Intracellular protein trafficking: it describes the way a protein reaching its destination to perform its

specific biological functions from its intracellular site of origin.

Broad factor: signal or coding sequence at the N-terminal end of the protein.

Signal hypothesis (by Blobel and Sabatini in 1971): They proposed that-

A. Proteins synthesized on membrane-bound polyribosomes contained an N-terminal

peptide extension (N-terminal signal peptide) which causes them to become attached to
the membranes of the ER (membrane bound polyribosomes ), and facilitates protein
transfer into the ER lumen.
B. On the other hand, polyribosomes synthesizing proteins lacking the signal peptide
would retain free movement in the cytosol (cytosolic polyribosomes ).

*** The one and only difference between the ER bound polyribosome and the free ribosome is that the
former synthesizes proteins carrying signal sequence.

Pathways for protein sorting: Two pathways-

A. Cytosolic branch and

B. RER branch

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The cytosolic branch: Proteins are synthesized in the free polyribosome.

Destination: mitochondria, nuclei, and peroxisomes by specific signals, or remain in the cytosol if they
lack a signal.

Preprotein: If there is need of trimming of the signal sequence once to get the biologically active
protein, the untrimmed protein will be called the preprotein.

Preproprotein: If there is need of trimming of the signal sequence twice to get the biologically active
protein, the untrimmed protein will be called preproprotein.

The RER branch: Proteins are synthesized in the membrane bound ribosome.

Destination: The ER, Golgi apparatus [GA], plasma membrane [PM]) as well as lysosomal enzymes, and
also those for export from the cell via exocytosis (secretion).

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Intermediary routes:

A. Proteins remain in the lumen of the endoplasmic reticulum (rough)

B. The secretory or exocytotic pathway: Proteins go to major transport route of intracellular
protein, the golgi apparatus.
a. Constitutive transport: Proteins destined for the GA, the PM, certain other sites, or for
secretion are carried in transport vesicles. [ continuous secretion]
b. Regulated secretion: Particularly prominent in the pancreas and certain other glands,
other proteins destined for secretion are carried in secretory vesicles.[secretion can be
controlled by switch on or off as per requirement.]

Role of the GA in protein’s pathways:

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 A. The processing of the oligosaccharide chains of membrane and other N-linked glycoproteins

destinations. The trans golgi network plays role.

Chaperons: Molecular chaperones are proteins which stabilize unfolded or partially folded
intermediates.

Function: allowing time to protein to fold properly, and prevent inappropriate interactions, thus
combating the formation of nonfunctional structures.

Mechanism of functioning:

Chaperonins: These are the second major class of chaperons.

Mechanism of functioning: they form a barrel-shaped cage to provide proper environment for the
protein folding.

Example: Hsp 60 in eukaryotes is equivalent to the GroEl in bacterium.

Characteristics of the chaperons:

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e
t
o
r
p
f
in
and also contains enzymes involved in O-glycosylation. All parts of the GA plays role in this
case.
B. The sorting of various proteins prior to their delivery to their appropriate intracellular

ld
Cytosolic protein sorting:

A. Contain an uptake signal, enabling them to taken up into the correct subcellular organelle.
B. If they are destined for the cytosol, they have no targeting signal.

Other name: posttranslational translocation.

Entry of protein into the mitrochondrial matrix:

1. The unfolded protein synthesized on cytosolic polyribosomes.

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 2. Containing a matrix-targeting sequence interacts with the cytosolic chaperone Hsp70.
3. Interacts with the mt outer membrane receptor translocon of the outer membrane (Tom) 20/22.
4. Transferred to the import channel Tom 40.
5. Transport across the inner mt membrane via a complex comprising Tim (translocon of the inner
membrane) 23 and Tim 17 proteins.
6. Inside of the inner mt membrane, it interacts with the matrix chaperone Hsp 70, which in turn
interacts with membrane protein Tim 44.
7. The hydrolysis of ATP by mt Hsp70 probably helps drive the translocation, as does the electronegative
interior of the matrix.
8. The targeting sequence is subsequently cleaved by the matrix protease.
9. The imported protein assumes its final shape, or may interact with an mt chaperonin prior to this.

