The first Endomemembrane System was discovered by Camillo Golgi

in the late 1800s.

Autoradiography (James Jamieson and George Palade of Rockefeller

University)
 Green Fluorescent Protein (GFP)
 This technology utilizes a gene isolated from a jellyfish that
encodes a small protein, called the GFP, which emits a green
fluorescent light.
 Biochemical analysis of
subcellular fractions
(Albert Claude and
Christian De Duve)
 Membranous vesicles
derived from the
endomembrane system
(primarily the
endoplasmic reticulum
and Golgi complex) form a
heterogeneous collection
of similar sized vesicles
referred to as
microsomes
 Cell-free system (George
Palade and Philip
Siekevitz)
The endomembrane system is composed of the
different inter-related membrane sacs within
the cytoplasm of the cell.
• Synthesis,
• Modification,
• Sorting and
• Transport.
• Biosynthetic pathway
• Secretory pathway
– Constitutive secretion
– Regulated secretion
 Who discovered
the ER?
 discovered by Belgian
biologist Albert Claude
(1899-1983).

 used the newly developed

electron microscope to explore
the interior of cells and along
with his associate Keith Porter
in 1945 observed the presence
of a “lace-work structure.”
 Endoplasmic
Reticulum
endoplasmic - “within the cytoplasm”
reticulum - Latin for a “a little net”
- extensive network of folded
membranes that extends from the
nuclear envelope to which it is
connected, throughout the cytoplasm.
- divided into two sub-compartments,
rough ER and smooth ER
 Synthesis of fatty acids and steroid hormones,
such as estrogens and testosterone.
 Detoxification in the liver of a wide variety of
organic compounds, including barbiturates and
ethanol. (oxygenases and cytochrome P450)
 Sequestering calcium ions within the cytoplasm
of cells.
 extensively developed in a number of cell
types, including those of skeletal muscle, kidney
tubules, and steroid-producing endocrine glands

FIGURE 1. The smooth ER (SER). Electron micrograph of a Leydig cell from the
testis showing the extensive smooth ER where steroid hormones
are synthesized.
Rough ER
continuous with the
outer membrane of the
nuclear envelope,
which also bears
ribosomes on its
cytosolic surface.
 Rough ER
- defined by the presence of
ribosomes bound to its cytosolic surface

- typically composed of a
network of flattened
sacs (cisternae)
Functions of the
Rough ER
 secrete large quantities of proteins,
such as the:

a. acinar cells of the pancreas

 Functions of the Rough ER

b. mucus secreting
cells of the lining of
the digestive tract
 Functions of the
Rough ER
 Rough ER is the starting point of the
biosynthetic pathways of:
proteins
carbohydrate chains
phospholipids
that journey through the membranous
compartments of the cell.
Rough ER vs. Smooth
ER
Ribosomes on its outer Not associated with
surface ribosomes

Site of synthesis of proteins Involved in lipid metabolism

destined for secretion
Protein Synthesis
On membrane-bound ribosomes:
a. Secreted proteins
b. Integral membrane proteins
c. Soluble proteins in ER, Golgi complex, lysosomes,
endosomes, vesicles and plant vacuoles

On free ribosomes:
a. Cytosolic proteins
b. Peripheral proteins
c. Proteins transported to the nucleus
d. Proteins to be incorporated in peroxisomes,
chloroplasts and mitochondria
1. Secretory proteins contain a signal sequence at their
N-terminus that directs the emerging polypeptide and
ribosome to the ER membrane.

2. The polypeptide moves into the cisternal space of the

ER through a protein-lined, aqueous channel in the ER
membrane. It was proposed that the polypeptide moves
through the membrane as it is being synthesized, that is,
cotranslationally
 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.
Synthesis of Integral Membrane Proteins on Membrane-Bound Ribosomes

Step 1. The nascent polypeptide enters the translocon just as if it were a

secretory protein.
Steps 2–3 show the synthesis of a transmembrane protein whose N-terminus
is in the lumen of the ER and C-terminus is in the cytosol.
In step 2, the translocon has opened laterally and expelled the
transmembrane segment into the bilayer.
Synthesis of Integral Membrane Proteins on Membrane-Bound Ribosomes

Step 3 shows the final disposition of the protein.

Steps 2a–4a show the synthesis of a transmembrane protein whose C-terminus is in

the lumen and N-terminus is in the cystosol.
In step 2a, the translocon has reoriented the transmembrane segment, in keeping
with its reversed positively and negatively charged flanks.
In step 3a, the translocon has opened laterally and expelled the transmembrane
segment into the bilayer.
Step 4a shows the final disposition of the protein.
• Start signal for internalization
• Stop signal for delocalization
 Membrane
Biosynthesis in the
• protein inserted into the lipid bilayer in a predictable
orientation determinedERby its amino acid sequence.
• The carbohydrate chains, which are first added in the
ER, provide a convenient way to assess membrane
sidedness because they are always present on the
cisternal side of the cytoplasmic membranes, which
becomes the exoplasmic side following the fusion of the
plasma membrane.
Synthesis of Membrane
Lipids
• Membrane proteins are synthesized with ER
except:
• Sphingomyelin and glycolipids (ER -> Golgi)
• Mitochondrial and chloroplast lipids are
synthesized by enzymes (Integral protein
active sites facing the cytosol)
• Some of the lipids are flipped into the
opposite leaflet by Fflippases.
Conversion of one type of
phospholipid to another -
most organelles have
enzymes that modify lipids
already present in membrane

