LU2 Cell Structure and Function

Lect. 3: Endoplasmic Reticulum (ER)

Relative Overall Volumes of Organelles

in Liver Cell (Hepatocyte)
Cytosol
Mitochondria
Rough ER cisternae
Smooth ER cisternae
plus Golgi cisternae
Nucleus
Peroxisomes
Lysosomes
Endosomes

54
22
9
6
6
1
1
1

Endoplasmic reticulum
An extensive lipid membrane system which

extends throughout the cell stretching from the

nuclear envelope all the way to the plasmalema.
These membranes form a network of
interconnecting flattened cisternae and vesicles
and they accounts for about 10% of the total cell
volume.
The E.R. plays a central role in lipid and
protein biosynthesis.

Endoplasmic reticulum

Small dots on the outer

surface of the membrane,
these are the
polyribosomes, and are the
sites of protein
synthesis.

The extent of ER varies

within cell types and

frequently, in specialized
cells with high metabolic
activity, there are areas
where these membranes are
densely packed

Electron Micrograph of ER

Electron Micrograph of ER

Rough Endoplasmic reticulum

The region of ER immediately

outside the nucleus has

ribosomes anchored to its
surfaces.

The function of RER is the

synthesis of secretory
proteins and membrane
proteins.

Protein synthesis is initiated by

binding of the mRNA

template to the ribosomes
attached to membranes.

Smooth Endoplasmic reticulum

Regions of the ER where there is a

noticeable absence of
ribososmes

SER is further away from the

nucleus and is more tubular

Its functions are

(i) membrane synthesis

(ii) transport of secretory
proteins from the lumen of ER
to the outside of the cell.
The SER is the site of enzymes

involved in lipid metabolism

Smooth ER

Proteins translocation into the lumen of

rough ER
Protein destined for the

rough endoplasmic
reticulum has a
hydrophobic signal
sequence
It is the first part of the
protein produced.

After the signal sequence

is completed, protein
synthesis is further
inhibited.

Proteins translocation into the lumen of

rough ER
The signal sequence is

recognized by a Signal
Recognition Particle
(SRP). This is then
bound to a receptor.
This is to allow the

interaction of the signal

sequence with a complex
(e.g. Sec61) on the rough
endoplasmic reticulum.

Proteins translocation into the lumen of

rough ER
This complex guides the

protein through a channel

like region. It also
consists of a docking
site for the ribosome.
The front end of protein

enters into the lumen of

the rough endoplasmic
reticulum.

Proteins translocation into the lumen of

rough ER
Protein synthesis resumes

and the rest of the protein

is inserted in the lumen.
A signal peptidase near

the inner surface of the

membrane cleaves the
signal sequence from
the growing peptide.

Proteins translocation into the lumen

of rough ER

Cotranslational
targeting of secretory
proteins to the ER

Step 1: As the signal sequence emerges from the ribosome, it is

recognized and bound by the signal recognition particle (SRP).
Step 2: The SRP escorts the complex to the ER membrane, where it binds
to the SRP receptor.
Step 3: The SRP is released, the ribosome binds to a membrane
translocation complex of Sec61 proteins, and the signal sequence is
inserted into a membrane channel.

Cotranslational
targeting of secretory
proteins to the ER

Step 4: Translation resumes, and the growing polypeptide

chain is translocated across the membrane.
Step 5: Cleavage of the signal sequence by signal peptidase
releases the polypeptide into the lumen of the ER.

Translocated Polypeptide
Chains Fold and Assemble in
the Lumen of the Rough ER

Protein folding in the ER

The molecular chaperone BiP binds to polypeptide chains as they
cross the ER membrane and facilitates protein folding and assembly
within the ER.
Binding immunoglobulin protein (BiP) is a molecular chaperone (or helper
proteins) that uses ATP/ADP cycling to regulate protein folding by the protein
disulfide isomerase (PDI) family of proteins.

The retranslocation & degradation of misfolded

ER proteins

Misfolded soluble proteins in the ER lumen (> 80% for some proteins)
are translocated back into the cytosol, where they are deglycosylated,
ubiquitylated, and degraded in proteasomes. Misfolded membrane
proteins follow a similar pathway. Misfolded proteins are exported
through the same type of translocator that mediated their import;

accessory proteins that are associated with the translocator allow it to

operate in the export direction.

Protein/Posttranslational Modifications (PTM)

Protein/Posttranslational Modifications (PTM)

Posttranslational Modifications (PTM) the chemical
modification of a protein after its translation
Proteolysis the directed degradation / digestion of proteins by
cellular enzymes called proteases or by intramolecular digestion
Glycosylation the enzymatic process that links saccharides to
produce glycans, either through or attached to proteins and lipids
Phosphorylation the addition of a phosphate group (PO4) to a
protein or organic molecule. It turns many enzymes on and off,
causing or preventing the mechanisms of diseases, such as cancer
and diabetes.

