Chapter 3 :

Endomembrane
System

PROTEIN BIOCHEMISTRY &

BIOTECHNOLOGY
BTP2223
By: MOHD AZMIR BIN ARIFIN
Contact No.: 019-238127
E-Mail: mazmir@ump.edu.my

OUTLINES

Basic biology and the structure of endomembrane

system
Endoplasmic reticulum (ER) structure and functions
Mechanisms of protein entry into ER lumen, protein
exit from ER, unfolded protein response & protein
degradation

The subcellular complexity of cell

Endomembrane
Composed of the different membranes that are suspended
in the cytoplasm within a eukaryotic cell.
Divide the cell into functional and structural
compartments, or organelles.
In eukaryotes : the nuclear membrane, the endoplasmic
reticulum (ER), the Golgi apparatus, lysosomes,
endosomes and the cell membrane*

Endomembrane system contains the following components:

Endoplasmic reticulum (ER):
Smooth ER
Rough ER
-

Golgi complex: Protein modification

Endosomes: Protein sorting between endo- and exoxytic routes

(membrane transport pathway)*
Lysosomes: Degradation processes, organelle turnover,
(containing hydrolytic enzymes capable of breaking down virtually
all kinds of biomolecules, including proteins, nucleic acids,
carbohydrates, lipids, and cellular debris )
Peroxisomes (Microbodies): beta-oxidation of fatty acids

Endoplasmic Reticulum (ER)

HISTORY
In the year 1945 -> The lace like membranes of the
endoplasmic reticulum were first seen in the
cytoplasm of chick embryo cells -> a team of
biologists, Keith R. Porter, Albert Claude, and Ernest
F. Fullman
1952- ER term used by Porter and Fullman in their
published article.

Endoplasmic Reticulum (ER)

Endoplasmic means "within the plasm" and reticulum
means "network".
A network of tubular and flat vesicular structures in
the cytoplasm is the endoplasmic reticulum
The tubules and vesicles interconnect with one
another
Its an internal delivery system of cell.
It makes up approximately 12% of cell volume.
Highly convoluted space is called the ER lumen or the
ER cisternal space.
The ER membrane separates the ER lumen from the
cytosol, and it mediates the selective transfer of
molecules between these two compartments.

STRUCTURE
Endoplasmic matrix: The space inside the tubules and
vesicles is filled with a watery medium that is different
from the fluid in the cytosol outside the ER.
ER membrane are constructed of lipid bilayer that contain
large amounts of proteins, similar to the cell membrane.
Electron micrographs show that the space inside the
endoplasmic reticulum is connected with the space
between the two membrane surfaces of the nuclear
membrane (perinuclear space)
Also it is connected with Golgi apparatus and cell
membrane

STRUCTURE
All eukaryotic cells have an ER except the red blood
cells and spermatozoa
The total surface area of this structure in some cellsthe liver cells, for instance-can be as much as 30 to
40 times the cell membrane area.
The functions of the endoplasmic reticulum vary
greatly depending on its cell type, cell function, and
cell needs.
The ER can even modify to change over time in
response to cell needs.

TYPES
2 types:
Rough ( granular) endoplasmic reticulum (RER)
Smooth ( agranular) endoplasmic reticulum (SER)
The quantity of RER and SER in a cell can slowly
interchange from one type to the other, depending on
changing metabolic needs.

RER
Rough ER is well developed in cells that are
active in protein synthesis.eg.
acinar cell of pancreas*
White blood cells that produce infection fighting
immune system proteins called antibodies have
highly developed RER
Ribosome: large numbers of minute granular
particles attached to the outer surfaces of many
parts of the rough endoplasmic reticulum.

RER
The
ribosomes
in
eukaryotes
measure
approximately 22 x 32 nm.
Each is made up of a large and a small subunit
called, the 60S and 40S subunits, on the basis of
their
rates
of
sedimentation
in
the
ultracentrifuge*
The ribosomes are complex structures,
containing many different proteins and at least
three ribosomal RNAs.
They are the sites of protein synthesis

RER
These proteins typically have a hydrophobic
signal peptide at one end.
The binding site of the Ribosome on RER is the
translocon formed by the heterotrimeric Sec61
complex*.
Free ribosomes are also found in the cytoplasm.

