PROTEIN SORTING AND TARGETING

BY

Rakesh HResearch Scholar

Department of Biotechnology

Sahyadri Science College,Shivamogga.

Kuvempu university.

Introduction:

Protein targeting

Protein targeting or protein sorting is the mechanism by which a cell

transports proteins to the appropriate positions in the cell or outside of it.

THE 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

TRANSCRIPTION

• INITIATION

•ELONGATION

•TERMINATION

TRANSLATION

● Initiation – the assembly of a

ribosome on an mRNA molecule.

● Elongation – repeated cycles

of amino acid addition.

● Termination – the release of

the new protein chain.

PROTEIN TARGETING

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.

• 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.

PROTEIN TRANSLOCATION

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.

TARGETING PATHWAYS

POSTTRANSLATIONAL TRANSLOCATION

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

CO TRANSLATIONAL TRANSLOCATIONSynthisised 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

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

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

The presequences of the targeting peptides areoften found at the N-terminal extension .

It is composed of between 6-136 basic andhydrophobic amino acids.

In case of peroxisomes the targeting sequence ison the C-terminal extension mostly.

signal sequences are removed from the finishedprotein by specialized signal peptidases once thesorting process has been completed

THE INTERNAL TARGETING PEPTIDES

the targeting peptides are often found at the with in

polypeptide chain, not at any end .

PROTEINS CAN MOVE BETWEEN

COMPARTMENTS IN DIFFERENT WAYS

Gated transport(Nucleus )

Transmembrane

transport(Mitochondria,

Peroxisomes,)

Vesicular transport (E.R)

GATED TRANSPORT

The protein trafficbetween the cytosol andnucleus occurs betweentopologically equivalentspaces, which are incontinuity through thenuclear porecomplexes.

The nuclear porecomplexes function asselective gates thatactively transportspecificmacromolecules andmacromolecularassemblies,

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.

VESICULAR TRANSPORT

Proteins from the ER to the Golgi

apparatus and proteins to E.R,

for example, occurs in this way.

transport intermediates— which

may be small, spherical transport

vesicles or larger, irregularly

shaped organelle fragments—

ferry proteins from one

compartment to another.

The transfer of soluble recognized

by a complementary receptor in

the appropriate membrane.

GATED TRANSPORT

THE TRANSPORT OF MOLECULES BETWEEN THE

NUCLEUS AND THE CYTOSOL

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 and outer nuclear membranes are continuous, they maintain

distinct protein compositions.

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.

Bidirectional traffic occurs continuously between the cytosol and the

nucleus.

The many proteins , histones, DNA and RNA polymerases, gene

regulatoryimported 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

IMPORT AND EXPORT OF PROTEINS

TO NUCLEUS Protein encodes a receptor

protein that is specialized for the

transport of a group of nuclear

proteins sharing structurally

similar nuclear localization

signals.

Nuclear import receptors do not

always bind to nuclear proteins

directly. Additional adaptor

proteins are sometimes used that

bridge between the import

receptors and the nuclear

localization signals on the

proteins to be transported.

Export -ribosomal subunits and

RNA molecules.

For import and export requires

energy

TRANSMEMBRANE TRANSPORT

MITOCHONDRIA AND CHLOROPLASTS

Mitochondria and chloroplasts are double-

membrane-enclosed organelles.

They specialize in the synthesis of ATP, using

energy derived from electron transport and

oxidative phosphorylation in mitochondria and

from photosynthesis in chloroplasts.

Both organelles contain their own DNA,

ribosomes , and other components required for

protein synthesis .

Their growth depends mainly on the import of

proteins from the cytosol.

THE TRANSPORT OF PROTEINS INTO

MITOCHONDRIA AND CHLOROPLASTS

•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 .

