Protein targeting or protein sorting is the mechanism by which a cell transports to the appropriate positions in the cell or outside of it. Both in prokaryotes and eukaryotes, newly synthesized proteins must be delivered to a specific sub-cellular location or exported from the cell for correct activity. This phenomenon is called protein targeting. Protein targeting is necessary for proteins that are destined to work outside the cytoplasm.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. In 1970, Günter Blobel conducted experiments on the translocation of proteins across membranes. He was awarded the 1999 Nobel Prize for his findings. He discovered that many proteins have a signal sequence, that is, a short amino acid sequence at one end that functions like a postal code for the target organelle.

1. PROTEIN
TARGETING
B Y H A I D E R A L I M A L I K

2. INTRODUCTION
Protein targeting
• Protein targeting or protein sorting is the mechanism by
which a cell transports to the appropriate positions in the cell
or outside of it.
• Both in prokaryotes and eukaryotes, newly synthesized
proteins must be delivered to a specific sub-cellular location
or exported from the cell for correct activity. This
phenomenon is called protein targeting.

4. CENTRAL
DOGMA
DNA synthesis maintains the genetic information and
passes this to the next generation
RNA synthesis (transcription) is a transfer of the
information from the DNA where it is stored into RNA
which can be transported and interpreted.
Ribosomes translate the nucleotides on the mRNA
into amino acid sequences producing a polypeptide.

5. PROTEIN TARGETING
• Protein targeting is necessary for proteins that are
destined to work outside the cytoplasm.
• 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.

6. • In 1970, Günter Blobel conducted experiments on
the translocation of proteins across membranes.
• He was awarded the 1999 Nobel Prize for his
findings. He discovered that many proteins have a
signal sequence, that is, a short amino acid
sequence at one end that functions like a postal
code for the target organelle.

7. TARGETING PATHWAYS

8. 1- POSTTRANSLATIONAL TRANSLOCATION
• Posttranslational translocation is the pathway which occurs
after the process of translation.
• Even though most proteins are co-translationally translocated,
some are translated in the cytosol and later transported to
their destination. This occurs for proteins that go to a
mitochondrion, a chloroplast, or a peroxisome.

9. 2- CO TRANSLATIONAL TRANSLOCATION
• In this pathway, transport of protein occurs during
translation which is not completed fully.
• Synthesized protein is transferred to an SRP receptor
on the endoplasmic reticulum (ER), a membrane
enclosed organelle. There, the nascent protein is
inserted into the translocation complex

11. 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.
• In the absence of targeting signals, a protein will remain
in the cytoplasm.

12. TYPES OF TARGETING PEPTIDES
• 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.
1. The pre-sequence
2. The internal targeting peptides

13. 1-PRESEQUENCES
• The pre-sequences of the targeting peptides are often
found at the N-terminal extension but in case of
peroxisomes the targeting sequence is on the C-terminal
extension mostly.
• Signal sequence is a short peptide (usually 16-30 amino
acids long) present at the N-terminus of the majority of
newly synthesized proteins that are destined towards
the secretory pathway.
• It is composed of between 6-136 basic and hydrophobic
amino acids.
• Signal sequences are removed from the finished protein by
specialized signal peptidases once the sorting process has
been completed.

14. 2-INTERNAL TARGETING PEPTIDES
• The targeting
peptides are often
found at the with
in polypeptide
chain, not at any
end .

15. Compartmental translocation of
proteins
There are three types of transport of proteins
through different compartments of cell.
a) Gated transport(Nucleus)
b)Transmembranetransport(Mitochondria,
Peroxisomes, chloroplast)
c) Vesicular transport (E.R)

16. GATED TRANSPORT
• The protein transfer from or to nucleus is aided by
nucleur pore.
• The nuclear pore complexes function as selective
gates that actively transport (with expenditure of
energy) specific macromolecules and
macromolecular assemblies.

17. PROTEIN TARGETING B/W CYTOSOL AND
NUCLEUS• The nuclear envelope encloses the DNA and defines the nuclear
compartment. This envelope consists of two concentric membranes that
are penetrated by nuclear pore complexes.
• The inner nuclear membrane contains specific proteins that act as
binding sites for chromatin and for the protein meshwork of the nuclear
lamina that provides structural support for this membrane.
• The inner membrane is surrounded by the outer nuclear membrane, which
is continuous with the membrane of the ER. Like the membrane of the ER
the outer nuclear membrane is studded with ribosomes engaged in
protein synthesis .
• The proteins made on these ribosomes are transported into the space
between the inner and outer nuclear membranes (the perinuclear
space), which is continuous with the ER lumen. with ribosomes engaged in
protein synthesis.
• Many proteins ,histones, DNA and RNA polymerases, gene regulatory
imported into the nuclear compartment from the cytosol. Proteins, and
RNA-processing proteins are selectively tRNAs and mRNAs are
synthesized in the nuclear compartment and then exported to the cytosol.