RER
The free ribosomes synthesize cytoplasmic proteins such
as hemoglobin and the proteins found in peroxisomes and
mitochondria.
The ribosomes that become attached to the endoplasmic
reticulum synthesize all transmembrane proteins, most
secreted proteins, and most proteins that are stored in the
Golgi apparatus, lysosomes, and endosomes.
The ribosomes bound to the RER at any one time are not a
stable part of this organelles structure as ribosomes are
constantly being bound and released from the membrane.

 Rough ER appears as
lumpy sheets of folded
membranes; the lumps
are the ribosomes.
They are not
permanently attached,
adhering only when
there is protein
manufacturing work to
be done

SER
Part of the endoplasmic reticulum has no attached ribosomes.
This part is also called the agranular endoplasmic reticulum.
The SER consists of tubules that are located near the cell
periphery.
These tubes sometimes branch forming a network that is
reticular in appearance.

SER
The network of SER allows increased surface area
for the action or storage of key enzymes and the
products of these enzymes.
SER is the site of lipid synthesis (including oils,
phospholipids and steroids), metabolizing of
carbohydrates, regulation of calcium concentration
and detoxification of drugs and poisons.

SER
It is found in abundance with Leydig cell
and cells of adrenal cortex*.
Smooth ER found in smooth and striated
muscle.
In brain cells it synthesizes male and
female hormones.

SARCOPLASMIC
RETICULUM
The sarcoplasmic reticulum (SR), from the Greek sarx,
("flesh)*
In skeletal and cardiac muscle, smooth ER is modified to
form sarcoplasmic reticulum.
The only structural difference between this organelle and
the smooth ER is the medley of proteins they have, both
bound to their membranes and drifting within the
confines of their lumens.
This fundamental difference is indicative of their
functions -> The ER synthesizes molecules, while the SR
stores and pumps calcium ions*.

FUNCTION
RER :
Predominant function is in the import of
nascent polypeptides into lumen*
Involves largely in folding, quality control and
export of completed proteins
Formation of Glycoprotein- Linking of sugars to
glycoprotein starts in the RER and is completed in
Golgi complex.
Synthesis of precursors- The RER produce
enzyme precursors for the formation of
lysosomes by Golgi Complex.
Smooth ER formation- The RER gives rise to the
smooth ER by loss of ribosomes.

FUNCTION
SER :
The smooth endoplasmic reticulum lacks ribosomes and
functions
in lipidmetabolism,carbohydratemetabolism,
anddetoxification and is especially abundant in mammalian
liver and gonad cells.
It also synthesizesphospholipids. Cells which secrete these
products, such as those in the testes, ovaries, and skin oil glands
have a great deal of smooth endoplasmic reticulum.
Detoxification-The SER brings about detoxification in the
liver , i.e., converts harmful materials(drugs, poisons) into
harmless ones for excretion by the cell.
Formationoforganelles-The SER produces Golgi apparatus ,
lysosomes and vacuoles.

FUNCTION
SER :
It also carries out the attachment of receptors on cell membrane
proteins and steroid metabolism.
In muscle cells, it regulates calcium ion concentration (SR)
The smooth endoplasmic reticulum also contains the
enzyme glucose-6-phosphatase, which converts glucose-6phosphate to glucose, a step in gluconeogenesis (carbohydrate
metabolism).

FUNCTION
SR :
Specializesinthestorageofcalcium
Calcium ions are pumped into the ER by ATP-dependent
calcium ATPases and are released in response to extracellular
signals to aid in muscle contraction
Binding of neurotransmitter molecules to receptors on the
surface of the muscle cell triggers a signal cascade that leads to
the release of calcium from the sarcoplasmic reticulum and
causes the contraction of muscle fibers.

