Intracellular trafficking and protein sorting

Intracellular Trafficking
and Sorting of Proteins
Apeksha Niraula
Junior Resident
Department of Biochemistry
BPKIHS

Contents
 Compartmentalization of cells
 Intracellular Trafficking
 Signal Sequences
 SRP. SRP Receptor, GTP, Sec 61 and Glycosylation
 Chaperones and roles in protein folding
 Endoplasmic Reticulum Associated and other Protein
Degradation
 Receptor Mediated Endocytosis
 Clinical Implications

Compartmentalization of Cells

All Eukaryotic Cells have Same basic sets of Membrane bound organelles.

Compartmentalization of Cells
Major Organelles
►Nucleus
►Cytosol
►ER
►Golgi Apparatus
►Mitochondria and Chloroplast
►Lysosomes
►Endosomes
►Peroxisomes

Protein Transport Mechanisms
1. Transport through pores (Nucleus).
2. Transport across membranes
(Chloroplast and Mitochondria[MT]).
3. Transport by vesicles (ER and Golgi).

Proteins Are Targeted by Signal Sequences to
Their Correct Destinations
 Many proteins carry signals (usually but not always specific
sequences of amino acids) that direct them to their
destination, thus ensuring that they will end up in the
appropriate membrane or cell compartment.
 These signals are a fundamental component of the sorting
system.

Two ways in which a sorting signal can be built into
a protein
a. Signal sequence
b. Signal patch

Signal Sequences/Patches
Direct Proteins to Final Destination
Signal patches direct proteins to:
1. Nucleus
2. Lysosomes
Signal Sequences direct proteins to:
1. ER proteins possess N-terminal signal of 5-10
Hydrophobic Amino acids
2. Mitochondrial proteins have alternating +positive
charged amino acids with hydrophobic Amino acids
3. Peroxisomal Proteins have 3 amino acids at C-terminus.

Polyribosomes 1. Cytosolic
2. Rough ER
Proteins
Mitochondrial
Nuclear
Peroxisomal
Cytosolic
ER membrane
GA Membrane
Plasma Membrane
Secretory
Lysosomal Enzymes

Protein Sorting Signal Sequences
 Signal sequences are a continuous stretch of amino acids
(15 to 60) within the protein to be sorted.
 Specific sequences direct the protein to the Nu, MT,
peroxisomes, or ER.
 Cytosolic proteins lack the signal sequence.

General Features of Protein Import to Organelles
Import of a protein into an organelle usually occurs in three stages :
Recognition, Translocation and Maturation.
Targeting sequences on the protein are recognised in the cytoplasm or
on the surface of the organelle.
Protein is generally unfolded for Translocation (maintained by
Chaperones).
Threading of the Protein through a membrane required energy and
organellar chaperones on the trans side of the membrane
Other proteins within the organelle catalyse folding of the protein,
often attaching cofactors or oligosachharides and assembling them
into active monomers or oligomers.

Transport of Proteins into Mitochondria

The Mitochondrion both imports and synthesizes
Proteins
 Contain many proteins
 13 Polypeptides (mostly membrane components of the
ETC) encoded by the Mitochondrial(mt) Genome.
 Majority (atleast several 100) are encoded by nuclear
genes, are synthesised outside the mitochondria on
cytosolic polyribosomes and must be imported.
 Matrix proteins must pass from cytoplasmic polyribosomes
through the outer and inner mitochondrial membranes to
reach their destination, called as Translocation.

 Proteins have an amino terminal leader sequence (Presequence),
20-50 amino acids in length.
 Contains many Hydrophobic and positively charged amino acids (
e.g: Lys or Arg)
 Translocation occurs Posttranslationally.
 Interactions with a number of cytosolic proteins that act as
Chaperones and as targeting factors occur prior to translocation.

 Tom – Translocase of Outer Membrane
 Tim- Translocase of Inner Membrane
 Tom 20/22, 40
 Tim 23/17,44.
 Proton-Motive force across the inner membrane is required to
import ; it is made up of the Electric Potential across the
membrane (inside negative) and the pH gradient.

Localization Signals Importins and Exportins are
involved in transport of macromolecules in and out
of the Nucleus
 Millions of macromolecules per minutes are transported
between the nucleus and the cytoplasm in an active eukaryotic
cell.
 Includes Histones, Ribosomal Proteins and ribosomal subunits,
transcription factors and mRNA molecules.
 Transport is Bidirectional; occurs through the Nuclear Pore
Complexes (NPCs).

Transport of Molecules between Nucleus and
Cytosol
Nuclear Envelope
►Two concentric membranes
- Outer membrane contiguous w/ER
- Inner membrane contains proteins
that
act as binding sites for chromatin and
nuclear lamina.
►Perforated by nuclear pores for selective
import
and export

Nuclear Pore Complex
►Mass of 125 million; ~50 different
proteins arranged in octagon.
►Typical mammalian cell 3,000-4,000
►Contains >1 aqueous channels
through which some molecules can
readily pass <5,000; molecular >
60,000 cannot pass.
►Functions ~Diaphragm
►Receptor proteins actively transport
molecules through Nuclear Pore.

