LYSOSOME

Functions of lysosomes
1
2
3
3. Controlled
Uptake of
nutrients
1. Digestive
2. Autophagic
1. Digestive
Optimal pH for function is low (pH 4.6 - 5.0)
H
+
-ATPase activity (1001000 times cytoplasm acidity)
Glycosylated interior (inner leaflet) protects
compartment from pH damage
Enriched with ~40 types of hydrolytic (degradative)
enzymes

2. Controlled uptake regulator
Endocytic particles (or bacteria) form endosomes which
are routed to the lysosome for degradation.
Some bacteria target and happily live in endosomes eg. Coxiella
burnetti (Q fever)

3. Autophagic (micro/macro types)
Organelle (macro) and ribosome (micro) turnover is
essential to remove damaged or malfunctioning cell
components (eg. mitochondria or chloroplasts)
Digestive
enzymes
Lysosome
Food vacuole
Plasma membrane
Digestion
Lysosome
Vesicle containing
damaged mitochondrion
Digestion
Mannose-6-phosphate (M6P) is added onto lysosomal
proteins in the cis-Golgi (two step reaction) permits their
identification later.

M6P is recognized by the M6P receptor (MPR) in the
TGN which sorts these proteins away from secreted
protein
Patients with I- cell disease are deficient in the enzymes that
convert mannose to M6P, or lack proper M6P receptors results
in lysosomes filled with undegraded cell structures/molecules

At TGN, lysosomal proteins are packaged into clathrin-
coated vesicles for transport to the lysosome
Mechanism for sorting lysosomal proteins
Lysosomal sorting using
clathrin coated vesicles
(CCV)
1
2
3 4
Cyto
TGN
Endocytosis involves the uptake of proteins and
other macromolecules at the plasma membrane.

Bulk materials are taken up by the cell in two
ways:
Within the membrane Proteins are concentrated
during uptake (receptor mediated endocytosis).
Within the fluid phase No increase in the
concentration of the molecules
Pinocytosis (cell drinking)
Phagocytosis (cell eating)
Lysosomes in endocytosis
Pinosomes are generated by the process of pinocytosis (cell
drinking).
No pseudopod formation plasma membrane pinching using
receptors and COP like proteins in the coated pits
Pinch sites
Pinososome
Endocytosis: Pinocytosis
Rab proteins
Fusion to early endosomes
Phagolysosome (low pH)
Phagosomes are generated by the process of phagocytosis
(cell eating).
Uptake of larger particles, dead cells and bacteria
Endocytosis: Phagocytosis
1) Internalize nutrients:
- Low density lipoprotein (LDL) (cholesterol)
- Fe
3+
TRANSFERRIN

2) Internalize molecules for storage:
- Vitellogenin synthesis in liver is transported via
blood and taken up by oocytes that have vitellogenin
receptors.

3) Removal of surface receptors:
Down-regulation" of receptors after stimulus.
Lysosomes in endocytosis:
Receptor-mediated endocytosis
4) Movement of proteins
across an epithelial layer
(transcytosis):
- Immunoglobulin G
(IgG) secretion in milk;
uptake in the gut of the
newborn (passive
immunity).
- Immunoglobulin
transport across
epithelium of gut

Lysosomes in endocytosis:
Receptor-mediated endocytosis





















Luminal
membrane
Basal
membrane
Epithelial cell Intestinal
Lumen
Blood or
Interstitial
fluid
Tight junction
IgG
F
c
region
endosome
Fc receptor
Epithelial cell

Many of the events in receptor mediated
endocytosis are similar to vesicle transport in the
secretory pathway.

Specific receptors are clustered together at sites
on the plasma membrane by binding to the coat
protein clathrin.
The cytoplasmic portion of receptors provide sites/
regions that recognize and determine which receptors
to internalize
1
Endocytosis step # 1: Formation of coated pits
Coat is formed from clathrin.
Three heavy chains and 3
light chains are assembled
into a triskelion.
Triskelions are assembled
into a basket-like structure on
the cytoplasmic face of the
vesicle.
Adaptor proteins connect
the cytoplasmic side of
receptors to clathrin.
2
3
1
Endocytosis step # 2: Coat assembly continues
until vesicle is formed and released into cytoplasm
Clathrin coated vesicles
used for both receptor-
mediated endocytosis
and for vesicle transport
from TGN to lysosome.
The adaptor proteins for
TGN are different from
those for plasma
membrane.


