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Indian Journal of Biochemistry & Biophysics
Vol. 45, June 2008, pp 149-156
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Protein translocation pathways across the inner and outer mitochondrial
membranes
Jaspreet Kaur and Gautam Kaul*
Department of Animal Biochemistry, National Dairy Research Institute, Karnal, Haryana 132001, India
Received 29 June 2007; revised 02 May 2008
Mitochondria import different proteins that are encoded by nuclear genes and synthesized in the cytosol. Separate
translocases in inner and outer mitochondrial membranes like TOM, TIM23, TOB/SAM, TIM22 complex facilitate
recognition, import and intramitochondrial sorting of preproteins. Various cytosolic factors as Hsp70 and auxillary factors
assist in targeting these preproteins to their destinations. Also, different protein components in the matrix participate in this
energetically driven translocation process in a reaction that depends upon membrane potential and matrix-ATP. This review
summarizes the present knowledge on import and sorting of mitochondrial precursor proteins with glance on unresolved
questions.
Keywords: Mitochondria, Translocation, Membrane potential, ATP hydrolysis.
Introduction
Mitochondria are not only of key importance for
the bioenergetics of eukaryotic cells, but have critical
functions in metabolism of various biomolecules
including amino acids and lipids and also play a role
in apoptosis1. They are essential for cell viability,
proliferation and actively participate in development
and differentiation processes. Mitochondria contain
about 800 proteins in yeast2 to 1500 different proteins
in humans. Although there is a complete genetic
system in the matrix, the innermost compartment of
mitochondria, only ~1% of all mitochondrial proteins
are encoded by organelle genome and synthesized in
the matrix2 and rest are encoded by nuclear genes.
Basic aspect of this protein sorting is the transfer of
nuclearly encoded and cytoplasmically synthesized
proteins both across and into the mitochondrial inner
and outer membranes. Many hundreds of different
mitochondrial proteins participate in these processes.
In contrast, only a few protein components are
encoded by mitochondrial DNA itself, synthesized on
organellar ribosomes and inserted from matrix side
into inner membrane3.
The translocase of outer mitochondrial membrane,
TOM complex represents a common entry gate for
__________________________
*Author for correspondence
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Fax: 0184- 2250042
E-mail: [email protected]
usually all the nuclear encoded mitochondrial
proteins. After making their way through TOM
complex, these precursor proteins are directed to
atleast four different import pathways: (i)
Presequence pathway: imports the matrix proteins
using TIM23 complex, (ii) Carrier pathway: imports
hydrophobic inner membrane proteins, (iii) TOB/SAM
or SAM/TOB pathway: the mitochondrial outer
membrane β-barrel proteins are inserted into
membrane
via
TOB/SAM
(topogenesis
of
mitochondrial outer membrane β-barrel proteins also
called as sorting and assembly machinery), and (iv)
recently identified specific protein import machinery
for small intermembrane space proteins4, but little is
known about this pathway.
Since mitochondria play essential roles in
eukaryotic cells, knowledge regarding the protein
import and biogenesis of this organelle is equally
important. In this review, presequence, carrier,
TOB/SAM import pathways as well as import of
preproteins in intermembrane space of mitochondria
are discussed.
Mitochondrial targeting signals
The targeting signals or sequences for all matrixtargeted and inner membrane (IM) proteins that
contain a single transmembrane anchor, as well as
some intermembrane space (IMS) proteins are
typically N-terminal extensions of about 15-40 amino
acids, rich in basic and hydroxylated amino acids,
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INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, JUNE 2008
which can form amphipathic α-helices5. However,
targeting information is lost in the mature sequences
of approximately 30% of mitochondrial-targeted
proteins6. Presequence of these precursor proteins
interacts directly with the receptors of translocase of
outer membrane (Tom20 and Tom22); one half of this
amphipathic helix having a hydrophobic surface, is
recognized by the binding groove within Tom207,
whereas the other half, which is positively-charged, is
recognized by the Tom22 of TOM complex.
The movement of preproteins through cytosol is
associated with cytosolic factors namely chaperones
and cytosolic Hsp70, an eukaryotic homologue of
bacterial DNaK is one of the chaperones considered
as an important factor in intracellular protein traffic.