Flow chart: steps of protein translocation in mitrochondrial matrix.

*** At the point of translocation the inner and outer mitrochondrial membrane are in close apposition
to each other.

Transport of macromolecule across the nucleus:

Molecules transported: histones, ribosomal proteins and ribosomal subunits, transcription factors, and
mRNA molecules.

Import of proteins: involving importins and Ran proteins

Characteristics of Ran:

A. Small monomeric nuclear GTPases.

B. Exist in either GTP-bound or GDP-bound states..

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 C. Regulated by guanine nucleotide exchange factors (GEFs), which are located in the nucleus, and
Ran GTPase-accelerating proteins (GAPs), which are predominantly cytoplasmic.
D. The GTP-bound state of Ran is favored in the nucleus and the GDP-bound state in the cytoplasm.
E. The GTP-bound state is active.

Mechanism of import:

1. A cargo molecule (C) in the cytoplasm interacts via its nuclear localization signal (NLS) to form a
complex with an importin (I). (This may be either importin α or both importin α and importin β.)
2. This complex interacts with Ran (R)·GDP and traverses the nuclear pore complex (NPC) into the
nucleoplasm.
3. In the nucleoplasm, Ran·GDP is converted to Ran·GTP by guanine nuclear exchange factor (GEF)
4. Causing a conformational change in Ran which releases the cargo molecule
5. The I-Ran·GTP complex then leaves the nucleoplasm via the NPC to return to the cytoplasm

Export of proteins: involving proteins exportins and Ran.

Mechanism: similar to that of importing containing Nuclear Export Signal (NES).

Export of the mRNA: involving protein mRNA exporter (**Ran is not used).

Features:

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 A. mRNA exporter is a heterodimeric molecule (ie, composed of two different subunits, TAP [also
called Nfx1] and Nxt-1)
B. This system appears to use the hydrolysis of ATP by an RNA helicase (Dbp5) to drive
translocation.

Mechanism: mRNA exporter carries RNP (ribonucleoprotein) molecules through the NPC (Nuclear
Protein Complexes).

*** RNP is the exporting form of the mRNA.

Protein importing into the peroxisome:

Proteins involved: Two peroxisomal–matrix targeting sequences (PTSs) have been discovered. One,
PTS1, is a tripeptide (ie, Ser-Lys-Leu [SKL], but variations of this sequence have been detected) located at
the carboxyl terminal of a number of matrix proteins, including catalase. Another, PTS2, is a nine amino
acid sequence the N-terminus and has been found in at least four matrix proteins (eg, thiolase).

*** Import of matrix proteins requires ATP, whereas import of membrane proteins does not.

Mechanism:

1. Synthesized on cytosolic polyribosomes, assumes its folded shape prior to import, and contains a C-
terminal peroxisomal-targeting sequence (PTS).
2. It interacts with cytosolic receptor protein Pex5.
3. The complex then interacts with a receptor on the peroxisomal membrane, Pex14.
4. In turn, the protein-Pex 14 complex passes to the Pex 2/10/12 complex on the peroxisomal
membrane and is translocated.
5. Pex 5 is returned to the cytosol. The protein retains its PTS in the matrix.

Zellweger Syndrome: it is apparent at birth and is characterized by profound neurological impairment.

Characteristics:

A. The number of peroxisomes can vary from being almost normal to being virtually absent
in some patients.

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 B. Accumulation of very-long-chain fatty acids.
C. Abnormalities of the synthesis of bile acids.
D. A marked reduction of plasmalogens.

Cause: Caused by mutations in genes encoding certain proteins—the PEX family of genes, also called
peroxins —involved in various steps of peroxisome biogenesis (such as the import of proteins described
above), or in genes encoding certain peroxisomal enzymes themselves.

Translocation of proteins into the Endoplasmic reticulum: requires N-terminal signal peptide.

