CELL BIOLOGY

Dr. Madani S. 2023/2024

The endomembrane system
The endomembrane system
The endomembrane system is a set of
membranes and various membrane-bound
organelles that are spread in the cytoplasm
of eukaryotic cells, working together to
coordinate some cellular processes like:
protein synthesis, modification, and
transport, lipid synthesis, detoxification, and
waste disposal.

The endomembrane system separates the

cell into different compartments
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
The components of the endomembrane system

The endomembrane system includes :

 Plasma membrane

 Nuclear membrane

 Vacuoles

 Lysosomes

 Golgi apparatus

 Vesicles

 Smooth and Rough endoplasmic

reticulum (ER)
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
 Are mitochondria,
chloroplasts and
peroxisomes part of the
endomembrane system?
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
Are mitochondria, chloroplasts and peroxisomes a part of the
endomembrane system?

No, mitochondria, chloroplasts, and peroxisomes are not considered part of the
endomembrane system. While they are all membrane-bound organelles, they have
distinct origins, functions, and biochemical environments compared to the
endomembrane system.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Endoplasmic Reticulum
The endoplasmic reticulum (ER) is a network of
membrane-bound cavities, shaped as tubules
and flattened sacs (cisternae), forming a
complex system within the cytoplasm.

The ER is divided into two compartments:

Lumen
• Luminal space or Lumen: The interior of the =cavity
ER, enclosed by the ER membrane.

• Extraluminal space: The cytoplasm

surrounding the ER, outside the ER
membrane
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
Endoplasmic Reticulum

There are two types of ER, each with distinct

functions:

 Rough endoplasmic reticulum (RER):

Studded with ribosomes, which are
responsible for protein synthesis. RER plays a
key role in protein secretion.

 Smooth endoplasmic reticulum (SER): Free

from ribosomes. SER is involved in lipid
synthesis, detoxification, and calcium
storage.
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
Ultastructure of the Endoplasmic Reticulum
TEM observations:

The ER membrane appears trilamellar, similar to the plasma membrane, but with a
thinner thickness of approximately 6 nm. This suggests that the ER membrane has a
similar basic structure as the plasma membrane

TEM Micrograph showing the rough endoplasmic reticulum TEM Micrograph showing the Smooth endoplasmic reticulum
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-202
Endoplasmic Reticulum isolation and analysis

Isolation:

The ER is isolated from the third pellet of Density

gradient ultracentrifugation in the form of small
vesicles called microsomes.

These microsomes can be rough or smooth,

depending on the presence of ribosomes.

Sucrose gradient centrifugation is used to separate

rough microsomes from smooth ones based on
their different densities.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Endoplasmic Reticulum isolation and analysis

Isolation:

To detach ribosomes coupled to the ER membrane,

rough microsomes are centrifuged in the presence
of a detergent.

Smooth microsomes are centrifuged in a sucrose

gradient to separate them according to their
densities and origins.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Endoplasmic Reticulum isolation and analysis

Membrane analysis:

Composition: The ER membrane is described as an "asymmetric fluid mosaic," it is

composed of approximately 70% proteins and 30% lipids.

Proteins: The main protein components include glycosyltransferases (involved in protein

glycosylation), P450 cytochromes (involved in detoxification and lipid metabolism), and
glucose 6-phosphatase (involved in glucose metabolism).

Lipids: Phospholipids are arranged in a bilayer structure.

Cholesterol: Cholesterol is present in smaller amounts

Carbohydrates: that are attached to proteins and lipids on the luminal side of the
membrane.
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
Endoplasmic Reticulum isolation and analysis

Content cavity:

Variability: The content of the ER lumen varies depending on the cell type and its
specific functions.

Examples:

• Exocrine pancreas cell: The lumen contains enzymatic proteins destined for secretion.

• Luteal cell: The lumen contains steroid hormones synthesized by the ER.