In addition, several other cytosolic factors may also
be considered as targeting factors as they bind
preproteins in a specific manner and deliver them to
the receptor components of TOM complex. Amongst
these, the best-characterized ones are mitochondrial
import stimulation factor (MSF) and aryl-hydrocarbon
receptor interacting protein which interacts with
preproteins and Tom207. MSF has shown two
activities: (i) it recognizes and forms stable complexes
with mature parts of mitochondrial precursors and
also facilitates depolymerizaton, and (ii) promotes
unfolding of preproteins3. Aggregated preadrenodoxin
(involved in electron transfer reaction) synthesized in
Escherichia coli8 becomes import competent in
presence of MSF. Both Hsp70 and MSF mediate
translocation of preproteins from cytosol to TOM
complex and involvement of each depends on the
nature of preprotein.
TOM or TOM40 complex
The holo-TOM complex has a size of ~450 kDa
and contains seven different proteins viz., the
receptors (Tom20 and Tom70) and five additional
proteins (Tom40, Tom22, Tom5, Tom6 and Tom7)
make up the TOM-core complex which acts as a
general insertion pore (GIP ~400 kDa)9-12 (Fig. 1).
Tom20 and Tom70 of TOM complex project into
cytosol and both have an N-terminal membrane
anchor and hydrophilic C-terminal domain of
~17 kDa and ~65 kDa, respectively13. Also, Tom22
extends an N-terminal domain of ~85 amino acid
residues into cytosol and has a single transmembrane
segment and a smaller C-terminal domain (~45
residues) facing the intermembrane space. Tom40
protein appears to be deeply embedded in the outer
membrane of mitochondria, although it does not have
many obvious contiguous hydrophobic sequence
segments.
In recent years, most of the homologous
components of TOM complex in mammalian system
have also been identified which include: Tom2014,
Tom2215, Tom7016, Tom4017 and Tom718. The yeast
GIP complex resolves as a ~400 kDa species on
native-PAGE, while the mammalian complex is a
~380 kDa species containing at least Tom40, Tom22
and Tom718. TOM complex plays an important role
during translocation of preproteins to subsequent
translocases in the import pathway19.
Role of different proteins of TOM complex
Tom20 and Tom70 act as receptors for the
precursor proteins20. Tom22 has various roles
including receptor functions21, helps in passage of
precursor proteins from the receptors to pore22, and
binds precursors at trans site23. Tom40 forms poreforming component of the complex and is essential
for cell viability. Different mutations generated by
altering 10 conserved regions of Neurospora crassa
Tom40 protein revealed that all these affect the ability
of altered Tom40 to be assembled into TOM complex
in isolated mitochondria and such mitochondria have
defects in importing preproteins to different subcomparments19. Although the receptors of TOM
complex (Tom20 and Tom22) preferentially
recognize presequences, Tom70 interacts mainly with
hydrophobic precursor proteins carrying internal
targeting signals (carrier pathway preproteins).
Tom6 plays a major role in the stability of TOM
complex25 and is required to promote assembly of
Tom40 and Tom22, but not for their interaction
during translocation10. Absence of Tom7 results in
more stable associations between Tom40, Tom20 and
Tom22, suggesting that it plays a role opposite to that
of Tom6 with respect to TOM stability10. Tom7 is
also involved in the trans site binding and passage of
precursors to TIM23 complex25. Mutants of N. crassa
Tom proteins lacking both Tom6 and Tom7 have an
extremely labile TOM complex and exhibit altered
growth phenotype20. Tom5 in Saccharomyces
cerevisiae maintains the structure of TOM complex
rather than acting in preprotein transfer26, whereas
single mutants lacking N. crassa Tom5 display no
apparent TOM complex abnormalities and has minor
role in maintaining TOM complex stability20.
Proteins of mitochondrial inner membrane contain
transmembrane α-helices, like proteins present in
most other membranes of eukaryotic cell. However,
KAUR & KAUL: PROTEIN IMPORT INTO MITOCHONDRIA
151
Fig. 1—A model for protein import into mitochondria [Preproteins are synthesized in the cytosol and are directed to the mitochondrial
surface by either N-terminal or internal targeting signals. These precursor proteins after passing through TOM complex are directed to
different import pathways: the presequence pathway which involves the TOM complex and TIM23 complex, the carrier pathway
requiring TOM complex and TIM22 complex. Translocation of mitochondrial outer membrane complex proteins, β-barrel proteins
requires SAM/TOB complex, in addition to TOM complex, whereas proteins with simple topology require only TOM complex. OM,
outer membrane; IM, inner membrane. Modified and produced from ref. 5]
outer membrane of mitochondria contains proteins
anchored in membrane by multiple β-strands9.