Type:

A. Co-transitional
B. Post-transitional

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Co-transitional pathway: entering of the protein into the endoplasmic reticulum during synthesis.

Steps-1: As the signal sequence emerges from the ribosome, it is recognized and bound by the signal
recognition particle (SRP).
Steps-2: The SRP escorts the complex to the ER membrane where it binds to the SRP receptor (SR).
Steps-3: The SRP is released, the ribosome binds to the translocon, and the signal sequence is inserted
into the membrane channel.
Steps-4: The signal sequence opens the translocon. Translation resumes and the growing polypeptide
chain is trans-located across the membrane.
Steps-5: Cleavage of the signal sequence by signal peptidase releases the polypeptide into the lumen of
the ER.

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Proteins attaching to the membrane of the endoplasmic reticulum: They follow the steps 1-4 of the
above mentioned process. If later it become necessary, they can transfer into the lumen of the
endoplasmic reticulum by following the last step.

Post-transitional translocation of the protein into the endoplasmic reticulum:

Protein involved: Binding immunoglobulin Protein (BiP) [In addition to its function in protein sorting to
the ER lumen, BiP promotes proper folding by preventing aggregation and will temporarily bind
abnormally folded immunoglobulin heavy chains and many other proteins, preventing them from
leaving the ER.]

Mechanism:

Step1: Proteins synthesized in the cytosol are prevented from folding by chaperone proteins such as
members of the Hsp70 family.
Step2: The N-terminal signal sequence inserts into the Sec61 translocon complex and the cytosolic
chaperones are released.
Step3: BiP interacts with the protein and the Sec62/63 complex and its bound ATP is hydrolyzed to ADP.
Step4: The protein is prevented from moving back into the cytosol by the bound BiP and successive
binding of BiP and ATP hydrolysis pulls the protein into the lumen.
Step5: 3. When the whole protein is inside, ADP is exchanged for ATP and BiP is released.

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Routes followed by the proteins to be inserted into the membrane of the endoplasmic reticulum:

A. Co-transitional insertion
B. Post-transitional insertion
C. Retention in the GA followed by retrieval to the ER
D. Retrograde transport from the GA

*** Orientation of proteins into the membrane

Type Orientation of insertion Example

l Cross the membrane once and have their amino termini in The LDL receptor and influenza
the ER lumen/cell exterior hemagglutinin
ll Cross the membrane once, but have their C-termini in the The asialoglycoprotein and
ER lumen/cell exterior transferrin receptors
lll A disposition similar to type I proteins, but do not contain a Cytochrome P450, an ER
cleavable signal peptide membrane protein
lV Cross the membrane a number of times (7 times for the G-protein-coupled receptors and
former and 12 times for the latter); they are also called glucose transporters
polytopic membrane proteins.

Co-transitional insertion of protein into the endoplasmic membrane:

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LDL receptor protein orientation in the membrane:

1. LDL receptor protein contains a highly hydrophobic segment which acts as a halt- or stop-
transfer signal and causes its retention in the membrane.
2. This sequence has its N-terminal end in the ER lumen and the C-terminal in the cytosol
3. The stop-transfer signal forms the single transmembrane segment of the protein and is its membrane-
anchoring domain.
4. The small patch of ER membrane in which the newly synthesized LDL receptor is located
5. Subsequently buds off as a component of a transport vesicle
6. Which eventually fuses with the plasma membrane so that the C-terminal faces the cytosol and the N-
terminal now faces the outside of the cell.

Orientation of asialoglycoprotein receptor:

1. The asialoglycoprotein receptor lacks a cleavable N-terminal signal peptide, but possesses an internal
insertion sequence, which inserts into the membrane but is not cleaved.
2. This acts as an anchor, and its C-terminus is extruded through the membrane into the ER lumen

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Post-transitional insertion of proteins into the endoplasmic reticulum: These proteins are sorted through
the lateral gate similar to that of the co-transitional insertion. Example- Cytochrome B 5.