Nuclear Localization Signal
 Generally comprised of two short sequences rich in Positively
charged amino acid Proline, Lysine, Arginine and Valine.
 Can be located anywhere.
 Thought to form loops or patches on protein surface.
 Resident, not cleaved.
 Transports proteins in folded state.
 Energy requiring process.

 Depending on NLS content, a cargo molecule interacts with
one of a family of soluble proteins called IMPORTINS.
Complex docks transiently at the NPC.
 Ran Plays a key critical regulatory role in the interaction
of the complex with the NPC and its translocation
through the NPC.
 Ran proteins Small monomeric nuclear GTPases
 Exist in either GTP bound or GDP bound
states.
 Regulated by Guanine Nucleotide Exchange
Factors(GEFs), located in Nucleus and Ran
GTPase accelerating proteins (GAPs)
[Cytosolic].

Exportins : Proteins similar to Importins.
 Involved in the export of many macromolecules( Proteins,
tRNA molecules, ribosomal subunits and certain mRNA
molecules) from the Nucleus.
 Cargo molecules for Export carry Nuclear Export Signals
(NESs).
 Family of Importins and Exportins are referred to as
Karyopherins.

The compartmentalization of Ran-GDP and Ran-GTP
Ran-GAP: cytosol->Ran-GDP
Ran-GEF: nucleus->Ran-GTP

Import of Proteins into Peroxisomes
 Carry Unique targeting sequences.
 Peroxisome Important organelle involved in aspects of the metabolism
of molecules ( FA, Cholesterol, Bile Acids, Plasmalogens,
Purines, Amino Acids).
 Bound by a single membrane and contains more than 50
Enzymes : Catalase and Urate Oxidase Marker
Enzymes for Peroxisome.
 Two Peroxisomal-matrix targeting sequences (PTSs) have been discovered.
I. PTS1 = Tripeptide; (Ser-Lys-Leu) [SKL] located at the Carboxy
terminal of the matrix proteins including Catalase.
Form complex with Pex5 (Cytosolic receptor).
II. PTS II = at the N-Terminus; has been found in atleast four matrix
proteins (e.g: Thiolase).
 These two sequences are not cleaved after entry into the matrix.

Zellweger Syndrome : Mutations in Genes involved
in the biogenesis of Peroxisomes
 Group of conditions that have overlapping signs and
symptoms including Neonatal adrenoleukodystrophy
(NALD), and infantile Refsum disease.
 Cause: Autosomal Recessive;mutations in 12 genes have
been found to cause the Zellweger spectrum.
These genes provide instructions for making a group
of proteins known as Peroxins, which are essential
for the formation and normal functioning of cell
structures called peroxisomes.

Signs and Symptoms:
Hypotonia
Feeding problems,
Hearing loss, loss of vision, and seizures.
 These problems are caused by the degeneration of myelin,
which is the covering that protects nerves and promotes the
efficient transmission of nerve impulses.

Signal Hypothesis explains how polyribosomes bind
to the Endoplasmic Reticulum
 Rough ER branch Second of the two branches involved in the
synthesis and sorting of proteins.
 Proteins are synthesised on membrane bound polyribosomes and are usually
translocated into the lumen of the rough ER prior to further sorting.
 Signal Hypothesis 1971, by Blobel and Sabatini
 Proposed that proteins synthesised on membrane bound polyribosomes
contained an N-terminal peptide extension (N-terminal Signal Peptide) which
mediate their attachment to the membranes of the ER and facilitated transfer
into the ER Lumen.
 While, proteins whose entire synthesis occurs on free polyribosomes would lack
this signal peptide.

 The evidence to support this hypothesis is : Mutant
proteins containing altered signal peptides in which
hydrophoboc amino acids are replaced by
hydrophilic ones, are not inserted into the lumen of
the ER.
 Non-Membrane proteins (e.g:-α Globin) to which
signal peptides have been attached by genetic
engineering can be inserted into the lumen of the
ER, or even secreted.

Revealing the ER Translocation Process….
Principal Components involved in ER translocation
N-terminal Signal peptide
Polyribosomes
SRP, Signal Recognition particle
SR, Signal Recognition Particle Receptor
Sec 61, The Translocon
Signal Peptidase
Associated Proteins (e.g:- TRAM and TRAP)

Steps in ER translocation
Step I : Emergence of Signal sequence from the ribosome and binds to SRP.
Step II : SRP- Ribosome Nascent Protein complex travels to the ER membrane
Binds to the SRP Receptor(SR)
Step III : SRP is released , translation resumes and the ribosomes binds to the
translocon (Sec 61 Complex) and the signal peptide inserts into the channel
in the translocon.
When translation is still occuring, the entry of the signal peptide into the
translocon and its further passage is termed Cotranslational Translocation.

Step IV : Signal peptide induces opening of the channel in the
translocon by binding to certain hydrophobic residues in
thus causing the plug to move.
Step V : Cleavage of the signal peptide by signal peptidase occurs
and the fully translocated polypeptide/protein is released
into the lumen of the ER.
 In Yeast, many proteins are targeted to the ER after their
translation is completed (Posttranslational Translocation) by
a process that doesnot require the SRP.
 Does involve Cytosolic Chaperones (Hsp70) to keep the
protein unfolded and also the luminal chaperone BiP, which
may “pull” the growing polypeptide into the ER Lumen.