18. IMPORT AND EXPORT OF PROTEINS
TO NUCLEUS
• The transport is bidirectional and occurs through the nuclear pore
complexes (NPCs). These are complex structures composed of aggregates
of about 30 different proteins.
• The nuclear envelope has hundreds of NPCs, located where the two
nuclear membranes meet.
• Each NPC has multiple copies of at least 30 different proteins called
nucleoporins.
• Most polypeptides destined for the nucleus have address labels, called
nuclear localization signals (NLSs), consisting of one or more short
internal sequences with basic amino acids.
• Importins and Ran (a monomeric G‐protein that can exist in either the
GTP‐bound or GDP‐bound conformation) help in import of proteins
containing NLS.
• Proteins similar to importins, referred to as exportins, are involved in the
export of many macromolecules (various proteins, tRNA molecules,
ribosomal subunits and certain mRNA molecules) from the nucleus. Cargo
molecules for export carry nuclear export signals (NESs).
• The family of importins and exportins are referred to as karyopherins.

19. TRANSMEMBRANE TRANSPORT
• Membrane-bound protein translocators
directly transport specific proteins across a
membrane from the cytosol into a space that is
topologically distinct.
• The transported protein molecule usually must
unfold to snake through the translocator.
• The initial transport of selected proteins from
the cytosol into the ER lumen or from the
cytosol into mitochondria.

20. PROTEIN TARGETING TO
MITOCHONDRIA
• Protein translocation across mitochondrial
membranes is mediated by multi-subunit protein
complexes that function as protein
translocators(TOM ,TIM 23,TIM22 ,OXA)
• TOM transports-mitochondrial precursor proteins,
nucleus encoded mitochondrial proteins.
• TIM23-proteins into the matrix space.
• TIM22-mediates the insertion of a subclass of inner
membrane proteins, including the carrier protein that
transports ADP, ATP, and phosphate.
• OXA-mediates the insertion of inner membrane
proteins .

21. PROTEIN TRANSPORT INTO THE
MITOCHONDRIA
• Two distinct translocation complexes are situated in the
outer and inner mitochondrial membranes, referred to as
TOM (translocase-of-the-outer membrane) and TIM
(translocase-of-the-inner membrane).
• Each complex is composed of a number of proteins, some
of which act as receptors (eg, Tom20/22 ) for the incoming
proteins and others as components (eg, Tom40 ) of the
transmembrane pores through which these proteins must
pass.
• Proteins must be in the unfolded state to pass through the
complexes, and this is made possible by ATP dependent
binding to several chaperone proteins including Hsp70.
• A proton-motive force across the inner membrane is
required for import; it is made up of the electric potential
across the membrane (inside negative) and the pH . The
positively charged leader sequence may be helped through
the membrane by the negative charge in the matrix. The

22. • Mit. Hsp70 ensures proper import into the matrix and
prevents misfolding while interaction with the mtHsp60–
Hsp10 system ensures proper folding.
• It is possible that the electric potential associated with
inner mitochondrial membrane causes a conformational
change in the unfolded preprotein being translocated
that this helps to pull it across.
• Furthermore, the fact that the matrix is more negative
than the intermembrane space may “attract” the
charged amino terminal of the preprotein to enter the
matrix.
• A number of proteins contain two signaling sequences—

23. PROTEIN TARGETING TO CHLOROPLAST
• In chloroplast, the targeting signal is
correspondent to Transit peptide(TP)
• The preprotein for chloroplasts may contain a
stromal import sequence or a stromal and
thylakoid targeting sequence. The majority of
preproteins are translocated through the Toc and
Tic complexes located within the chloroplast
envelope.
• The signal sequence (transit peptide) binds with
target protein along with chaperone cystolic
Hsp70. This is the signal to move the
polypeptide through Toc complex.
• Stromal peptidase cleave the target sequence
and pull the rest of polypeptide inside.

24. TRANSLOCATION OF PROTEIN IN
CHLOROPLAST
• The vast majority of chloroplast proteins are synthesized as
precursor proteins (preproteins) in the cytosol and are imported
post-translationally into the organelle.
• Preproteins that contain a cleavable transit peptide are recognized
in a GTP-regulated manner by receptors of the outer-envelope
translocon, which is called theTOC complex.
• The preproteins cross the outer envelope through an aqueous pore
and are then transferred to the translocon in the inner envelope ,
which is called the TIC complex.
• The TOC and TIC translocons function together during the
translocation process.
• Completion of import requires energy, which probably comes from
the ATP-dependent functioning of molecular chaperones in the
stroma.
• The stromal processing peptidase then cleaves the transit
sequence to produce the mature form of the protein, which can fold
into its native form.

25. VESICULAR TRANSPORT
• Proteins from the ER to the Golgi apparatus and
proteins to E.R, for example, occurs in this way.