Other routes for the protein to be inserted into the membrane of the endoplasmic reticulum:

KDEL containing protein insertion:

1. KDEL-containing proteins first travel to the GA in vesicles coated with coat protein II (COPII) known as
anterograde vesicular transport.
2. In the GA they interact with a specific KDEL receptor protein, which retains them transiently
3. They then return to the ER in vesicles coated with COPI (retrograde vesicular transport).
4. Where they dissociate from the receptor, and are thus retrieved.

Protein not containing KDEL insertion: Pass to the Golgi and then return, by retrograde vesicular
transport, to the ER to be inserted therein.

Endoplasmic reticulum the quality control center of the cell: It retains the unfolded proteins embedded
in its membrane by the help of the chaperons and enzymes before assuming final destination.

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The saga of misfolded protein in the endoplasmic reticulum:

ER stress: Accumulation of misfolded in the ER is called the ER stress.

Unfolded protein response (UPR): Sensing of the level of misfolded protein and initiation of intracellular
signaling mechanism to compensate for the stress condition and restore the endoplasmic reticulum
homeostasis.

Initiator of the UPR response: ER stress sensor is the trans-membrane protein embedded in the
membrane of the ER initiates the UPR responses.

Principal effects cause by the ER stress sensor:

(1) Transient inhibition of translation to reduce the amount of newly synthesized proteins,
(2) Induction of a transcription leading to increased expression of ER chaperones and
(3) Increased synthesis of proteins involved in degradation of misfolded ER proteins.

Ultimate result of the UPR response: The UPR increases the ER folding capacity and prevents a buildup
of unproductive and potentially toxic protein products, in addition to other responses to restore cellular
homeostasis.

Destruction: If impairment of folding persists, cell death pathways (apoptosis) are activated.

Degradation of the misfolded protein:

Energy source for the degradation: The energy for translocation appears to be at least partly supplied by
p97, an AAA-ATPase (one of a family of ATPases Associated with various cellular Activities).

Simplified pathway of protein degradation:

1. A target protein which is misfolded undergoes retrograde transport through the ER membrane
into the cytosol.
2. Where it is subjected to polyubiquitination
3. Following polyubiquitination, it enters a proteasome, inside which it is degraded to small

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 peptides that exit and may have several fates
4. Liberated ubiquitin molecules are recycled.

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Proteins related to Endoplasmic Reticulum Associated Destruction (ERAD): Sec61, Derlin 1 and the ERAD
E3 ligases, Hrd1 and Doa10, are potential ERAD channel candidates.

Cellular pathways for protein degradation:

A. Major pathway (involves ubiquitin and is ATP-dependent)

B. Minor pathway (involves lysosomal proteases and does not require ATP)

The gospel of ubiquitin: Ubiquitin is a small (76 amino acids), highly conserved protein that plays a key
role in marking various proteins for subsequent degradation in proteasomes.

Functions of ubiquitin:

A. Cell-cycle regulation (degradation of cyclins)

B. DNA repair
C. Inflammation and the immune response
D. Muscle wasting, viral infections, and many others.

Role of enzymes and Mechanism of ubiquitination:

Enzyme Function Numbers Mechanism

s
E1 Activation Only one Step1: The C-terminal COO− group of ubiquitin
(Ub) is first linked in a thioester bond to an SH
group of the activating enzyme (E1).
E2 Conjugation Several in number( having Step2: The activated ubiquitin is transferred to an
family) SH group of the conjugating enzyme.
E3 Ligation Several hundred in Step3: The transfer of ubiquitin from E2 to an ε-
number( having sub- amino group on a lysine of the target protein is
family) then catalysed by a ligase enzyme.

***It has been estimated that a minimum of four ubiquitin molecules must be attached to commit a
target molecule to degradation in a proteasome.

***Ubiquitin can be cleaved from a target protein by deubiquitinating enzymes and the liberated
ubiquitin can be reused.

***Additional rounds of ubiquitination then build up the polyubiquitin chain. (LYS Pr, target protein.)

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A glimpse to THE PROTEOSOME & degradation of the ubiquitinated proteins:

Proteosome: Proteosomes are protein complex with a relatively large cylindrical structure and are
composed of four rings with a hollow core containing the protease active sites, and one or two caps or
regulatory particles that recognize the polyubiquinated substrates and initiate degradation.

