Biomedical Engineering Reference
In-Depth Information
also called presequence translocase [
2
]. Proteins destined for the
matrix require a membrane potential across the inner membrane as
an initial energy source for insertion of the N-terminal presequence
into the inner membrane pore provided by the TIM23 complex.
Completion of matrix import furthermore requires chemical energy
in the form of ATP-hydrolysis, catalyzed by the matrix heat shock
protein of 70 kDa (mtHsp70, Ssc1 in yeast), which provides the
driving force for the unfolding of preprotein domains and the trans-
location of the bulk polypeptide chain across the membrane [
3
].
MtHsp70 represents the core component of the
P
resequence-
Translocase-
A
ssociated
M
otor (PAM). The motor complex is
recruited to the inner face of the inner membrane import complex
by an interaction with the peripheral membrane protein Tim44.
Further essential PAM-components are the nucleotide exchange
factor Mge1 (
M
itochondrial
G
rp
E
homolog), as well as the ATPase-
stimulating J-domain protein Pam18, and its partner Pam16. In the
matrix, the presequence is cleaved off (often by the
m
atrix-
p
rocess-
ing
p
eptidase, MPP), resulting in the mature-sized protein. Folding
of imported proteins is initially promoted by mtHsp70, which sta-
bilizes, and thereby protects unfolded proteins until they acquire
their native conformation (assisted by the Hsp60 chaperonin sys-
tem) or in the case of multimeric proteins, assemble into the respec-
tive protein complexes [
4
].
Due to its convenience, especially with regards to genetic
manipulation, the yeast
Saccharomyces cerevisiae
represents an ideal
model organism for the study of the mitochondrial biogenesis pro-
cesses typical for eukaryotic cells. Although research in the recent
years has revealed signifi cant additional functions of mitochondria
in mammalian cells compared to yeast, the preprotein import
machineries are remarkably conserved. An experimental analysis of
the mitochondrial protein import fi rst requires the isolation of
functionally intact mitochondria, as well as an assessment of their
structural integrity. The isolation of yeast mitochondria includes an
enzymatic digestion of the cell wall, followed by a mechanical dis-
ruption of the resulting spheroblast. Enrichment of the mitochon-
drial fraction is achieved by subsequent differential centrifugation
of the crude cell lysate. The structural integrity of the mitochon-
drial preparation is assessed by checking the membrane potential
across the inner mitochondrial membrane, which is of particular
importance for the comparative analysis of mitochondria derived
from different yeast strains. The assay described here is based on
the reversible interaction of a fl uorescent dye with membranes in a
membrane potential-sensitive manner [
5
,
6
]. Secondly, an appro-
priate precursor protein, consisting of a mitochondrial signal
sequence and a reporter component has to be generated. The pre-
cursor can be either synthesized as a radiolabeled polypeptide by
translation in a cell-free system, or isolated from
E. coli
cells as a
recombinant protein. The import reaction in vitro is fi nally performed
by combining intact mitochondria with precursor polypeptides