Recently, it has been revealed that TOM complex is
not sufficient to integrate β-barrel proteins into outer
membrane and also not able to assemble its own
precursor proteins into functional complexes. This led
to the discovery of topogenesis of mitochondrial outer
membrame β-barrel proteins (TOB) complex, also
called the sorting and assembly machinery (SAM)
complex (TOB/SAM complex).
Mitochondria lacking protein Sam37 (formerly
named Tom37) are strongly impaired in Tom40
assembly, the channel protein of outer mitochondrial
membrane, whereas other known import pathways,
presequence and carrier pathways are not affected27.
Since mitochondrial targeting signals are diverse in
their nature, it is puzzling that they converge at the
same import machinery for outer membrane
translocation and subsequent sorting to their
appropriate sub-mitochondrial compartments5 by
different import routes.
TOB/SAM complex
The mitochondrial TOB/SAM complex is derived
from the bacterial Omp85 complex, but to date no
detailed structural analysis of a mitochondrial β-barrel
protein has been acheived28. The founding subunit of
SAM complex is Sam37/Mas37/Tom3727 (Fig. 1). For
membrane integration of mitochondrial β-barrel
proteins, Sam37 is required which is a subunit of
SAMcore complex (~200 kDa). As compared to the
integration of β-barrel proteins, simple outer
membrane proteins with α-helical transmembrane
segment e.g. Tom20 can be inserted into outer
mitochondrial membrane with help of TOM complex
alone27.
The two additional subunits of SAMcore complex
are Sam50/Tob55 and Sam35/Tob38. Sam50
assembles as an integral outer membrane protein with
a predicted β-barrel domain29, whereas Sam35 and
Sam37 behave as peripheral membrane proteins that
are exposed on mitochondrial surface28,30-32. The
central subunit of SAM complex is represented by
Sam50, which in its purified form forms ring-shaped
particles with a large channel31. N-terminal domain of
Sam50/Tob55 is exposed to the intermembrane space
and represents interaction site for β-barrel precursors
before insertion in SAM/TOB complex2,33.
Sam35 and Sam37 function codependently in the
biogenesis of β-barrel proteins. Sam35 is required for
SAM complex to bind outer membrane substrate
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INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, JUNE 2008
proteins and its destabilization inhibits substrate
binding by Sam50, whereas Sam37 seems to assist the
release of substrates from SAM comlex28. This
SAMcore complex can further associate with a fourth
subunit, the morphology component Mdm10 to form
SAMholo complex. SAMcore complex is required for
biogenesis of all β-barrel proteins of outer membrane
,whereas Mdm10 and SAMholo complex play a
selective role in β-barrel biogenesis as they promote
assembly of Tom40, but not porin.
Tom7, a conserved subunit of TOM complex
functions antagonistically to Mdm10 in the biogenesis
of Tom40 and porin, suggesting that its role is not
limited to TOM complex. Tom7 also functions in the
mitochondrial protein biogenesis by a new
mechanism, segregation of a sorting component,
leading to a differentiation of β-barrel assembly34. In
yeast, assembly of Tom40 and porin in outer
membrane requires Tom13 in addition to SAMcore
complex29.
TIM complex or TIM23 complex/translocase
TIM23 complex can be structurally and
functionally sub-divided into two parts — membrane
integrated translocation channel and import motor,
which is located at the matrix face of channel35
(Fig. 1). Although membrane integrated part of this
translocase is sufficient for insertion of some
preproteins into the inner membrane, but complete
translocation of preproteins into matrix requires a
combination of both subunits. Without motor section,
preproteins cannot translocate into matrix. The
components of membrane integrated part of TIM23
complex include Tim17, Tim21, Tim23 and Tim50,
while import motor of translocase comprises five
proteins: Tim14, Tim16, Tim44, Mge1 and mtHsp70
(mitochondrial heat-shock protein 70)35. An integral
23 kDa protein of yeast inner membrane, N-terminal
half of Tim23 is hydrophilic in nature, whereas Cterminal half is hydrophobic. Tim17, also an integral
membrane protein shares sequence similarity with the
hydrophobic portion of Tim233. Tim23 also contains
an additional N-terminal domain, not present in
Tim17 which has a receptor function and also
responds to membrane potential.
