2. Protein sorting
Protein targeting or protein sorting is the biological mechanism by which
proteins are transported to the appropriate destinations in the cell or
outside of it. Proteins can be targeted to the inner space of an organelle,
different intracellular membranes, plasma membrane, or to exterior of
the cell via secretion. This delivery process is carried out based on
information contained in the protein itself. Correct sorting is crucial for
the cell; errors can lead to diseases.
Targeting signals
Targeting signals are the pieces of information that enable the cellular
transport machinery to correctly position a protein inside or outside the
cell. This information is contained in the polypeptide chain or in the
folded protein. The continuous stretch of amino acid residues in the
chain that enables targeting are called signal peptides or targeting
peptides. There are two types of targeting peptides, the presequences
and the internal targeting peptides. The presequences of the targeting
peptide are often found at the N-terminal extension and is composed of
between 6-136 basic and hydrophobic amino acids. In case of
peroxisomes the targeting sequence is on the C-terminal extension
mostly. Other signals, known as signal patches, are composed of parts
which are separate in the primary sequence. They become functional
3. when folding brings them together on the protein surface. In addition,
protein modifications like glycosylations can induce targeting.
Targeting signal for mitochondria
The mitochondrial targeting signal is a 10-70 amino acid long peptide
that directs a newly synthesized proteins to the mitochondria. It is found
at the N-terminus and consists of an alternating pattern of hydrophobic
and positively charged amino acids to form what is called an
amphipathic helix. Mitochondrial targeting signals can contain additional
signals that subsequently target the protein to different regions of the
mitochondria, such as the mitochondrial matrix.
Like signal peptides, mitochondrial targeting signals are cleaved once
targeting is complete.
4. Protein sorting in mitochondria
Mitochondria are multifunctional double-membrane-bound organelles
that arose from a bacterial endosymbiont during the evolution of
eukaryotic cells. Known as the powerhouses of the cell, mitochondria
harbor the oxidative phosphorylation machinery for ATP synthesis, but
also a large number of biosynthetic pathways. Moreover, they are
intimately involved in complex cellular processes, like calcium
homeostasis and programmed cell death. As a relic of their evolutionary
origin, mitochondria contain their own genetic material and machineries
to manufacture their own RNAs and proteins. However, the small
circular mitochondrial genome encodes only a few proteins (8 and 13
polypeptides in yeast and humans, respectively). All remaining
mitochondrial proteins (approximately 99%) are encoded by the nuclear
genome and synthesized on cytosolic ribosomes in their precursor
forms. To acquire their mature, functional state these precursor proteins
need to be efficiently targeted and imported into mitochondria and
sorted to the correct submitochondrial compartment: outer membrane,
intermembrane space (IMS), inner membrane, and matrix. The inner
mitochondrial membrane is further subdivided into the inner boundary
membrane, which is closely opposed to the outer membrane, and large
tubular invaginations, termed cristae membranes. Within the four
mitochondrial compartments, sophisticated translocation, sorting, and
assembly machineries serve to establish incoming precursors in a
5. functional state within the context of their new environment.
Most mitochondrial proteins are synthesized as cytosolic precursors
containing uptake peptide signals. Cytosolic chaperones deliver
preproteins to channel linked receptors in themitochondrial membrane.
The preprotein with presequence targeted for the mitochondria is bound
by receptors and the General Import Pore (GIP) (Receptors and GIP are
collectively known as Translocase of Outer Membrane or TOM) at the
outer membrane. The preprotein is translocated through TOM as hairpin
loops. The preprotein is transported through the intermembrane space
by small TIMs (which also acts as molecularchaperones) to the TIM23
or 22 (Translocase of Inner Membrane) at the inner membrane. Within
the matrix the targeting sequence is cleaved off by mtHsp70.Three
mitochondrial outer membrane receptors are known: TOM20, TOM22
and TOM70.
• TOM70: Binds to internal targeting peptides and acts as a docking
point for cytosolic chaperones.
• TOM20: Binds presequences
• TOM22: Binds both presequences and internal targeting peptides
• The TOM channel (TOM40) is a cation specific high conductance
channel with a molecular weight of 410 kDa and a pore diameter of
21Å.
6. The presequence translocase23 (TIM23) is localized to the mitochondial
inner membrane and acts a pore forming protein which binds precursor
proteins with its N-terminus. TIM23 acts a translocator for preproteins for
the mitochondrial matrix, the inner mitochondrial membrane as well as
for the intermembrane space. TIM50 is bound to TIM23 at the inner
mitochondrial side and found to bind presequences. TIM44 is bound on
the matrix side and found binding to mtHsp70.
The presequence translocase22 (TIM22) binds preproteins exclusively
bound for the inner mitochondrial membrane.
Mitochondrial matrix targeting sequences are rich in positively charged
amino acids and hydroxylated ones.
Proteins are targeted to submitochondrial compartments by multiple
signals and several pathways.
Targeting to the outer membrane, intermembrane space, and inner
membrane often requires another signal sequence in addition to the
matrix targeting sequence.
7. Fig: Overview of mitochondrial protein sorting pathways
Cytosolic chaperones deliver precursor proteins to the organelle in a
translocation-competent state. Some α-helical proteins are inserted into
the outer membrane with the help of Mim1. Virtually all other precursors
initially traverse the outer membrane via the TOM complex and are
subsequently routed to downstream sorting pathways. Biogenesis of
outer membrane β-barrel proteins requires the small TIM chaperones of
the IMS and the SAM complex. Cysteine-containing IMS proteins are
imported via the MIA pathway. Metabolite carriers of the inner
mitochondrial membrane are transferred by the small TIM chaperones to
8. the TIM22 complex, which mediates their membrane integration.
Presequence-containing precursors are directly taken over from the
TOM complex by the TIM23 machinery that either inserts these proteins
into the membrane or translocates them into the matrix in cooperation
with the import motor PAM. OM, outer membrane; IMS, intermembrane
space; IM, inner membrane, Δψ, membrane potential across the inner
mitochondrial membrane.