Functions of the proteosome:

A. Degradation of ubiquitinated protein both misfolded and short lived and

B. The proteasome plays an important role in presenting small peptides produced by degradation
of various viruses and other molecules to MHC class I molecules, a key step in antigen
presentation to T lymphocytes.

Mechanism of degradation:

1. The regulatory particle recognizes the ubiquitinated protein which are unfolded by ATPases present
in the regulatory particles or caps.
2. Protease active sites in the core of the proteosome attack peptide bonds and degrade the protein.
3. Peptides are released into the cytosol for further degradation by cytosolic peptidases.

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Transport vesicle in intracellular protein trafficking:

***Clathrin is used in vesicles destined for exocytosis , in some of those carrying cargo to lysosomes.

***COPI and COPII, the vesicles involved in retrograde transport (from the GA to the ER) and
anterograde transport (from the ER to the GA), respectively, however, are clathrin-free. Transport and
secretory vesicles carrying cargo from the GA to the PM are also clathrin-free.

Key steps of vesicle’s life cycle:

A. Budding
B. Tethering
C. Docking
D. Fusion

Mechanism of vesicle transport:

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 Step 1:Sar1 is activated when GDP exchanged for GTP and it becomes embedded in the ER membrane
to form a focal point for bud formation.
Step 2: Coat proteins bind to Sar1·GTP and cargo proteins become enclosed inside the vesicles.
Step 3: The bud pinches off, formatting a complete coated vesicle. Vesicles move through cells along
microtubules or actin filaments.
Step 4: The vesicle is uncoated when bound GTP is hydrolyzed to GDP by Sar1.
Step 5: Rab molecules are attached to vesicles after switching of Rab.GDP to Rab.GTP, a specific GEF
(see Table 49–9). Rab effector proteins on target membranes bind to Rab·GTP, tethering the vesicles
to the target membrane.
Step 6: v-SNAREs pair with cognate t-SNAREs in the target membrane to form a four helix bundle
which docks the vesicles and initiates fusion.
Step 7: When the v- and t-SNARES are closely aligned, the vesicle fuses with the membrane and the
contents are released. GTP is then hydrolyzed to GDP, and the Rab·GDP molecules are released into
the cytosol. An ATPase (NSF) and α-SNAP (see Table 49–9) dissociate the four-helix bundle between
the v- and t-SNARES so that they can be reused.
Step 8: Rab and SNARE proteins are recycled for further rounds of vesicle fusion.

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 ***In synaptic vesicles one v-SNARE is designated synaptobrevin. Botulinum B toxin is one of the
most lethal toxins known and the most serious cause of food poisoning. One component of this toxin is a
protease that binds synaptobrevin, thus inhibiting release of acetylcholine at the neuromuscular
junction and possibly proving fatal.

***The fungal metabolite brefeldin A prevents GTP from binding to ARF, and thus inhibits formation of
COPI vesicles.

General points about membrane assembly:

1. Asymmetry of proteins and lipids are maintained during membrane assembly:

a. The inside of the vesicles after fusion becomes the outside of the plasma membrane, and
the cytoplasmic side of the vesicles remains the cytoplasmic side of the membrane.
b. The enzymes responsible for the synthesis of phospholipids reside in the cytoplasmic
surface of the cisternae (the sac-like structures) of the ER.
c. They probably self-assemble into thermodynamically stable bimolecular layers, thereby
expanding the membrane and perhaps promoting the detachment of so-called lipid vesicles
from it.
d. It has been proposed that these vesicles travel to other sites, donating their lipids to other
membranes.
e. Cytosolic proteins that take up phospholipids from one membrane and release them to
another (ie, phospholipid exchange proteins ).

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 2. Lipids and proteins turnovers at different rates at different membrane: Turnover rates of lipids
and proteins are independent.

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