presequences. Polytopic
outer and inner membrane proteins, by contrast, carry internal
signals that are usually distributed throughout the mature protein.
