Univ of Medea, Cell bio.

, 1st Y, Boukhalfa

ENDOMEMBRANE SYSTEM

Only found in eukaryotic cells, the endomembrane system is a collection of

membrane-bound cytoplasmic cavities that communicate with one another by
vesicles or canalicles. This system's various components include the Golgi
apparatus, the endoplasmic reticulum, the plant vacuole, lysosomes, phagosomes,
and endosomes.

a. ENDOPLASMIC RETICULUM :
1. DEFINITION :

It is a group of membranes that enclose cavities in form of tubules or cisternae. It can

have ribosomes, like the Rough Endoplasmic Reticulum (RER), which is connected
to the nuclear envelope, or it can have none, like the Smooth Endoplasmic Reticulum
(SER).

Figure 1 : rough and smooth endoplasmic reticulum

2. ULTRASTRUCTURE:

TEM: the ER membrane is trilamellar and thinner (6 nm) than the plasma membrane.

SEM: there are globular intramembrane particles.

 Univ of Medea, Cell bio., 1st Y, Boukhalfa

3. CHEMICAL COMPOSITION :
3.1. Isolation :
 The ER is isolated from the 3rd pellet of the UCD in the form of rough or
smooth microsomes (small vesicles). Centrifugation in a sucrose density
gradient separates rough from smooth microsomes.
 Centrifugation of the rough microsomes in the presence of a detergent
detaches the ribosomes attached to the membranes.
 Centrifugation of the smooth microsomes in a sucrose concentration gradient
separates them according to their density and origin (SER, Golgi apparatus
and plasma membrane)
3.2. Analysis results :
 Membrane : Glycosyl transferase, cytochrome P450, and glucose 6-
phosphatase make up about 70% of its protein content, whereas 30% is
made up of lipids. The phospholipids create a bilayer with a high proportion
of unsaturated fatty acids (high fluidity). There are trace amounts of
cholesterol. A tiny portion of carbohydrates are located at the luminal
surface and are linked to lipids and proteins.
 Cavity contents: It is specific to each cell type. For example, the RER
cavity of the exocrine pancreas cell contains enzymatic proteins, the SER
cavity of the luteal cell contains steroid hormones and the sarcoplasmic
reticulum cavity contains Ca++.

4. FUNCTIONS :
4.1. Function of Rough Endoplasmic Reticulum (RER) :
 Synthesis and translocation of proteins : Protein synthesis always begins
in the cytosol with initiation and the start of elongation. Two types of protein
can be synthesised in the RER: soluble or luminal proteins and hydrophobic
proteins, which are inserted into the membrane
a. Luminal proteins: this takes place in several steps :

Step l: the signal sequence consists of the first hydrophobic amino acids located at
the Nterminus of the protein. It enables the synthesised protein to be addressed. As
soon as it emerges from the ribosome, this sequence is recognised by a
ribonucleoprotein complex in the cytosol known as the Signal Recognition Particle
 Univ of Medea, Cell bio., 1st Y, Boukhalfa

(SRP), which binds to the signal sequence and causes a temporary halt in
translation.

Step 2: this receptor captures the ribosome-mRNA-SRP complex, promoting its

interaction with the translocon (translocation protein complex). The large subunit of
the ribosome is attached to the REG membrane at the level of its receptor. The
translocon then opens up via a hydrophilic channel, allowing the protein being
synthesised to be translocated (passed) into the lumen, allowing translation to
resume. The SRP then separates from its receptor and from the signal sequence.

Step 3: The protein continues to elongate and enters the translocon channel.

Step 4: When the protein enters the lumen (or cavity) of the RER, a signal peptidase
located on the luminal side of the RER membrane cuts the signal sequence.

Step 5: As the protein appears in the lumen, it is taken up by chaperone proteins

involved in translocation and post-translational modifications. The protein released
into the lumen of the RER is a luminal protein.

Figure 2 : Proteosynthesis and translocation of a luminal protein.

 Univ of Medea, Cell bio., 1st Y, Boukhalfa

b. Membrane proteins:

Proteins destined for membranes are also addressed by the signal sequence; but the
translocation of these proteins is blocked by a very hydrophobic sequence which
destabilises the translocon, so that the protein remains inserted in the RER
membrane: this is a membrane protein.