Internal transport pathway in cells

Gated transport*
Via protein channels or pores
Eg: nuclear import and export
Transmembrane transport
Also known as translocation
Via protein translocation channel
Eg: Protein entry into the ER
Vesicular transport
Via membrane-bound intermediates
(vesicles)
Eg: ER to Golgi

Overview of sorting of nuclear-encoded proteins in

eukaryotic cells
mRNAs are
translated on
cytosolic
ribosomes
Ribosomes
synthesizin
g ER
proteins

Proteins
without ER
signal are
sent to
cytosol

Those proteins
with organellesp uptake sqn
Nascent proteins are
will be sent to
directed to
several
RER by the signal sqn
destinations
Translation
completed here

Proteins moved
to golgi
complex via
vesicles from
whence they
are transported
to several
destinations

Partially
folded
protein

Fully
folded

Fully
folded

Protein translocation
In the early 1970s, Gnter Blobel, in collaboration with David Sabatini
and Bernhard Dobberstein of Rockefeller University proposed a theory
They suggested the following:
many proteins have a signal sequence at their N-terminus that
functions like a postal code for the target organelle*.
The translation of mRNA into protein by a ribosome takes place
within the cytosol.
If the synthesized proteins "belong" in a different organelle, they
can be transported there in either of two ways depending on the
protein:
Co-translational translocation (translocation during the process
of translation),
Post-translational translocation (translocation after the process
of translation is complete).

Synthesis of Secretory, Lysosomal, or Plant Vacuolar Proteins on

Membrane-Bound Ribosomes

Step 1. As the signal sequence emerges from the ribosome,

it is bound by a signal recognition particle (SRP) that
arrests further synthesis.
Step 2. This mediates the binding of the complex to the RER
membrane by the SRP receptor.
Step 3. The SRP is released from the membrane.
Step 4. The nascent polypeptide passes through a protein-lined
pore in the ER membrane and into the ER lumen.

The signal sequence allows the recognition particle to bind

to the ribosome
causing the ribosome to bind to the RER
pass the new protein through the ER membrane
Signal sequence is cleaved by signal peptidases
release

the new PP into the ER lumen

Co-translational entry into the ER

Many proteins are made on ribosomes attached to
cytosolic surface of RER membranes
Nascent polypeptides attached to ribosomes are
directed to the ER through interaction of a signal
sequence on the new protein with a cytosolic
molecule termed signal recognition particle (SRP)
A typical signal sequence: +H3N-Met-Met-Ser-Phe-

Val-Ser-Leu- Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-ThrGlu-Ala-Glu-Gln-LeuThr-Lys-Cys-Glu-Val-Phe-Gln (hydrophobic, negatively

charged,
SRP is composed of 6 distinct polypeptides and a
positively
small 7Scharged)
RNA molecule. Its binding briefly halts

translation until the

complex is brought to a
translocon channel on the RER membrane*

Co-translational entry into the ER (contd)

SOLUBLE PROTEINS
The peptide moves
through the
translocation channel
into the lumen of the
ER.
The signal peptide
sequence remains
attached to the
membrane.
It is later cleaved off
by a signal peptidase.
Leaving the protein
free in the lumen of
the ER.

Co-translational entry into the ER (contd)

MEMBRANE PROTEINS

Key point is that the orientation

of a protein in the membrane is
established
when it is first
inserted into the membrane. This
orientation of the protein persists
all of the way to its final
destination. That is, the cytosolic
side of membrane remains on
the cytosolic side throughout all
processes.
As membrane proteins are being
translated, they are translocated
or transferred into the ER until a
hydrophobic membrane crossing
domain is encountered. This
serves as a 'stop transfer' signal
and leaves the protein inserted
in the ER membrane.
The
hydrophobic
transmembrane domain holds the
protein
in
the
membrane
because of the very strong

Import of a membrane protein. This figure illustrates the

case of a protein being incorporated in the membrane of
the endoplasmic reticulum. The blue sheath-like
component shown in the figure is the transport complex
that moves the protein through the membrane. This
example is a single pass membrane protein that contains a
single membrane crossing domain.