• Sarcoplasmic reticulum: The lumen of this specialized ER stores calcium ions (Ca++)
for muscle contraction.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Protein synthesis and targeting: Palade experiment

Using pancreatic tissue from rats,

Palade employed a radioactive
labeling technique to trace the
path taken by newly synthesized
proteins destined for the
extracellular environment. This
technique is called Pulse-chase
technique

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Protein synthesis and targeting : Palade experiment

Pulse: Cells are incubated with a

medium containing labeled amino
acids

Chase: after the pulse phase, the

labeled precursors are removed,
and the cells are incubated in a
non-radioactive medium for
various time intervals.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Rough Endoplasmic Reticulum Function: Palade experiment

Palade's experiment revealed the

following key findings:

Proteins destined for secretion are

synthesized on the RER.

They are then packaged into

transport vesicles and transported
to the Golgi apparatus.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Protein synthesis and targeting : Palade experiment

Within the Golgi, proteins are

modified and sorted into specific
secretory vesicles.

Finally, these vesicles fuse with the

plasma membrane and release
their contents via exocytosis.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Protein synthesis

Step 1: Transcription

• Transcription occurs in the nucleus of the cell.

• The enzyme RNA polymerase binds to the DNA promoter region of a gene.

• RNA polymerase unwinds the DNA double helix and reads the template strand, making
a complementary RNA molecule called messenger RNA (mRNA).

• The mRNA molecule is processed and spliced to remove introns (non-coding sequences)
and leave only exons (coding sequences).

• The mature mRNA molecule then exits the nucleus through nuclear pores and enters
the cytoplasm.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Protein targeting

There are different classes of ribosomes that assist in translation.

The free poly-ribosomes directly release the growing peptide chain into the cytoplasm
after the complete protein has been synthesized upon the termination.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Protein targeting

Many proteins, after being synthesized on ribosomes in the cytosol, need to be transported
across a membrane to reach their final destinations within the cell. This process, known as
protein translocation, is essential for diverse cellular functions.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Protein targeting

Whereas the membrane-bound polyribosomes do not release the protein chain directly
into the cytoplasm, instead they divert the growing polypeptide chain into the lumen of
ER for further post-translational modifications. In this case translation and translocation
happen simultaneously

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Rough Endoplasmic Reticulum Function: Lumen protein translocation

Steps of Protein Translocation into the ER

Lumen:

Step 1: Signal Sequence Recognition

The N-terminal signal sequence of the

nascent polypeptide chain emerging from the
ribosome is recognized by the Signal
Recognition Particle (SRP) in the cytosol.

The SRP binds to the signal sequence and the

ribosome, causing a temporary pause in
translation.
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
Rough Endoplasmic Reticulum Function: Lumen protein translocation

Step 2: SRP-mediated Targeting

The SRP-ribosome-mRNA complex is then

directed to the ER membrane by interacting
with the SRP receptor on the cytosolic side of
the membrane.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Rough Endoplasmic Reticulum Function: Lumen protein translocation

Step 3: Translocation Initiation

The ribosome binds to the translocon, a

protein complex embedded in the ER
membrane that forms a channel for protein
translocation.

The translocon opens a hydrophilic channel,

allowing the nascent polypeptide chain to
pass through while translation resumes.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Rough Endoplasmic Reticulum Function: Lumen protein translocation

Step 4: Signal Sequence Cleavage

As the protein enters the ER lumen, the signal

sequence is cleaved by a signal peptidase
located on the luminal side of the ER
membrane.

This cleavage releases the mature protein

into the ER lumen.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Rough Endoplasmic Reticulum Function: Lumen protein translocation

Step 5: Chaperone Binding and Protein

Folding

Chaperone proteins in the ER lumen bind to

the translocated protein, facilitating its proper
folding and preventing aggregation.