Role of different proteins of TIM23 complex
Tim17 and Tim23 form the translocation channel
of TIM23 complex through which precursor proteins
usually with an N-terminal cleavable presequence
cross the hydrophobic barrier of inner membrane in
unfolded state36. Tim21 promotes coupling of the two
translocator complexes (TIM23 and TOM) during
translocation process. A single transmembrane
domain anchors Tim21 in the mitochondrial inner
membrane and it exposes its C-terminal domain into
intermembrane space. The C-terminal domain of
Tim21 does not to bind to any of the TIM23
components but specifically interacts with TOM
complex. It has been proposed that Tim21 binds to
trans site of TOM complex, thus keeping two
translocases in close contact37. Tim50 facilitates
protein transfer from TOM40 complex to TIM23
comlex36.
Mitochondrial Hsp70 (mtHsp70) in matrix
functions as import motor to drive vectorial
translocation and unfolding of precursor proteins in
cooperation with its partner proteins, mitochondrial
associated motor and chaperone (MMC) proteins.
Tim44 provides anchor for this motor (mtHsp70), so
that it can bind to the polypeptide emerging out from
TIM23 channel. The cochaperone Mge1 is nucleotide
exchange factor for mtHsp70 and enhances its
ATPase activity38. A new member of this translocator
system Tam41 (translocation assembly and
maintenance 41) has been identified from the yeast is
a peripheral inner mitochondrial membrane protein
facing matrix and not a constituent of TIM23
complex. It does not promote motor function of
mtHsp70 but is instead involved in the maintenance
of TIM23 complex integrity36. Roles of Tim14 and
Tim16 are discussed later.
Insertion of preproteins into TIM23 channel
depends mainly on the presence of membrane
potential across inner membrane which plays a dual
role in import process. Firstly, it activates the channel
protein itself and secondly it exerts an electrophoretic
effect on positively-charged presequence, thereby
driving presequence to matrix side7. Although, the
exact mechanism of import motor is still not
understood, it is clear that several rounds of ATPdependent binding to and release from the mtHsp70
lead to complete translocation of polypeptides into
matrix of mitochondria. The components of TIM23
translocase are highly conserved throughout
eukaryotic kingdom35.
Pathways of preprotein translocation across and into the outer
membrane
After translocation through β-barrel protein the
Tom40 pore, presequence binds to intermembrane
space domain of receptor Tom223. Tom40 itself
KAUR & KAUL: PROTEIN IMPORT INTO MITOCHONDRIA
interacts with preproteins in transit. Preproteins which
follow the presequence pathway have a cleavable
presequence and are guided across outer membrane by
a chain of binding sites, including the cytosolic
receptors Tom20, Tom22, Tom5 and Tom40 which
form translocation channel and intermembrane space
domain of Tom227. Other small proteins Tom6 and
Tom7 are required for assembly and stability of TOM
complex (Fig. 1), but they do not interact with the
precursor proteins21.
Tom70 recognizes the precursors of hydrophobic
carrier proteins of inner membrane such as the
ADP/ATP carrier. These precursors possess multiple
internal targeting sequences but lack presequence. The
cytosolic chaperones, particularly Hsp70 and Hsp90
classes bind these hydrophobic precursors and prevent
their aggregation in the cytosol. Heat-shock proteins
then specifically interact with Tom70 and deliver the
carrier precursors to this receptor. These heat-shock
proteins are essential in mitochondrial biogenesis, as
revealed in Caenorhabditis elegans which develops
early aging or progeria-like phenotypes, if HSP6 (a
nematode orthologue of mtHsp70) is reduced by RNA
interference39.
Several
Tom70
molecules
simultaneously bind to one precursor molecule which
is subsequently transferred to the import pore, Tom407.
The effective internal diameter of protein import
channel in mitochondrial outer membrane appears to be
between 20-26 Å during translocation40.