Post-translation
translocation
Some proteins, however, are imported into the ER after

their synthesis has been completed.

To function in post-translational translocation, the
translocator needs accessory proteins that feed the
polypeptide chain into the pore and drive translocation .

Cotranslational
targeting of
secretory
proteins to
the ER

Posttranslational
translocation
of
proteins into
the ER

In prokaryotes this requires certain cofactors such as SecA

and SecB. This pathway is poorly understood in eukaryotes,
but is facilitated by Sec62 and Sec63, two membrane-bound
proteins.

Post-translational translocation of secretory proteins at the yeast ER. Hsp70

chaperones maintain the translocation competence of post-translational
precursors prior to them encountering the ER membrane. The polypeptide
binds the Sec61/62/63/71/72 complex and inserts into the ER translocation
channel. Sec63 recruits the luminal chaperone Kar2 to the luminal face of the
membrane where it is suggested to promote the unidirectional movement of
the protein being imported by acting as a molecular ratchet*.

BACTERIA vs ARCHEA vs
EUCARYOTES

Protein folding and glycosylation in the ER

About half of all eukaryotic proteins are glycosylated
Almost all proteins entering the secretory pathway are
glycosylated
Immediately on entering the ER lumen, proteins are
glycosylated on arginine residues (N-Linked)
The precursor, oligosaccharide is held in the ER lumen
by the lipid called dolichol*
The oligosaccharide is transferred to the emerging
protein by the enzyme oligosaccharyl transferase (OST)

Protein Glycosylation in the

RER

Quality control in the ER

Glycosylation is used as a marker for the state
of protein folding*
The first phase involves the trimming of the
two terminal residues
The proteins (now with a single terminal
glucose) undergo
folding while bound to
molecular chaperones, such as calnexin
(transmembrane) and calreticulin (soluble)
Disulphide bonds are formed with the help of
protein disulphide isomerase (PDI)
The glucosyl transferase (GT) enzyme is able to
recognise the unfolded proteins

 The ER-membrane-bound chaperone protein calnexin

binds to incompletely folded proteins containing one
terminal glucose on N-linked oligosaccharides, trapping
the protein in the ER.
Removal of the terminal glucose by a glucosidase
releases the protein from calnexin.
A glucosyl transferase is the crucial enzyme that
determines whether the protein is folded properly or not:
if the protein is still incompletely folded, the enzyme
transfers a new glucose from UDP-glucose to the Nlinked oligosaccharide, renewing the protein's affinity for
calnexin and retaining it in the ER.
The cycle repeats until the protein has folded completely.

The role of glycosylation in

the ER
protein folding

Exiting the ER misfolded proteins

Despite all the help from chaperones, many protein molecules
(more than 80% for some proteins) translocated into the ER
fail to achieve their properly folded state.
Such proteins are exported from the ER back into the cytosol,
where they are degraded
Once the misfolded protein has reached the cytosol, its
oligosaccharides are removed.
Deglycosylation is catalyzed by an N-glycanase, which
removes the oligosaccharide chains by cleaving the amide
bond between the carbonyl group and the amino group of the
original asparagine to which the oligosaccharide was attached.
The deglycosylated polypeptide is rapidly ubiquitylated by
ER-bound ubiquitin-conjugating enzymes and is then fed into
proteasomes, where it is degraded*

The export & degradation of

misfolded ER
protein

 Proteins that are consistently unable to be folded are

eventually dislocated from the ER in a process termed
ER-associated degradation (ERAD)
There is strong evidence that the unfolded protein
passess back through the translocon into the cytosol
In the cytosol, the N-glycanase enzyme removes the
carbohydrate side chain (oligosaccharide chains )
The small ubiquitin (8.5 kDa) is added to the dislocated
proteins to mark them for degradation by the
proteosome