These chaperones may also assist in post-

translational modifications, such as disulfide
bond formation and glycosylation.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Rough Endoplasmic Reticulum Function: Membrane protein translocation

Membrane Protein Translocation Steps:

While protein translocation into the ER lumen follows
similar steps as described previously, the process for
membrane proteins involves additional steps for their
insertion into the membrane:
1. Signal Sequence Recognition and Targeting:
Similar to luminal proteins, the N-terminal signal
sequence of the nascent polypeptide chain is
recognized by the SRP. The SRP-ribosome-mRNA
complex is then targeted to the ER membrane
through the SRP receptor. CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
Rough Endoplasmic Reticulum Function: Membrane protein translocation

2. Translocation Initiation and Hydrophobic

Segment Insertion:
The ribosome binds to the translocon complex on the
ER membrane.
As the protein is translated, a hydrophobic segment
within the sequence interacts with the translocon,
initiating its insertion into the ER membrane.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Rough Endoplasmic Reticulum Function: Membrane protein translocation

3. Signal Sequence Cleavage and Stop-Transfer

Sequence Recognition:
While the N-terminal signal sequence is cleaved by a
signal peptidase, a stop-transfer sequence within
the protein is recognized by the translocon.
This stop-transfer sequence determines the length
of the protein inserted into the membrane and the
orientation of the protein's N- and C-termini.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Rough Endoplasmic Reticulum Function: Membrane protein translocation

4. Transmembrane Domain Insertion and Protein

Folding:
Depending on the location of the stop-transfer
sequence, the remaining hydrophobic segments of
the protein are inserted into the ER membrane,
forming transmembrane domains.
Chaperones in the ER lumen assist the protein in
folding and ensuring proper integration into the
membrane.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Rough Endoplasmic Reticulum Function
Post-translational Modifications of Proteins:
1. N-Glycosylation:
It involves the addition of a complex carbohydrate chain, called an oligosaccharide, to
specific asparagine (N) residues within the protein sequence that is translated. The
transfer of the oligosaccharide is done by an oligosaccharidyl transferase enzyme. This
glycosylation plays crucial roles in protein folding, stability, targeting, and interactions with
other molecules.
2. Changes in Carbohydrates :
The ER enzymes immediately modify the initial oligosaccharide tree, adding or removing
specific sugar residues. This further diversifies the glycan structure and its functional
properties.
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
Rough Endoplasmic Reticulum Function
Post-translational Modifications of Proteins:
3. Establishment of disulfide bridges:
Formation of covalent bonds between the sulfur atoms of cysteine residues occurs within
the ER, contributing to protein folding and stability.
4. Association between proteins and membrane lipids:
For integral membrane proteins, specific hydrophobic segments interact with the
phospholipid bilayer, anchoring them within the ER membrane.
This association plays a vital role in protein localization, function, and signal transduction.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Rough Endoplasmic Reticulum Function
Post-translational Modifications of Proteins:
5. Exit from the ER by vesicular transport:
Proteins destined for other cellular
compartments are packaged into transport
vesicles that bud from the ER membrane.
These vesicles then fuse with the Golgi apparatus
for further processing and sorting.
The ER-Golgi intermediate compartment (ERGIC)
acts as a sorting station, ensuring proper
trafficking of proteins.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Smooth Endoplasmic Reticulum Function
1. Synthesis of phospholipids:
The SER is the primary site of phospholipid synthesis, which are essential building blocks of
cellular membranes.
Transmembrane enzymes embedded in the SER membrane are responsible for this process,
with their active sites facing the cytosol.
Flipases, also integrated into the SER membrane, facilitate the transport of newly
synthesized phospholipids from the cytosolic leaflet to the luminal leaflet of the
membrane.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Smooth Endoplasmic Reticulum Function
2. Synthesis of steroid hormones:
The SER plays a crucial role in synthesizing steroid hormones like cortisol, estrogen, and
testosterone in specific cell types like the adrenal glands and gonads.
P450 cytochrome enzymes, with their active sites facing the cytosol, utilize cholesterol as a
precursor molecule within the SER to generate steroid hormones.
3. Detoxification:
The SER contributes to detoxification by metabolizing harmful substances like drugs,
toxins, and metabolic byproducts.
P450 cytochrome enzymes within the SER hydroxylate these harmful substances, making
them water-soluble for easier transport and elimination by the organism.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Smooth Endoplasmic Reticulum Function
4. Accumulation and release of Ca++:
The SER acts as a reservoir for storing calcium ions (Ca2+), essential for various cellular
processes.
Ca2+ is actively transported into the SER lumen by a Ca2+-ATPase pump against its
concentration gradient.
Calsequestrin, a binding protein within the SER lumen, stores Ca2+.
The release of Ca2+ back into the cytosol is controlled via calcium channels, playing a
critical role in muscle contraction and other Ca2+-dependent processes.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus

The Golgi apparatus, also known as the Golgi

complex, is a collection of flattened,
membrane-enclosed sacs (cisternae) and
associated vesicles. It resides in the
cytoplasm of eukaryotic cells, usually located
near the nucleus and the centrosome. Golgi apparatus

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus
Using the silver nitrate stain, the Golgi apparatus becomes visible under a light
microscope as clusters of dark, irregularly shaped "scales" scattered near the nucleus.

Golgi
apparatus
Golgi apparatus Golgi apparatus

Nucleus

Golgi apparatus
Golgi apparatus

Micrographs showing the golgi apparatus of nerve cells highlighted with silver nitrate stain , observed with
the light microscope
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
The Golgi apparatus
When viewed in the transmission electron microscope, the Golgi apparatus appears as
stacks of smooth, flat membranous sacs, known as cisternae, which are slightly curved
and , dilated at the periphery, surrounded by vesicles of various sizes.
Dictyosomes: Each stack of 4 to 8 cisternae is referred to as a dictyosome. There are
typically around 20 of these in a single cell.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus
Within each dictyosome, the cisternae are functionally distinct:
 Cis saccules: Situated closest to the endoplasmic reticulum, these
receive newly synthesized proteins.
 Median saccules: These intermediate cisternae perform
modifications and processing of the proteins.
 Trans saccules: Located furthest from the ER, these saccules
package and sort the processed proteins for their final destinations.
 Trans-Golgi Network: The trans face of the Golgi apparatus
connects to a network of tubules and vesicles called the TGN. This
serves as a sorting and distribution center for the finished proteins
and lipids.
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
The Golgi apparatus

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus ultrastructure
• Tripartite structure: the Golgi membrane appears tripartite in transmission electron
microscopy (TEM): outer leaflet membrane is smooth, while the inner leaflet
membrane is rough with dense proteins.
• Globular particles: Scanning electron microscopy (SEM) reveals the presence of
integrated globular particles on the Golgi membrane. These are likely protein
complexes with specific roles in protein processing and sorting, adding another layer of
complexity beyond simply embedded and peripheral proteins.
• The thickness of the membranes is variable and intermediate between those of the
GER (or RER) and the plasma membrane (cis saccule: 6nm and trans saccule: 7,5nm).

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus isolation and analysis

Isolation:

The Golgi apparatus is isolated from the third pellet

of Density gradient ultracentrifugation in the form
of small vesicles called microsomes.

 Rough grinding : gives microsomes in the third

pellet.