 Post-translational modifications of proteins :

Glycosylation : corresponds to the attachment of an oligosaccharide molecule to a

protein (RER) or a lipid in the SER There are two types: N-glycosylation, which takes
place in the RER, and O-glycosylation, which takes place in the Golgi apparatus.

N-Glycosylation : N is the most common and asparagine is the amino acid of the
protein that will be glycosylated. Dolichol is a fatty acid synthesised in the cytoplasm.
It is inserted into the membrane of the ER onto which a tree of 14 sugar residues is
grafted: 2 Nacetylglucosamines, 9 mannoses and 3 glucoses. The polysaccharide is
transferred by a glucosyl transferase to the amino acid Asparagine in the polypeptide
chain

Sugar modification: the oligosaccharide tree is immediately modified by RER

enzymes.

Establishment of disulphide bridges: bonds are formed between sulphur amino

acids.

Exit from the ERG via vesicular transport: these are vesicles which bud from the
RER and fuse with the proximal saccule (cis side) of a dictyosome (Golgi apparatus).
These vesicles have a membrane derived from the RER and can therefore transport
luminal proteins and integrated membrane proteins. The intermediate compartment
between the RER and the Golgi apparatus is called the "Endoplasmic Reticulum-
Golgi Intermediate Compartment" or ERGIC. Depending on certain sequences, the
transmembrane or luminal proteins known as "RER resident" (RER specific) cannot
leave the REG. If they have been mistakenly transported into the ERGIC, their return
to the RER is assured.
 Univ of Medea, Cell bio., 1st Y, Boukhalfa

Figure 3 : Protein N-glygosylation in the endoplasmic reticulum

Figure 4 : Vesicular transport of RER proteins to the Golgi Cis

4.2. Functions of Smooth Endoplasmic Reticulum (SER) :

 Biosynthesis of phospholipids : The SER is the main supplier of
phospholipids for cell membranes. Phospholipids are synthesised by
transmembrane enzymes whose active site is directed towards the cytosol,
 Univ of Medea, Cell bio., 1st Y, Boukhalfa

then distributed in the bilayers by flipases. These flipases, integrated into

the LER membrane, switch them from the cytosolic hemimembrane to the
luminal hemimembrane
 Steroid hormone synthesis : The SER synthesises steroid hormones
using cytochrome P450, the active site of which is located on the cytosolic
side of its membrane. Its precursor is pregnenolone, produced by the
tubular cristae mitochondria via cytochrome P450.
 Detoxification : Thanks to the cytochrome P450 of the SER, toxic
products are hydroxylated. These products become water-soluble and are
easily transported and eliminated by the body.
 Accumulation and release of Ca++ : Ca++ crosses the SER membrane
with the help of an ATPase (Ca++ pump). It is stored in the cavity by a
binding protein (Calsequestrin) and then released into the cytosol during
muscle contraction via calcium channels.

b. GOLGI APPARETUS :

1. DEFINITION : is an organelle found in eukaryotic cells. It plays a crucial role in the

modification, sorting, and packaging of proteins and lipids that have been
synthesized in the endoplasmic reticulum.

2. STRUCTURE AND ULTRASTRUCTURE :

 Structure :

Under a light microscope, after impregnation with silver nitrate, the Golgi apparatus
appears as small scales near the nucleus.

 Ultrastructure:

T.E.M : a stack of saccules and small vesicles. Each stack of 4 to 8 saccules is a

dictyosome (on average 20 dictyosomes per cell). Each dictyosome comprises cis
saccules (close to the ER), the input side supplied by the ER, median saccules and
trans saccules (output side) in continuity with a network of canaliculi called the Trans
Golgi Network (TGN).
 Univ of Medea, Cell bio., 1st Y, Boukhalfa

The thickness of the membranes is variable and intermediate between those of the
RER and the plasma membrane (cis saccule 6nm and trans saccule 7.5nm)

Figure 5 : Ultrastructure of golgi apparatus

3. CHEMICAL COMPOSITION :

 Isolation technique:

- Brutal crushing of an organ fragment + UCD gives microsomes in the 3rd pellet.