β-Barrel proteins represent a distinct class of
mitochondrial outer membrane proteins41, but little is
known about how these newly synthesized proteins
sort in eukaryotic cell, integrate into lipid bilayers and
assemble into oligomeric structures33. Like other outer
membrane proteins, β-barrel proteins such as porin and
Tom40 first translocate across TOM complex and in a
manner dependent on chaperones in intermembrane
space, pass on to TOB/SAM complex for their
insertion into outer membrane28,33. In the
intermembrane space, small TIM complexes, essential
Tim9-Tim10 complex and non-essential Tim8-Tim13
complex promote transfer of β-barrel precursors to
TOB/SAM complex. These small proteins have
chaperone-like functions which probably shield
hydrophobic segments of the precursor proteins against
aggregation in aqueous intermembrane space, keeping
β-barrel precursors in a competent state for their
insertion.
Biogenesis of Tom40, pore-forming channel of
outer mitochondrial membrane involves the
153
movement of precursors in unfolded state to SAMcore
complex to form an assembly intermediate I (~250
kDa) which further assembles into mature TOM
complex by passing through assembly intermediate II
(~100 kDa)42. How this assembly intermediate II
separates from SAMcore complex is under investigation.
Mdm10 also associates with SAM to form a larger
complex of ~350 kDa and plays a specific role in
assembly of TOM complex, probably by functioning as
a scaffold, where the late steps of assembly of the
multi-subunits TOM complex takes place2.
Transfer of preproteins from TOM to TIM 23:
Translocation of preproteins across the inner mitochondrial membrane
Majority of presequence carrying proteins are
imported into the matrix by cooperation of
presequence translocase of inner membrane, TIM23
complex and the associated motor (presequence
associated motor, PAM). Protein import into matrix
requires two forms of energy – an electrochemical
gradient (positive outside) across inner membrane
which exerts an electrophoretic force on the positively
charged N-terminal targeting sequence43 and
hydrolysis of ATP in matrix44. Ability of generating
strong translocating driving force solely depends upon
ATP-driven import motor and membrane potentialincreases the interaction of preprotein with motor41.
Precursor proteins are imported with help of TOM
complex and TIM23 complex into matrix (Fig. 1).
This ATP-driven import motor consists of five
proteins, three essential proteins–mtHsp70, peripheral
inner membrane protein Tim44 and a nucleotide
exchange factor of matrix termed mitochondrial GrpE
(Mge1)38 and two cochaperones Pam18/Tim14 and
Pam16/Tim16.
Molecular mechanism of the import motor involves
the following steps: (i) mtHsp70 with bound ATP
interacts with Tim44 and preproteins, but with low
affinity (ii) Pam18/Tim14 stimulates ATP hydrolysis
which in turn leads to the ADP-bound form of
mtHsp70, stabilizing its interaction with Tim44 and
preproteins. (iii) Mge1, a soluble matrix protein then
promotes the release of ADP-from mtHsp70. PAM,
thus operates as a multi-step motor, but the exact
order of events during reaction cycle of mtHsp 70 is
not fully understood7. TOM and TIM23 complexes do
not appear to form stable super complex in the
absence of precursor proteins35.
TIM23 channel is also tightly regulated, so as to
maintain the permeability barrier of inner
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INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, JUNE 2008
mitochondrial membrane. In absence of precursor
proteins, it remains in its inactive state, but regains its
active state, when precursor proteins are present and
opens up for translocation of polypeptide chains. Both
Tim50 and presequence act in an antagonistic manner
in this process. Whereas the hydrophilic IMS domain
of Tim50 promotes oligomerization and voltagedependent closure of channel, presequences selectively
override Tim50-induced closure and activate the
channel, thereby crossing the channel45. This process is
very complicated under in vivo conditions and there is
no clear-cut answer to the question as how energy
sources of mitochondria, membrane potential and
matrix ATP cooperate to drive preprotein import.
A new model for the mechanism of import motor
called “entropic pulling” has been proposed that
postulates generation of an active import-driving force
based on thermodynamic principles and molecular
geometries during translocation. According to this
model, translocation force is generated by the
excluded-volume constraint between mtHsp70 and
membrane and requires efficient interaction of
mtHsp70 and Tim44 for efficient translocation and
unfolding of proteins46.
