Introduction

● The endoplasmic reticulum ER is a membrane-bound organelle found in eukaryotic cells. It is

involved in various essential cellular processes, including protein synthesis, lipid metabolism,
and calcium storage.
● The ER is a network of interconnected tubules, vesicles, and cisternae flattened sacs. It can be
divided into two regions:
○ Rough endoplasmic reticulum RER
○ Smooth endoplasmic reticulum SER
● The RER is studded with ribosomes on its outer surface, giving it a "rough" appearance. The
ribosomes on the RER synthesize proteins that are either inserted into the ER membrane or
secreted from the cell.
● The SER lacks ribosomes and has a more "smooth" appearance. It is involved in lipid
synthesis, metabolism, and detoxification reactions.

Protein Sorting Pathways in Eukaryotic Cells

 ● Protein sorting refers to the process by which newly synthesized proteins are directed to their
appropriate cellular locations within eukaryotic cells.
● Proteins destined for specific cellular locations contain targeting signals or sorting signals
within their amino acid sequences.
● Targeting signals can be located at the N terminus, C terminus, or within the protein itself.
● Signal sequences can be recognized by specific receptor proteins or translocation machinery.

Secretory Pathway

● The secretory pathway is a major protein-sorting pathway in eukaryotic cells.

● It involves the synthesis, processing, and trafficking of proteins from the endoplasmic
reticulum ER to the Golgi apparatus, and ultimately to the plasma membrane or other
secretory vesicles.
● The process begins with the binding of a signal sequence on the protein to a signal
recognition particle SRP, which targets the ribosome nascent chain complex to the ER
membrane.
● The protein is then translocated across the ER membrane through a protein translocation
channel called the translocon.
● Once inside the ER, the protein undergoes folding, modification, and quality control processes.
● Proteins destined for secretion or incorporation into the plasma membrane are packaged into
transport vesicles that bud off from the ER and fuse with the Golgi apparatus.

Endocytic Pathway

● The endocytic pathway involves the internalization of proteins from the plasma membrane
and their trafficking to various intracellular compartments.
● Endocytosis can occur through clathrin-mediated or caveolin-mediated processes.
● Internalized proteins are sorted within early endosomes, which mature into late endosomes
and eventually fuse with lysosomes for degradation or recycling.

Mitochondrial and Nuclear Targeting

● Proteins destined for mitochondria or the nucleus have specific targeting sequences.
● Mitochondrial proteins have mitochondrial targeting sequences that are recognized by
receptors on the mitochondrial outer membrane.
● Nuclear proteins contain nuclear localization signals that direct them to the nucleus through
nuclear pore complexes.

Vesicular Transport and Sorting

● Protein sorting can also occur through vesicular transport between organelles.
 ● Cargo proteins are packaged into transport vesicles that bud off from one organelle and fuse
with the target organelle.
● Vesicular transport involves specific coat proteins e.g., clathrin, COPI, and COPII that help
shape the vesicles and select cargo proteins.

Signal Hypothesis

The signal hypothesis explains how proteins are directed to different cellular compartments,
including the endoplasmic reticulum ER, Golgi apparatus, plasma membrane, mitochondria, and
nucleus. It highlights the importance of specific amino acid sequences and their recognition by cellular
machinery in achieving proper protein sorting and localization.

Signal recognition particles SRP

 ● Signal recognition particles SRPs are ribonucleoprotein complexes involved in the targeting of
proteins to the endoplasmic reticulum ER in eukaryotic cells and the plasma membrane in
prokaryotic cells.
● SRPs play a crucial role in the cotranslational protein targeting pathway.
● It is composed of six discreet polypeptides and a 300-nucleotide RNA.
● P54 binds to the ER signal sequences, while all other proteins bind to the RNA.

Synthesis of Secretory Proteins & Their Cotranslational Translocation Across ER Membrane

● Step 1, 2: Once the ER signal sequence emerges from the ribosome, it is bound by a signal
recognition particle SRP.
● Step 3: The SRP delivers the ribosome/nascent polypeptide complex to the SRP receptor in
the ER membrane. This interaction is strengthened by the binding of GTP to both the SRP and
its receptor.
● Step 4: Transfer of the ribosome/nascent polypeptide to the translocon leads to the opening of
this translocation channel and insertion of the signal sequence and adjacent segment of the
growing polypeptide into the central pore. Both the SRP and SRP receptors, once dissociated
from the translocon, hydrolyze their bound GTP and then are ready to initiate the insertion of
another polypeptide chain.
● Step 5: As the polypeptide chain elongates, it passes through the translocon channel into the
ER lumen, where the signal sequence is cleaved by signal peptidase and is rapidly degraded.
● Step 6: The peptide chain continues to elongate as the mRNA is translated toward the 3' end.
Because the ribosome is attached to the translocon, the growing chain is extruded through the
translocon into the ER lumen.
 ● Step 7, 8: Once the translation is complete, the ribosome is released, the remainder of the
protein is drawn into the ER lumen, the translocon closes, and the protein assumes its native
folded conformation.

Translocons Sec61

Translocons are protein complexes found in the membranes of endoplasmic reticulum ER in

eukaryotic cells and plasma membranes in prokaryotic cells.

● Translocons consist of multiple protein subunits that form a functional complex.

● The main component of translocons is the Sec61 complex, which is composed of three
subunits: α, β, and γ in eukaryotes.
 ● The Sec61 complex forms a protein-conducting channel that spans the ER membrane.
● Other associated proteins, such as Sec62, Sec63, and BiP binding immunoglobulin protein,
assist in translocation processes.