- Low pH of the endosome
releases ligand (cargo) from
the plasma membrane
receptor.
- Ligand/endosomes moves on
to fuse with the lysosome.
- Some membrane receptors
recycle back to plasma
membrane.
3
4
Endocytosis steps # 3 and 4: Uncoating and fusion
of the vesicles with endosomes
Example: Cholesterol uptake

Cholesterol is carried with apo-B
protein as LDL particle.
LDL receptor internalizes LDL.
Familial hyper-cholesterolemia
leads to elevated blood
cholesterol:
Mutations to LDLR gene (encodes
the LDL receptor)
Mutations to apoB gene
Tf transferrin
TfR transferrin receptor
Example: Iron uptake
Iron is released from transferrin
in endosome
Peroxisomes
Peroxisomes can be formed either
by de novo synthesis or
growth/division.

Membrane bound: proteins within it
have homology to proteins of the ER
Enriched with oxidative enzymes

Specialized organelles that function
in two ways:
a and b-oxidation breaks down fatty
acid chains
- Toxic oxygen species formation/
breakdown
Peroxisomes
Urate oxidase is not found in
humans uric acid can build
up leading to the illness called
gout.
Peroxisomes have 32
unique proteins called PEX
that function as:
- Protein import machinery
- Enzymes
- Receptors
VACUOLE
Vacuoles: various maintenance functions
Vacuoles are large vesicles that have a variety of functions.
Some protists have contractile vacuoles that help to
eliminate water from the protist.
In plants, functions of vacuole include: digestion, osmotic
regulation, storage of pigments and/or defensive
compounds.

Contractile
vacuoles
Nucleus
Central vacuole
Chloroplast
Nucleus
Vacuoles are a
predominant feature of
plant cells.
Lysosomes are
predominately found in
animal cells and rarely in
plant cells.

Vacuoles are membrane
bound compartments that
have similar functions as
lysosomes. They contain:
Many acid hydrolases
V-type H
+
-ATPases
Vacuoles can occupy as much
as 90% of the volume of many
plant cells.
http://amit1b.wordpress.com/10-the-living-cell-gallery/
1. Defensive
Toxic chemical repository
cyanide containing
glycosides
glucosinolates
2. Vacuoles are storage
units
Stores solutes and
macromolecules such as
ions, sugars, amino acids,
proteins, and
polysaccharides, organic
acids
Vacuole
Golgi
ER
chloroplast
mitochondria
Functions of vacuoles in plant cells
3. Intracellular
digestion
pH ~ 25
Organic acids assist
in maintaining low pH

The vacuolar
membrane is
referred to as the
tonoplast
Specific proteins:
Tonoplast intrinsic
proteins (TIP)
Vacuole
Turgor pressure
[Solute]
H
2
O
H
2
O
Plant cells have high
osmotic pressure
The Mitochondrion:
Aerobic Respiration
Aerobic respiration
Aerobic respiration is a biological/metabolic process
where molecular oxygen (O
2
) is used to generate
energy in the form of ATP by breaking down a carbon
source
In most cases, lipids (fatty acids), carbohydrates, and
proteins are converted to pyruvate.
Pyruvate is broken down into CO
2
and water (H
2
O) by
the tri-carboxylic acid (Krebs) cycle (TCA).
The process generates electrons (e
-
) and NADH used
to drive the electron transport chain and create a proton
gradient used to synthesize ATP.

C
6
H
12
O
6
+ O
2
6 CO
2
+ 6 H
2
O + energy
Aerobic respiration
Glucose
O
2
present
Pyruvate
Glycolysis
Fermentation
Lactate
NADH
NAD
+
NAD
+
NADH
O
Plasma membrane
NAD
+
2ATP
O
2
absent
Pyruvate
Acetyl-CoA
TCA
Cycle
3NADH, 2FADH
2
Electron transport chain
36ATP
Cytoplasm
NAD
+
NADH
CO
2
5 e
-
pairs
OH
OH
OH
OH
2CO
2
+ H
2
O
O
O
O
-
(Ethanol)
CH
2
OH
HC
CH
2
OPO
3
2-
OH
Glycerol 3-P
H
3
C
COO
-
Fatty acid
Fermentation is useful for aerobic respiration replenishes NAD
+
supply
Mitochondria can self-replicate by fission.