26. PROTEIN TARGETING IN
ENDOPLASMIC RETICULUM
• All eukaryotic cells have an endoplasmic reticulum (ER). Its membrane
typically constitutes more than half of the total membrane of an
average animal cell.
• The ER is organized into a netlike labyrinth of branching tubules and
flattened sacs extending throughout the cytosol , to interconnect.
• The ER has a central role in lipid and protein biosynthesis.
• Its membrane is the site of production of all the transmembrane
proteins and lipids for most of the cell’s organelles( the ER itself, the
Golgi apparatus, lysosomes, endosomes, secretory vesicles, and the
plasma membrane).
• The ER membrane makes a major contribution to mitochondrial and
peroxisomal membranes by producing most of their lipids.
• Almost all of the proteins that will be secreted to cell exterior plus those
destined for the lumen of ER, Golgi apparatus, or lysosomes are initially

27. PROTEIN TARGETING IN
ENDOPLASMIC RETICULUM
• Most nascent proteins are transferred across
the ER membrane into the lumen by the co-
translational pathway, so called because the
process occurs during ongoing protein
synthesis.
• The process of elongation of the remaining
portion of the protein being synthesized
probably facilitates passage of the nascent
protein across the lipid bilayer.
• It is important that proteins be kept in an
unfolded state prior to entering the conducting
channel—otherwise, they may not be able to
gain access to the channel.

28. • The signal sequence emerges from the ribosome and binds to the
signal recognition particle (SRP).
• The SRP-ribosome-nascent protein complex travels to the ER
membrane, where it binds to the SRP receptor (SRP-R). The SRP
guides the complex to the SRP-R, which prevents premature
expulsion of the growing polypeptide into the cytosol.
• The SRP is released, translation resumes, the ribosome binds to the
translocon (Sec 61 complex), and the signal peptide inserts into the
channel in the translocon. SRP and both subunits of the SRP-R can
bind GTP which must be in the GTP form in both complexes to
enable them to interact.
• When interaction occurs, GTP is hydrolyzed, SRP dissociates from
SRP-R and is released, and the ribosome binds to the translocon,
allowing the signal peptide to enter it.
• The signal peptide induces opening of the channel in the translocon
by binding to certain hydrophobic residues in it, thus causing the
PROTEIN TARGETING IN
ENDOPLASMIC RETICULUM

29. PROTEIN TARGETING IN
ENDOPLASMIC RETICULUM
• The growing polypeptide is then fully translocated across the
membrane, driven by its ongoing synthesis. The translocon consists of
three membrane proteins (the Sec61 complex) that form a protein-
conducting channel in the ER membrane through which the newly
synthesized protein may pass.
• The channel opens only when a signal peptide is present, preserving
conductance across the ER membrane when it closes.
• Closure of the channel when proteins are not being translocated
prevents ions such as calcium and other molecules leaking through it,
and causing cell dysfunction.
• Cleavage of the signal peptide by signal peptidase occurs, and the fully
translocated polypeptide/protein is released into the lumen of the ER.
The signal peptide is presumably degraded by proteases.

31. PROTEIN TARGETING IN
ENDOPLASMIC RETICULUM
• Ribosomes are released from the ER membrane and dissociate into their two types
of subunits.
• Secretory proteins and soluble proteins destined for organelles distal to the ER
completely traverse the membrane bilayer and are discharged into the lumen of
the ER.
• Many secretory proteins are N-glycosylated. N-Glycan chains, if present, are added
by the enzyme oligosaccharide protein transferase as these proteins traverse the
inner part of the ER membrane — a process called co-translational glycosylation.
• Those ER resident proteins that escape from the ER are returned to the ER by
vesicular transport.
• (A) The KDEL receptor present in vesicular tubular clusters and the Golgi apparatus,
captures the soluble ER resident proteins and carries them in COPI-coated
transport vesicles back to the ER. Upon binding its ligands in this low-pH
environment, the KDEL receptor may change conformation, so as to facilitate its
recruitment into budding COPI-coated vesicles.
• (B) The retrieval of ER proteins begins in vesicular tubular clusters and continues
from all parts of the Golgi apparatus.

32. TARGETING OF SECRETARY PROTEINS

33. SUMMARY
• Both in prokaryotes and eukaryotes, newly synthesized
proteins must be delivered to a specific subcellular location
or exported from the cell for correct activity. This
phenomenon is called protein targeting.
• Secretory proteins have an N-terminal signal peptide which
targets the protein to be synthesized on the rough
endoplasmic reticulum (RER).
• During synthesis it is translocated through the RER
membrane into the lumen. Vesicles then bud off from the
RER and carry the protein to the Golgi complex, where it
becomes glycosylated.
• Other vesicles then carry it to the plasma membrane.
Fusion of these transport vesicles with the plasma
membrane then releases the protein to the cell exterior.

34. REFERENCES
• Biochemistry, Third Edition ( David Hames & Nigel Hooper,
)
• Molecular Biology, Third Edition ( Phil Turner, Alexander
McLennan,Andy Bates & Mike White)
• Palade G (1975) Intracellular aspects of the process of
protein synthesis.Science 189, 347–358.
• Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore,
D., Darnell, J., 2000, Molecular Cell Biology,
• 4th Ed., W.H. Freeman.
http://bcs.whfreeman.com/lodish5e/
• Lehninger principles of Biochemistry, Fourth edition , David
L. Nelson, Michael M. Co