 Gentle grinding: gives stacked saccules in the

third pellet.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus: membrane composition
Lipid Composition:
Intermediate characteristics: The Golgi membrane's lipid content, chain length, saturation,
and fluidity fall between the ER and the plasma membrane. This allows the Golgi to
maintain a balance between flexibility for protein processing and structural stability for
efficient transport.
The intermediate fluidity might facilitate controlled movement and interaction of proteins
within the Golgi while still providing a sturdy platform for processing and sorting.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus: membrane composition
Protein Composition:
Intermediate protein levels: Similar to lipids, protein levels in the Golgi membrane are
intermediate between the ER and the plasma membrane. This reflects the Golgi's role in
both receiving and exporting proteins.
Two protein categories:
Common proteins: Shared with the ER, including enzymes like glycosyl-transferase and
cytochrome b5-reductase, involved in basic protein processing functions.
Golgi-specific proteins: Include sulfo-transferases and phosphatases, essential for Golgi-
specific modifications like sulfation and dephosphorylation, crucial for protein sorting and
trafficking.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus: membrane composition
Carbohydrate Composition:
Low carbohydrate content: Sugars are present mainly on the luminal surface and in
negligible amounts compared to the other components.
Possible role: These surface sugars likely play a role in protein glycosylation, targeting, and
interaction within the Golgi lumen.
Molecular Architecture:
Asymmetric fluid mosaics: Like other biological membranes, the Golgi membrane is a
mosaic of lipids and proteins with varying distributions and functions, contributing to its
asymmetric nature and dynamic fluidity.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus: Cavity composition
Cavity Contents:
Similar products to the ER: The Golgi lumen contains similar molecules as the ER cavity,
but in different concentrations. This reflects the selective transport and modification of
proteins and lipids during their transit through the Golgi.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus: Function

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). Vesicles from the different compartments
carry out this transport.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus: Function
2. Definitive protein glycosylation
Carbohydrate modifications: during protein transport
from the cis saccules to the medial and then trans
saccules, the glycoproteins undergo modifications to
their N-linked oligosaccharide trees (N-Glycosylated).
O-Glycosylation: it happens in the medial and trans
saccules. O-linked glycosylation typically includes the
addition of sugar residue at the free hydroxyl group
(OH) of serine or threonine (amino acids with two OH
groups), it is mediated by the glycosyl transferases
(specific Golgi enzymes).
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
The Golgi apparatus: Function
3. Mannose phosphorylation:
Certain sugar molecules (mannose) on the N-linked
sugar chains of some proteins get a phosphate
group added (phosphorylated) becaming mannose
6-phosphate (Man-6-P)
This modification happens in the "cis cisternae",
the first compartment of the Golgi apparatus.
Only specific proteins, called acid hydrolases
destined for lysosomes, undergo this change.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus: Function
4. Sulfation: specific proteins undergo Sulfation, the
addition of sulfate (SO42-) groups, by sulfotransferases in
the trans Golgi cisternae. This modification can influence
protein function, interactions, and stability.
5. Glycolipid Formation: Some lipids synthesized in the
smooth endoplasmic reticulum (SER) travel to the Golgi
apparatus for further processing. Within the Golgi,
specific enzymes called glycosyltransferases add sugar
molecules (primarily galactose) to these lipids, forming
glycolipids. There are different types of glycolipids,
depending on the specific lipid and sugar combination.
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
The Golgi apparatus: Function
6. Calcium storage and release
7. Autophagosome formation: the Golgi,
particularly its Trans-Golgi Network (TGN),
might contribute membranes for
autophagosome formation. Cisternae from
the TGN could detach and envelop targeted
organelles. Autophagy, the cellular recycling
process.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus: Function
8.Protein sorting and maturation
These two phenomena occur simultaneously within
the Golgi apparatus, with sorting primarily happening
in the Trans-Golgi Network (TGN). Forming vesicles at
the TGN have a protein-based cytoplasmic coat,
named "coated vesicles." This coat recognizes
specific "address tags" on the transported
substances, concentrating them in a designated area
within the budding vesicle. This sorting process,
facilitated by the coat, simultaneously allows the
membrane to bud into mature vesicles.
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
The Golgi apparatus