As membranes bud, some

phospholipids preferentially
included in forming vesicle,
others excluded

Phospholipid-transfer proteins
move specific phospholipids
between membrane
compartments through
aqueous cytosol & may move
them from ER to other
organelles (mitochondria,
chloroplasts)
• have key roles in function of many glycoproteins (e. g., binding
sites in their interactions with other macromolecules)
• aid in proper folding of protein to which they are attached
Sugar sequences that comprise glycoprotein
oligosaccharides are highly specific
Sugar sequences from purified glycoprotein are consistent &
predictable

How is oligosaccharide sugar

sequence assembled?
glycosyltransferases
• The first seven sugars (five mannose and two NAG residues) are
transferred one at a time to the dolichol-P on the cytosolic side of the
ER membrane
• The dolichol with its attached oligosaccharide is then
flipped across the membrane and the remaining sugars (four mannose
and three glucose residues) 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 which then
flips across the membrane
• It will donate its sugar to the growing end of the oligosaccharide chain
• Once the oligosaccharide is completely assembled, it is transferred
enzymatically to an asparagine residue (within the sequence N-X-S/T)
of the nascent polypeptide.
• The dolichol-PP is flipped back across the membrane and is ready to
begin accepting sugars again
• Proper protein folding
• Protein maturation
• lead to total absence of N-glycosylation
cause death of embryos prior to implantation
• Congenital Diseases of Glycosylation
(CDGs)
• leading to partial glycosylation pathway
disruption in ER also cause serious inherited
disorders affecting nearly every organ system
• diversify in more complex organisms (evolution
accompanied by diversification of the CHO
sequences on proteins)
• is recognized & bound by monitoring enzyme (GT)
• it adds a single glucose back to one of the mannose residues
• GT recognizes incompletely folded or misfolded proteins
• Once the glucose residue is added, the tagged glycoprotein is
recognized by the same chaperones giving it another chance to
fold properly
• The added glucose is removed & GT checks the protein
• If 3D structure is right, protein continues on its way;
• if not, glucose is added & process repeats until eventually, the
glycoprotein has folded correctly or it remains misfolded & is
destroyed
Quality control: ensuring that
misfolded proteins do not
proceed forward
• First visualization by GFP
• exit sites of RER cisternae are smooth-surfaced & serve as places
where the first transport vesicles in biosynthetic pathway are
formed
• transport vesicles fuse to each other to form larger vesicles &
interconnected tubules in region between ER & Golgi complex
• This region is called ERGIC
(endoplasmic reticulum Golgi intermediate compartment)
& the vesicular-tubular clusters that form there are called VTCs
• VTCs move farther away from the ER toward Golgi complex; other
studies indicate that this movement occurs along tracks composed
of microtubules
• Discovered by Camillo Golgi
• flattened, disk-like membranous cisternae with dilated
rims & associated vesicles & tubules (smooth
membranes so found with smooth microsomes
• individual Golgi stacks often interconnected to form
ribbonlike complexs,
• In plants single Golgi stack is called dictyosome
• Golgi cisternae polarized –
• cis face (entry face closest to ER)
• trans face (exit face at opposite end of stack; closer to
plasma membrane)
Cisternal maturation model (up to mid-
1980s)
• Cisternae mature & change in composition as they
move through Golgi complex; each cisterna matures
into next cisterna along stack (origin of name)
• Each cisterna was thought to physically move from the
cis to the trans end of the stack, changing in
composition as it progressed

New model favored (mid-1980s until late-1990s)

• Vesicular Transport Model
• Cargo (secretory, lysosomal, membrane proteins) is
shuttled through Golgi stack from CGN to TGN in
vesicles that bud from one compartment & fuse with
neighboring one farther along stack
ERGIC
Until mid-1990s, it was assumed that transport
vesicles always moved in forward (anterograde)
direction, from cis origin to trans destination, but
new evidence says that……

Some move in backward (retrograde) direction

from trans donor to cis acceptor membrane
• Glycosylation
 the formation of a vesicle by budding from the membrane.
 The cytoplasmic surfaces of transport vesicles are coated
with proteins.
1. clathrin-coated vesicles
are responsible for the uptake of extracellular molecules from
the plasma membrane by endocytosis as well as the transport of
molecules from the trans Golgi network to lysosomes.
2. COPII
bud from the ER and carry their cargo forward along the secretory
pathway, to the Golgi apparatus.
3. COPI
bud from the ER-Golgi intermediate compartment or the Golgi
apparatus and function in the retrieval pathways that serve to retain
resident proteins in the Golgi and ER.
Clathrin
(blue)

A protein
called coat
protein II
(COPII;
green)

A
different
protein
called coat
protein I
(COPI; red)
• Anterograde transport
• ER to Golgi Complex
• Retrograde transport
• Golgi complex to ER
• Contains retrieval tags
– (KDEL) lys-asp-glu-leu
• Materials from
GC to
endosomes,
lysosomes, and
vacuoles