- Advanced grinding (gentle) + UCD gives saccules stacked in the 3rd pellet.

 Analysis results :
1. Membranes : They contain lipids and proteins. There are proteins common to
the membranes of the endoplasmic reticulum and the Golgi apparatus (Glycosyl-
transferase, Cytochrome b5-reductase) and proteins specific to the Golgi
membranes (Sulpho-transferase, Phosphatase).Glucides : the quantity of sugar
is negligible, they are located on the luminal side.
 Univ of Medea, Cell bio., 1st Y, Boukhalfa

2. Cavity contents: Same products as in the ER cavities, but in different

concentrations

4. FUNCTIONS :

The Golgi apparatus is crucial for processing and distributing biomolecules within the
cell and to the external environment :

1. Transport :

ERGIC delivers proteins, glycoproteins, lipids and glycolipids to the Golgi-cis, which
are then transported to the Golgi-medium and then to the Golgi-trans to reach the
trans-Golgi network (TGN). This transport is carried out by vesicles from the different
compartments.

2. Definitive GLycosylation of proteins :

Modifications to sugars: during the transport of proteins from cis to medial and
then trans saccules, glycoproteins undergo modifications to their N-linked (N-
glycosylated) oligosaccharide trees.

O-Glycosylation: O-linked oligosaccharides (O-glycosylated) are built up in the

medial and trans saccules. These oligosaccharides are built up, ose by ose, on the
protein at the free hydroxyl group (OH) of serine or threonine (amino acids with two
OH groups), thanks to glycosyl-transferases (specific Golgi enzymes).

3. Mannose phosphorylation :

The mannoses of the sugar tree are phosphorylated to mannose 6-P in the cis
cisternae. Only glycoproteins (acid hydrolases) destined for lysosomes undergo this
change.

4. Sulphation : Addition of an SO 2- group by sulpho-transferases in the

cisternae of the Golgi-trans.
5. Calcium storage and release
 Univ of Medea, Cell bio., 1st Y, Boukhalfa

6. Formation of macroautophagy vacuoles :

Although the Golgi apparatus is not the primary site of macroautophagy vacuole
formation, it plays an important role in providing materials and proteins necessary
for autophagosome formation and function.

7. Sorting and maturation :

These two phenomena take place at the same time. Sorting takes place at the level
of the transglobular network (TGN). The vesicles or tubules/canalicles being formed
have a cytoplasmic coat formed by proteins and are called covered vesicles or
covered tubules/canalicles. This coat recognises the signals carried by the substance
to be transported and enables it to be concentrated in a restricted area (sorting). It
also allows the membrane to bud (Figures 6 and 7). The vesicles or
tubules/canalicles can follow two routes:

Secretory pathways : Constitutive secretion pathway: this allows proteins to be

permanently discharged by exocytosis into the extracellular environment (e.g.
secretion of proteins making up the extracellular matrix). Constitutive secretion
material destined for the plasma membrane is transported in tubules/canalicles with a
 Univ of Medea, Cell bio., 1st Y, Boukhalfa

cytosolic coating made up of proteins from the FAPP (Four Phosphate Adaptator
Protein) family.

Regulated secretion pathway: the secretory material, whether hormonal (e.g.

insulin) or exocrine (e.g. pancreatic enzymes), undergoes maturation during transport
in vesicles covered by a coat of clathrin and adaptor proteins. The vesicles increase
in size to form mature secretion grains. This type of secretion takes place after a
signal, as required.

Endosomal, lysosomal pathway : This involves proteins with mannose 6P (M6P),

which has been phosphorylated at the cis-saccule. These luminal proteins recognise
a membrane receptor and the whole is sent to the lysosomes via an intermediate
endosomal compartment characterised by a relatively acid pH (5 to 6). The vesicles,
destabilised by the acidic pH, lose their 6P mannose receptor, which is recycled to
the TGN. The result is a vesicle with no M6P receptor, containing acid hydrolases
and with an acidic pH lumen. Whichever route the vesicles or tubules/canalicles take,
they lose their coat before reaching their destination.