PROTEIN TRANSPORT INTO THE MITOCHONDRIA

Import of Mitochondrial Proteins

►Post-translational: Unfolded polypeptide chain

1. precursor proteins bind to receptor proteins of TOM

2. interacting proteins removed and unfolded polypetide is fed into

translocation channel

►Occurs contact sites joining IM and OM - TOM transports mito targeting signal across

OM and once it reaches IM targeting signal binds to TIM, opening channel complex thru

which protein enters matrix or inserts into IM

PROTEIN TRANSPORT INTO THE MITOCHONDRIA

Import of Mitochondrial Proteins

►Requires energy in form of ATP and H+ gradient and assitance of hsp70

-release of unfolded proteins from hsp70 requires ATP hydrolysis

-once thru TOM and bound to TIM, translocation thru TIM requires

electrochemical gradient

PROTEIN TRANSPORT INTO THE MITOCHONDRIA

Protein Transport into IM or IM Space Requires 2 Signal Sequences

1. Second signal =hydrophobic sequence; immediately after 1st signal sequence

2. Cleavage of N-terminal sequence unmasks 2nd signal used to translocate protein from

matrix into or across IM using OXA

3. OXA also used to transport proteins encoded in mito into IM

4. Alternative route bypasses matrix; hydrophobic signal sequence = “stop transfer”

CHLOROPLAST The preprotein for chloroplasts

may contain a stromal importsequence or a stromal andthylakoid targeting sequence.The majority of preproteins aretranslocated through the Tocand Tic complexes locatedwithin the chloroplast envelope.

In the stroma the stromalimport sequence is cleaved offand folding as well as intra-chloroplast sorting to thylakoidscontinues.

Proteins targeted to theenvelope of chloroplastsusually lack cleavable sortingsequence.

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.

Most proteins that are destined for the

thylakoid membrane,

Preproteins that contain a cleavable transit

peptide are recognized in a GTP-regulated

manner12 by receptorsof 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.

THE ENDOPLASMIC RETICULUM All eukaryotic cells have an endoplasmic reticulum (ER). Its membrane

typically constitutes more than half of the total membrane of an averageanimal cell.

The ER is organized into a netlike labyrinth of branching tubules andflattened 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 proteinsand 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 andperoxisomal membranes by producing most of their lipids.

almost all of the proteins that will be secreted to the cell exterior plus thosedestined for the lumen of the ER, Golgi apparatus, or lysosomes are initiallydelivered to the ER lumen.

TRANSLOCATION OF PROTIENS IN E.R

VESICULAR TRANSPORT

UTILIZATION OF DIFFERENT COATS IN

VESICULAR TRAFFIC

VESICULAR TRANSPORT FROM ER TO GC

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.

TARGETING OF SECRETARY PROTEINS

THE GOLGI APPARATUS

The Golgi apparatus is integral inmodifying, sorting, and packagingthese macromolecules for cellsecretion (exocytosis) or use withinthe cell.

Post office; it packages and labelsitems(a mannose-6-phosphate labelto proteins destined for lysosomes)which it then sends to different partsof the cell.

glycosylation refers to the enzymaticprocess thatattaches glycans to proteins, lipids, orother organic molecules.

Glycosylation is a form of co-translational and post-translationalmodification

Five classes of glycans are produced:

N-linked glycans attached to nitrogen of asparagine or arginine side-chains. N-linked glycosylation requires participation of a special lipidcalled dolichol phosphate.

O-linked glycans attached to the hydroxy oxygen of serine,threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains, orto oxygens on lipids such as ceramide

phospho-glycans linked through the phosphate of a phospho-serine;

C-linked glycans, a rare form of glycosylation where a sugar is addedto a carbon on a tryptophan side-chain

Glypiation, which is the addition of a GPI anchor that links proteins tolipids through glycan linkages.

PROTEIN TRAFFICKING OR SITE SPECIFIC TRANSPORT

SUMMARY

Both in prokaryotes and eukaryotes, newly synthesizedproteins must be delivered to a specific subcellularlocation or exported from the cell for correct activity. Thisphenomenon is called protein targeting. Secretoryproteins have an N-terminal signal peptide which targetsthe protein to be synthesized on the rough endoplasmicreticulum (RER). During synthesis it is translocatedthrough the RER membrane into the lumen. Vesiclesthen bud off from the RER and carry the protein to theGolgi complex, where it becomes glycosylated. Othervesicles then carry it to the plasma membrane. Fusionof these transport vesicles with the plasma membranethen releases the protein to the cell exterior.

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