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus: secretion pathways
1. Constitutive Secretion Pathway:
• Function: Continuous delivery of essential proteins to the extracellular environment.
• Cargo: Structural proteins (collagen, fibronectin), enzymes, etc.
• Sorting: No specific signals, simple and consistent glycosylation.
• Vesicles: Various types of coats (including coatomers) or uncoated.
• Origin and destination: TGN to plasma membrane for exocytosis.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus: secretion pathways
2. Regulated secretion pathway:
• Function: Controlled release of specific proteins in response to signals.
• Cargo: Examples like insulin and pancreatic enzymes illustrate different types.
• Sorting: Specific "address tags" differentiate from constitutive cargo.
• Maturation: Protein modifications, concentration, and formation of large granules.
• Stimulus and release: Triggered by specific signals for precise delivery.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Golgi apparatus: secretion pathways
3. Endosomal and Lysosomal Pathways: The Mannose-6-Phosphate Route
This pathway directs proteins destined for lysosomal degradation, marked with
mannose-6-phosphate (M6P) added in the cis Golgi, to their final destination. The M6P
tag acts as a recognition signal for the M6P receptor, which packages these proteins into
coated vesicles. These vesicles are transported to late endosomes with an acidic pH (5-
6). This acidity triggers dissociation of the M6P tag from the receptor, which recycles
back to the TGN for further use. The M6P-stripped vesicles, now containing lysosomal
enzymes and other proteins, lose their coats and merge with lysosomes for protein
degradation.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Endosomes
Early Endosomes:
₋ The main sorting machine of the endocytic
pathway
₋ Relatively small
₋ Often located in the periphery of the
cytoplasm near the plasma membrane
₋ Tubular and vacuolar in composition
₋ Weakly acidic (pH 5.9-6.8)
₋ Low Ca2+ concentration
₋ Lack acid hydrolases

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

Endosomes
Late Endosomes:
₋ May fuse with lysosomes to degrade cargo
₋ Round or oval-shaped
₋ Shuttle between peripheral cytoplasm and
perinuclear area
₋ Lumen often contains variously sized vesicles
₋ More acidic than early endosomes (pH 4.9-6.0)
₋ Increased Ca2+ concentration compared to early
endosomes
₋ Replete with acid hydrolases

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Lysosome
Lysosomes are membrane-enclosed organelles that contain
an array of enzymes capable of breaking down all types of
biological molecules or cell debris
Ultrastructure:
Lysosomes appear as spherical or elliptical vesicles with
diverse sizes ranging from 0.05 to 0.5 micrometers in
diameter, depending on the ingested material. Their unique
membrane, containing proton pumps and resistant to their
own enzymes, protects the cell from the potent digestive
activity within. Cytochemical techniques using enzymes like Micrographs showing the Lysosome
under transmission electron microscope
acid phosphatase help identify these vital organelles.
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
The Lysosome: membrane composition
Like other membranes, lysosomes have a basic phospholipid bilayer structure,
providing a barrier and maintaining selective permeability.
Proteins:
Proton pumps: These ATP-powered pumps maintain an acidic (around pH 5)
environment within the lysosome lumen, which is essential for optimal enzyme activity.
Lysosomal membrane proteins: These glycoproteins serve diverse functions, including
mediating lysosomal movement, fusion with other organelles, and interaction with
cytosolic proteins.
Mannose-6-phosphate receptors (M6PRs): These receptors recognize molecules
labeled with mannose-6-phosphate tags and target them for lysosomal degradation.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Lysosomes : cavity content