Glycosylation in the Rough Endoplasmic Reticulum

Most proteins made on RER are glycosylated and thus become
glycoproteins, whether integral proteins of membrane, soluble
lysosomal or vacuolar enzymes or parts of ECM
Carbohydrate groups have key roles in function of many
glycoproteins (e.g., binding sites in their interactions with
other macromolecules); also aid in proper folding of protein to
which they are attached

Sugar sequences that comprise glycoprotein

oligosaccharides are highly specific, and

Sugar sequences from purified glycoprotein are consistent

& predictable - how?

How is oligosaccharide sugar sequence assembled?

catalyzed by a family of membrane-bound enzymes

Glycosyltransferases
Glycosyltransferases transfer specific monosaccharide
from a nucleotide sugar.
Donor is always a nucleotide sugar: GDP-mannose, GDPfucose, UDP-galactose, UDP-N-acetylglucosamine;
acceptor of transferred sugar is growing end of
carbohydrate chain.
Sequence of sugar transfer during oligosaccharide
assembly depends on the sequence of action of
glycosyltransferases participating in process.

How is oligosaccharide sugar sequence assembled?

Glycosyltransferase sequence, in turn, depends on the
location of specific enzymes within the various secretory
pathway membranes.
Thus, sugar arrangement in oligosaccharide chains of a
glycoprotein depends on the spatial localization of
certain enzymes in this assembly line.

How is oligosaccharide sugar sequence assembled?

Carbohydrate chains are attached to protein by Nlinkages (asparagine N atom) of both soluble &
integral membrane proteins.
These oligosaccharides differ in average size, sugar
composition & path of synthesis & also share properties
like their high specificity.
N-linked basal (core) chain segment is assembled on lipid
carrier not protein then transferred as a block to specific
asparagine residues of polypeptide as it enters RER by
oligosaccharyltransferase.

The asparagine-linked (N-linked)

precursor oligosaccharide that is
added to most proteins in the rough
ER membrane

How is oligosaccharide sugar sequence assembled?

Lipid carrier is dolichol phosphate embedded in membrane
(hydrophobic molecule built from >20 isoprene units) &
sugars are added one at a time by membrane-bound
glycosyltransferases.
This part of glycosylation process is essentially
invariant/remains unchanged.
In mammalian cells, it starts with transfer of Nacetylglucosamine 1-phosphate & then transfer of another Nacetylglucosamine, then 9 mannose & 3 glucose units in a
precise pattern.
This block of 14 sugars is then transferred by
oligosaccharyltransferase from dolichol phosphate to nascent
polypeptide as it is being translocated into ER lumen.

 The first seven sugars are transferred one at a time to the

dolichol-PP on the cytosolic side of the ER membrane (steps 1 &

2).
At this stage, the dolichol with its attached oligosaccharide flips
across the membrane (step 3), and the remaining sugars are
attached on the luminal side of the membrane.

 These latter sugars are attached one at a time on the cytosolic

side of the membrane to the end of a dolichol phosphate

molecule (steps 4 & 7), which then flips across the membrane
(steps 5 & 8) and donates its sugar to the growing end of the
oligosaccharide chain (steps 6 & 9).

 Once the oligosaccharide is completely assembled, it is

transferred enzymatically (oligosaccharyltransferase ) to an

asparagine residue of the nascent polypeptide (step 10).
The dolichol-PP flips back across the membrane (step 11) and
is ready to begin accepting sugars again (steps 12 & 13).

The synthesis of the

oligosaccharide starts on the
cytosolic side of the ER
membrane and continues on the
lumenal face after the
(Man)5(GlcNAc)2 lipid
intermediate is flipped across
the bilayer by a transporter
protein. All the subsequent
glycosyl transfer reactions on
the lumenal side of the ER
involve transfers from dolicholP-glucose and dolichol-Pmannose; these activated, lipidlinked monosaccharides are
synthesized from dolichol
phosphate and UDP-glucose or
GDP-mannose (as appropriate)
on the cytosolic side of the ER
and are then thought to be
flipped across the ER
membrane. GlcNAc = Nacetylglucosamine; Man =
mannose; Glc = glucose.

Glycosylation in the Rough Endoplasmic Reticulum

Mutations that lead to total absence of Nglycosylation cause death of embryos prior to
implantation
Mutations leading to partial glycosylation pathway
disruption in ER also cause serious inherited disorders
affecting nearly every organ system.
These diseases are called Congenital Diseases of
Glycosylation (CDGs) and they are usually identified
through blood tests that detect abnormal glycosylation of
serum proteins.

Glycosylation in the Rough Endoplasmic Reticulum

Example: One of these diseases, CDG1b can be managed through a
remarkably simple treatment.
It results from deficiency of the enzyme phosphomannose
isomerase (catalyzes conversion of fructose-6-phosphate to
mannose-6-phosphate).
Its reaction is a crucial reaction in the pathway that makes
mannose available for incorporation into oligosaccharides.
The disease can be managed by giving patients oral
supplements of mannose; first tested in boy who was dying
from uncontrolled gastrointestinal bleeding (a usual
complication of the disease).
Within months of taking mannose supplements, the child was
living a normal life.