Figure 7 : Covered vesicles or tubules/canalicles involved in permanent vector

membrane flow
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LYSOSOMAL COMPARTMENT

1. Definition : Lysosomes are haloplasmic organelles formed by the association

of hydrolase vesicles from the TGN with degradation compartments,
endosomes, phagosomes and autophagic vacuoles.
2. ULTRASTRUCTURE :

They appear as vesicles of variable shape and size, from 0.05 to 0.5µm in diameter,
depending on the material ingested. They can be identified by cytochemical
techniques thanks to an enzyme present in their membrane, acid phosphatase, which
forms an electron-dense lead phosphate precipitate.

3. Analysis results :

Membrane : is a special membrane because most of these proteins are unusually

highly glycosylated on the luminal side to protect it from the acid hydrolases
contained in the cavity. They are different types :

- Enzymatic (ex : acid phosphatase) and non-enzymatic proteins, permeases,

transporters of small cytosolic materials to be degraded and end products of the
digestion of macromolecules (e.g. amino acids, nucleotides, simple sugars, etc.).

- Proton (H+) ATPases that pump H+ into the cavity and maintain a very acidic pH
in the lysosome.

Contents of the cavity :

The cavity contains around 40 types of acid hydrolases, digestive hydrolytic

enzymes which break down proteins, nucleic acids, oligosaccharides and
phospholipids. Their activity is optimal at pH=5.

4. ORIGIN OF THE MATERIALS TO BE DEGRADED AND BIOGENESIS OF

THE LYSOSOME :

Depending on their origin, the materials to be degraded follow different routes to the
lysosome stage. They can have two origins:
 Univ of Medea, Cell bio., 1st Y, Boukhalfa

Extracellular: heterophagic route In this case, the materials to be degraded are

ingested by endocytosis (phagocytosis, pinocytosis or receptor-mediated
endocytosis), and are contained in phagosomes in the 1st case or in early
endosomes at neutral pH (7.4) in the other two cases. However, their pH gradually
decreases due to the H+ ATPases supplied by the fusion of secretory golgenic
vesicles originating from the TGN and containing a number of acid hydrolases in their
cavity. The early endosomes are then transformed into late endosomes at pH 6.5.
Many of the golgi vesicles flowing from the TGN continue to fuse with the late
endosomes, increasing the number of H+ ATPases and acid hydrolases, allowing the
late endosomes to be converted into lysosomes at pH5. Golgi vesicles with
membranes rich in H+ ATPases and acid hydrolases in their cavity fuse with the
phagosome, which also converts directly into a lysosome at pH5.

Intracellular: autophagic pathways Microautophagy: Direct entry (diffusion) into the

lysosome of small soluble molecules from the cytosol (e.g. peptides) via permeases.

Macroautophagy : Formation of an autophagic vacuole (macro-autophagosome)

formed by the sequestration of spent organelle(s) and a little cytosol by a membrane
cisterna of the TGN. This membrane cistern contains a number of acid hydrolases in
its cavity, which become active following a change in intra-cavity pH, apparently
thanks to the presence of H+ ATPases in the membrane of this cisterna. Digestion
therefore begins in the autophagosome and ends in the lysosome, into which it has
been converted..

Crinophagy : This is a form of autophagy which involves the elimination of grains of

secretion (regulated secretion) which are no longer needed (e.g. prolactin).
 Univ of Medea, Cell bio., 1st Y, Boukhalfa

Figure 8 : pathways by which materials to be degraded enter the lysosome.

5. DESTIN OF LYSOSOMES :

Aged lysosomes, lacking functional acid hydrolases, constitute residual bodies. Most
often, these residual bodies release their contents outside the cell by exocytosis: this
is cellular defecation, but they can also persist in the cell for the rest of their lives.

6. FUNCTION OF LYSOSOMES :

All families of biological molecules are broken down into elementary metabolites,
which are transported back to the cytosol by proteins in the lysosomal membrane.
Lysosomes thus play an essential role in cell digestion, the destruction of foreign
bodies and therefore in cell nutrition