Protein insertion machinery of the inner membrane
(TIM22 complex)
Substrate proteins for TIM22-mediated translocation
contain multiple hydrophobic segments that can be
inserted into inner membrane. Import of ADP-ATP
carrier (AAC) protein of inner mitochondrial
membrane has been characterized47. This 300 kDa
complex consists of a pore-forming subunit Tim22
along with Tim18 and Tim54 that play unknown
function and small Tim proteins Tim9, Tim10 and
Tim1248.
The general import of inner membrane proteins is
summarized here (Fig. 1). After recognition by Tom70
and translocation by TOM pore across outer membrane
as a loop, these precursor proteins are bound by Tim9Tim10 (70 kDa) complex49. Non-essential Tim proteins
(Tim8-Tim13 complex) also support the transfer of
selected inner membrane proteins, whereas Tim9–
Tim10 complex docks precursor proteins to the TIM22
complex. These Tim proteins contain the “twin CX3C”
motif that is required for formation of 70 kDa
complex50. Function of this motif is not fully known,
but it seems to be important for Zn2+ coordination in
these small Tim proteins. It has also been suggested
that cysteine present in this motif helps to form
juxtapositional intramolecular disulfide bonds51. These
proteins can bind zinc but insertion of intramolecular
disulfide bonds is requisite for assembly of complex52.
An intermembrane space protein Hot13 is also required
for efficient assembly of these Tim proteins into TIM
complexes. Thus, small TIM complexes have a
specialized assembly and the local redox state of these
complexes mediate translocation of inner membrane
proteins51.
Precursor is then translocated to the core of insertion
machinery, channel forming protein Tim2253 which
also shares homology with Tim23. Indeed, like
presequence translocase, Tim22 channel is also
activated by membrane potential, but no ATP-driven
machinery is required in this process. Membrane
potential is sufficient for both initial insertion of
precursor polypeptide into translocase and for
completion of transport i.e. the lateral release of protein
into inner membrane7.
Tanslocation of proteins to intermembrane space
(IMS)
Precursors destined for intermembrane space as
cytochromes b2 and c1 contain a bipartite targeting
sequence and N-terminal directs precursors to TIM23
complex. After cleavage by matrix processing
peptidase (MPP), precursor arrests at the “stop
transfer” domain in translocon54. The inner membrane
protease (IMP) consisting of complex Imp1/Imp2 and
facing intermembrane space mediates a second
cleavage, thereby arresting cytochromeb b2 in
intermembrane space55. This translocation also requires
hydrolysis of matrix ATP and electrochemical
membrane potential ∆ψ. For proteins lacking Nterminal and not requiring electrochemical membrane
potential for their translocation thereby bypassing
TIM23, their import mechanisms depends upon –
energy gain of protein around the cofactor and
association of a protein with high affinity binding site
such as another protein56.
Polynucleotide phosphorylase (PNPase), which is an
exoribonuclease as well as poly(A) polymerase that
regulates RNA levels is also localized in the
intermembrane space and not in cytosol or matrix as
considered earlier57 . Import of PNPase depends on ∆ψ
and MPP cleavage of presequence. Translocation of
PNPase into intermembrane requires i-AAA
proteaseYme1 which has a chaperone-like activity56.
This new Yme1 in protein translocation gives an idea
of the diverse protein translocation pathways of
intermembrane space which needs investigation.
KAUR & KAUL: PROTEIN IMPORT INTO MITOCHONDRIA
Conclusions and Perspective
During recent years, important insights have been
revealed into the machineries involved in targeting and
sorting of proteins to mitochondria. However, inspite
of rapid progress in our understanding of the
mechanisms of protein import into mitochondria, many
questions still remain to be answered. For example,
general import pathways have been revealed on the
basis of detailed analysis of import of various proteins,
which depends upon one or two of translocators TOM,
TIM23 and TIM22 complexes during transloction. But
there may be more specific pathways for several
mitochondrial proteins, including those synthesized
inside mitochondria that remain to be elucidated. More
than 30 proteins have been identified as translocator
components, indicating that pathways of import and
sorting are much more complex than previously
envisaged. Identification and characterization of
SAM/TOB pathway led to several surprising twists in
the analysis of organelle biogenesis. Important areas
for future studies will be: the characterization of
internal targeting and sorting signals of precursor
proteins, structural analysis of membrane integrated
transport complexes and import machinery mechanism.
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