Post-translational Translocation Across ER Membrane

● Once the N terminal segment of a protein enters the ER lumen, the signal sequence is cleaved
by signal peptidase.
● The Sec63 complex, in coordination with BiP ATP, prevents the backsliding of the polypeptide
and aids in its translocation into the ER.
● The binding of BiP ADP molecules acts as a ratchet, drawing the entire polypeptide into the
ER.
● Subsequent exchange of ADP for ATP releases the polypeptide, allowing it to fold into its
native conformation.
● The recycled BiP ATP is then available for another interaction with Sec63.

N-Glycosylation

N glycosylation is a post-translational modification process that involves the attachment of a glycan

oligosaccharide chain to specific asparagine residues Asn within a protein.
Process

● Initiation:
○ The synthesis of the glycan chain begins in the endoplasmic reticulum ER, where a
precursor molecule called dolichol phosphate Dol P is synthesized.
○ Dolichol phosphate is a strongly hydrophobic lipid, containing 75 95 carbon atoms,
that is embedded in the ER membrane.
○ Two N acetylglucosamine GlcNAc and five mannose residues are added one at a time
to a dolichol phosphate on the cytosolic face of the ER membrane.
● Assembly:
○ A series of enzymatic reactions occur on Dol P, resulting in the stepwise addition of
sugar residues to form a lipid-linked oligosaccharide LLO precursor.
○ The nucleotide sugar donors in these reactions are synthesized in the cytosol. Note
that the first sugar residue is attached to dolichol
by a high-energy pyrophosphate linkage.
○ Tunicamycin, which blocks the first enzyme in this pathway, inhibits the synthesis of all
N-linked oligosaccharides in cells.
● Transfer:
○ The LLO precursor is then flipped across the ER membrane and transferred en bloc to
the protein target by an oligosaccharyltransferase OST complex.
○ After the seven residue dolichol pyro phosphoryl intermediate is flipped to the luminal
face, the remaining four mannose and all three glucose
residues are added one at a time.
○ In the later reactions, the sugar to be added is first transferred from a
nucleotide sugar to a carrier dolichol phosphate on the cytosolic face of the ER.
○ The carrier is then flipped to the luminal face, where the sugar is transferred to the
growing oligosaccharide, after which the “empty” carrier is flipped back to the cytosolic
face.

Disulfide Bond Formation

 ● Protein disulfide isomerase PDI contains an active site with two closely spaced cysteine
residues that are easily interconverted between the reduced dithiol form and the oxidized
disulfide form.
● Numbered red arrows indicate the sequence of electron transfers. Yellow bars represent
disulfide bonds.
● Formation of disulfide bonds:
○ In the formation of disulfide bonds, the ionized S form of a cysteine thiol in the
substrate protein reacts with the disulfide S S bond in oxidized PDI to form a
disulfide-bonded PDI substrate protein intermediate.
○ A second ionized thiol in the substrate then reacts with this intermediate, forming a
disulfide bond within the substrate protein and releasing reduced PDI.
● Rearrangement of disulfide bonds:
○ Reduced PDI can catalyze the rearrangement of improperly formed disulfide bonds by
similar thiol-disulfide transfer reactions.
○ In this case, reduced PDI both initiates and is regenerated in the reaction pathway.
● These reactions are repeated until the most stable conformation of the protein is achieved.

Role of N glycosylation in ER Protein Folding

For Unfolded Proteins

● N glycosylation plays a crucial role in recognizing and targeting unfolded or misfolded

proteins for ER-associated degradation ERAD.
● ER chaperones, such as calnexin and calreticulin, recognize the glycans attached to unfolded
proteins and assist in their folding.
● If an unfolded protein fails to achieve its proper conformation, it is retrotranslocated to the
cytosol and targeted for degradation by the proteasome through the ERAD pathway.
For Incompletely Folded Proteins

● N glycosylation serves as a quality control checkpoint for incompletely folded proteins.

● ER chaperones recognize the glycans on incompletely folded proteins and facilitate their
further folding and maturation.
● The presence of N glycans helps retain incompletely folded proteins in the ER, providing
additional time for them to reach their correct conformation.
● If the incompletely folded protein fails to achieve its native structure, it may undergo ERAD for
degradation.

For Normally Folded Proteins

● N glycosylation contributes to the stability, trafficking, and functionality of normally folded

proteins in the ER.
● The addition of specific glycans can be required for the proper folding, stability, or targeting of
certain proteins.
● Glycans attached to normally folded proteins can influence their interactions with other
proteins, participate in cell-cell recognition processes, and modulate receptor-ligand
interactions.
● Additionally, N glycans on proteins act as a protective barrier, shielding hydrophobic regions
and preventing their aggregation or inappropriate interactions.

Export and Degradation of Misfolded ER Proteins

Retrotranslocation to the Cytosol:

● Once misfolded proteins are identified, they are retrotranslocated or extracted from the ER
lumen or membrane to the cytosol.
 ● This process involves the action of protein complexes such as the ER membrane-associated
protein degradation ERAD machinery, which consists of various components, including E3
ubiquitin ligases.

Ubiquitination:

● In the cytosol, misfolded proteins are recognized by E3 ubiquitin ligases, which transfer
ubiquitin molecules to specific lysine residues on the misfolded proteins.
● The addition of ubiquitin molecules acts as a "tag" that marks the misfolded proteins for
degradation.

Proteasomal Degradation:

● The polyubiquitinated misfolded proteins are targeted to the 26S proteasome, a large
multi-subunit complex responsible for protein degradation.
● The 26S proteasome recognizes the polyubiquitin chains on the misfolded proteins, unfolds
them, and degrades them into smaller peptides.
● The peptides generated from proteasomal degradation can be further processed and
presented by major histocompatibility complex MHC molecules for immune surveillance.