SRP, SRP Receptor,GTP, Sec 61, and Glycosylation
 SRP Contains Six Proteins and has a 7s RNA
 Both RNA molecule and its proteins play various
roles (Such as binding other molecules) in its
functions.
SR ER Membrane Protein
 Composed of α and β subunits.
 SRP and both subunits of the SR can bind GTP; must be in GTP
form to interact.

When SRP and SR binds hydrolysis of GTP is stimulated.
SRP is released and ribosomes bind to the translocon allowing
the signal peptide to enter it.
 SRP and SR act as GTPase- accelerating protein (GAPs).
When GTP is hydrolysed to GDP they dissociate
SRP and SR can be regarded as Molecular Matchmakers.

Translocon Consists of Three membrane proteins (The Sec61
complex) that forms a protein conducting channel
in the ER membrane thorugh which the newly
synthesised proteins may pass.
 These channel appears OPEN only when a signal peptide is present
preserving conductance across the ER membrane when it closes.
 The insertion of the signal peptide into the conducting channel,
while the other end of the parent protein is still attached to
ribosomes is called COTRANSLATIONAL INSERTION.

Proteins follow several routes to be inserted into or
attached to the membranes of the Endoplasmic
Reticulum
I. Cotranslational Insertion

II. Synthesis on Free Polyribosomes and Posttranslational
attachment to the Endoplasmic Reticulum Membrane
 e.g:- Cytochrome b5 directly enters the ER membrane
subsequent to translation, assisted by several Chaperones.
III. Other routes include retention in the GA with retrieval to the
ER and also retrograde transport from the GA
 Proteins with KDEL aa sequence (Lys-Asp-Glu-Leu) at their
carboxy terminal.
 KDEL- containing proteins first travel to the GA in COPII
transport vesicles and interact with KDEL receptor protein,
retained transiently.
They then return in COPI transport vesicles to ER , dissociates
and retrieved.

 HDEL (H= Histidine) serve a similar purpose.
 The above mentioned Proteins result in net localization of soluble
proteins to the ER lumen.
 Certain Non-KDEL containing proteins also pass to the golgi and
then return, by Retrograde Vesicular Transport to the ER.

The ER functions as the Quality Control compartment of
the cell
After entering the ER, newly synthesized proteins attempt to
fold with the assistance of chaperones and folding enzymes,
and their folding status is monitored by chaperones and also
the enzymes.
Chaperones
 Proteins that prevent faulty folding and unproductive interactions of
other proteins.
 Also called Heat Shock Protein (Hsp)
 Found in Cytosol, Mitochondria, and Lumen of ER.
 Bind predominantly to hydrophobic regions of unfolded proteins
and prevent their aggregation.
 Shows ATPase activity.

 Misfolded or incompletely folded proteins interact with
chaperones, which retain them in the ER and prevent them
from being exported to their final destinations.
 If such interactions continue for a prolonged period of time,
the misfolded proteins are usually disposed of by
Endoplasmic reticulum associated degradation (ERAD).
 This avoids a harmful build-up of misfolded proteins.
 Cystic fibrosis- genetic disease; Retention of misfolded
proteins occurs in the ER, and in some cases, the retained
proteins still exhibit some functional activity.

 Ubiquitin is key molecule in protein degradation
 Tages the protein to be degraded; Polyubiquitination
 Polyubiquitinated proteins delivered to Proteasome where it is degraded.

Transport Vesicles are Key Players in Intracellular
Protein Traffic
 Anterograde Transport From ER to Golgi (COPII)
 Retrograde Transport From Golgi to ER ( COPI)
 COPI and COPII are Clathrin- free.
 Transport and secretory vesicles carrying cargo from the GA to PM are also
Clathrin free.
 Vesicles involved in Endocytosis are coated with Clathrin

Models of Transport Vesicles involve SNAREs and
other factors
Vesicles Heart of Intracellular Transport of many
proteins.

Vesicles Functions
COPI Involved in intra-GA transport
and retrograde transport from
the GA to the ER
COPII Involved in export from the ER to
either ERGIC or the GA
Clathrin Involved in transport in post-GA
locations including the PM, TGN
and endosomes
Secretory Vesicles Involved in regulated secretion
from organs such as the pancreas
(eg, secretion of insulin)

The Golgi Apparatus has two major functions:
1. Modifies the N-linked oligosaccharides and adds O-linked oligosaccharides.
2. Sorts proteins so that when they exit the trans Golgi network, they are
delivered to the correct destination.

Transport Vesicle Fusion is Mediated by Proteins

Energy requirements:
•GTP hydrolysis by dynamin
accompanies pinching off.

Multivesicular body
Late endosome
pH 6 induces dissociation

Proteins are targeted to different compartments in
different ways

Intracellular trafficking and protein sorting

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Intracellular trafficking and protein sorting