Biomedical Engineering Reference
In-Depth Information
These receptors are part of the translocase of the outer membrane,
the TOM complex [
8
-
10
]. This complex appears to interact with
all mitochondrial preproteins and either facilitates their insertion
into the outer membrane, or their translocation through the outer
membrane into the IMS.
Most of the preproteins destined for the inner membrane or
the matrix contain cleavable N-terminal targeting sequences, the
so-called presequences that are hydrophobic on one face and posi-
tively charged on the other [
11
,
12
]. These targeting signals can be
predicted on the basis of the primary sequence of proteins by the
use of specific algorithms like MITOPRED [
13
], MitoProt II
[
14
], TargetP [
15
], or Predotar [
16
]. Presequences are recognized
at the level of the inner membrane by the TIM23 translocase com-
plex. This translocase mediates the cargo's translocation across and
the insertion into the inner membrane using ATP and the mem-
brane potential as energy sources. In the matrix, the presequences
are proteolytically removed by the mitochondrial processing pepti-
dase (MPP), and the proteins fold into their native structures by
the assistance of mitochondrial chaperones.
Some preproteins that are destined for the inner mitochondrial
membrane or the IMS contain bipartite signal sequences. In these
proteins, the N-terminal matrix-targeting signals are followed by
hydrophobic sorting signals that direct the preproteins into the
inner membrane. Once inserted, the cargo may remain anchored
in the inner membrane or are released into the IMS after a further
processing event.
Another group of inner membrane proteins is sorted by the
second Tim machinery, the TIM22 complex [
17
-
20
]. Substrates
of the TIM22 pathway lack presequences and contain patches of
targeting information that is scattered throughout the sequence of
the mature polypeptide. These proteins, such as members of the
metabolite carrier family, cross the TOM channel, are ushered
through the IMS by specific chaperones, and eventually inserted in
the inner membrane by the TIM22 complex in a membrane
potential- dependent manner.
Some soluble IMS proteins are imported independently of
ATP hydrolysis or the membrane potential. The import of these
proteins seems to be purely driven by the use of cysteine oxidation,
which is facilitated by two specialized IMS components, Mia40
and Erv1 [
21
-
24
].
To investigate the different protein import machineries and
pathways, in vitro reconstituted systems are adequate tools. To per-
form such experiments, preproteins labeled in vitro with [
35
S] can
be imported into isolated mitochondria. These assays reveal charac-
teristics and requirements of protein import and were highly effec-
tive in the past to characterize the import process of mitochondrial
preproteins. In this chapter, a standard protocol for reconstituted