It contains around 40 types of acid

hydrolases, which degrade proteins,
nucleic acids, oligosaccharides, and
phospholipids. These enzymes function
optimally in an acidic environment
around pH 5.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Lysosome
Extracellular materials for degradation enter the cell through various endocytic pathways
like phagocytosis (ingesting bacteria), pinocytosis (small molecules), or receptor-mediated
endocytosis. These materials initially reside in phagosomes or early endosomes with a
neutral pH. However, fusion with Golgi-derived vesicles containing H+ ATPases and acid
hydrolases gradually acidifies these compartments. Early endosomes transform into late
endosomes (pH 6.5) and eventually mature into lysosomes (pH 5) through continued
fusion with TGN vesicles. Notably, phagosomes can directly convert into lysosomes by
fusing with Golgi vesicles rich in lysosomal components.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Lysosome
Microautophagy:
Degrades small molecules (peptides, amino acids)
directly through lysosome channels (permeases).
Fast and energy-efficient.
Macroautophagy:
Encases large waste (organelles, proteins) in
double-membraned vesicles (autophagosomes).
Autophagosomes contain pre-packaged acid
hydrolases for initial digestion.
Fuses with lysosomes for complete breakdown and
resource recovery.
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
The Lysosome

Crinophagy:
Targets specific waste - unused/outdated
secretory granules (e.g., prolactin).
Granules have markers that guide them
directly to lysosomes for degradation.
Maintains efficient disposal of
specialized waste products

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Plant cell vacuole

Vacuoles are large, membrane-bound sacs found in

plant cells. They are the largest organelle in a plant
cell, often occupying up to 90% of the cell volume.
Function:
• Maintains turgor pressure (internal pressure) that
keeps the plant cell rigid and upright.
• Stores nutrients, waste products, and pigments.
• Helps in cell expansion and growth.
• Aids in plant defense against pathogens.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Plant cell vacuole

Structure:
• The vacuole is bound by a single membrane
called the tonoplast.
• Filled with a watery fluid called cell sap
containing various solutes, sugars, ions, and
organic molecules.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024

The Plant cell vacuole
Ultrastructure:
Transmission Electron Microscope (TEM):
• Trilamellar structure: This confirms the tonoplast is a typical cell membrane, consisting
of two dark leaflets (dense layers) sandwiching a lighter middle layer.
• Asymmetrical layers: The different thicknesses of the cytosolic and luminal dense
layers suggest variations in protein and lipid composition on each side of the
membrane.
• Fibrous coating: This could be a layer of glycoproteins or polysaccharides associated
with the luminal side of the tonoplast.
Scanning Electron Microscope (SEM):
• Globular particles are highlighted after freeze-fracture
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
The Plant cell vacuole
Isolation:
1. Plasmolysis and cell wall digestion: Shrink and weaken cells, then digest cell walls to
access the protoplast.
2. Homogenization and centrifugation: Break down cell content and separate lighter
protoplasts from heavier debris.
3. Protoplast isolation and stress treatment: Extract protoplasts and stabilize their
membranes, inducing vacuole separation.
4. Vacuole isolation and bursting: Collect vacuoles and break them open to release cell
sap.
5. Gradient density ultracentrifugation and fractionation: Separate the vacuole lysate
into denser tonoplast membranes and lighter cell sap.
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
The Plant cell vacuole
Composition
Tonoplast: the specific types of phospholipids and sterols present in the tonoplast can
vary depending on the plant species and function. Some have roles in maintaining
membrane fluidity and integrity, while others might be involved in specific transport
processes.
The diverse protein population of the tonoplast includes:
Proton pumps: Actively transport protons into the vacuole, creating an acidic
environment.
Channel proteins: Facilitate the selective transport of ions and other solutes across the
membrane.
Enzymes: Involved in various processes like storage molecule breakdown, detoxification,
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
The Plant cell vacuole
Composition
Tonoplast:
The fibrous carbohydrate coating on the luminal side of the tonoplast is thought to play
roles in cell wall biosynthesis, solute accumulation, and interactions with other organelles.
Cell Sap:
Nutrients: Sugars, amino acids, and organic acids.
Waste products: Pigments, secondary metabolites, and breakdown products of cellular
processes.
Defense compounds: Toxins and other chemicals that deter herbivores and pathogens.
Signaling molecules: Involved in plant communication and responses to environmental
stimuli.
CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024
The Plant cell vacuole: Transport mechanisms across the tonoplast.

CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024