Functions:
1. Synthesis of ATP via the oxidation of
pyruvate: most ATP is produced by
oxidative phosphorylation.
- The more energy a cell needs the
more mitochondria (skeletal muscle)
- Mitochondria are localized near sites
where ATP requirement is greatest
Example: near baso-lateral surface
of gut epithelium, where Na
+
/K
+

ATPase activity is highest
2. -oxidation of fatty acids (peroxisomes
also participate in this).
3. Apoptosis (programmed cell death).
Controlled
Cell death
3
b-
oxidation
TCA
cycle
Acetyl-CoA
Acyl-CoA
2
1
ATP
Mitochondria
Structure of mitochondria
Mitochondrial outer membrane is distinct from the inner
membrane.
The outer membrane is permeable to small molecules.
Enriched with porins that form non-specific channels in
outer membrane (permeable to molecules < 10 kDa)
Outer membrane
Intermembrane space
lumen
Cyto side
Intermembrane side
Matrix
H
+
ADP + Pi
ATP
MITOCHONDRIAL PORIN
Structure of mitochondria: Outer membrane
Mitochondria inner membrane:
Contains high abundance of cardiolipin make membrane
less permeable to protons.



Contains high abundance of protein (approx. 75% of mass):
electron transport chain (ETC) complexes and ATP
synthases.
Is highly convoluted joins to double layered membrane
sheets called as cristae.
Is highly impermeable.

Both the outer and inner membranes have distinct protein import
system.
Structure of mitochondria: Inner membrane

Metabolite enriched: TCA cycle intermediates
Soluble electron carriers eg. cytochrome c (cyt. c)
High proton concentration: slightly acidified space (pH ~56)

Intermembrane space
Matrix
H
+
ADP + Pi ATP
Cyt.c
H
+
H
+
H
+
H
+
H
+
H
+
H
+
H
+
H
+
H
+
H
+
H
+
ATP
H
+
H
+
H
+
ADP
Inner
Membrane
(IM)
Outer
Membrane
(OM)
Structure of mitochondria: Intermembrane space

Mitochondrial DNA (mtDNA),
RNA, ribosomes:
Self-replicating and maternally
inherited.
Can be circular or linear

Protein: very high abundance
Pyruvate and fatty acid
oxidations
TCA/Krebs cycle
Mostly encoded by nuclear
genome.



Structure of mitochondria: Matrix
NADH
NADH FADH
2

ATP ATP
ATP
CYTOPLASM
Glycolysis
Electrons
carried by NADH
Glucose Pyruvate
Pyruvate
Oxidation
Citric Acid
Cycle
Oxidative
Phosphorylation
(electron transport
and chemiosmosis)
Mitochondrion
Substrate-level
phosphorylation
Substrate-level
phosphorylation
Oxidative
phosphorylation
ATP production
Major energy reserves in cells:
polysaccharides and fats



Starch granules
in potato tuber cells
Glycogen granules
in muscle
tissue
Glycogen
Glucose
monomer
Starch
Cellulose
Hydrogen bonds
Cellulose
molecules
Cellulose microfibrils
in a plant cell wall
Fatty acids
Glycerol
Fat (triglyceride)
Glycolysis: break-down of glucose
to pyruvate
Each glucose produces: 2 NADH + 2
ATP

Lipolysis:
Triglycerides fatty acids
Glycerol-3-phosphate shuttle:
triglycerides glycerol glycerol-3-
phosphate
(In mitochondria: glycerol-3-phosphate
dihydroxyacetone-3-phosphate to
make FADH
2
)
Making ATPs Step 1:
Breaking down energy sources in the cytoplasm

Making ATPs Steps 2:
Acetyl-CoA production and TCA cycle
Fatty acids and pyruvate are
transported into mitochondria via
permeases.
Oxidation:
Pyruvate acetyl-CoA
Fatty acids acetyl-CoA (-
oxidation)
Fatty acids produce much more
acetyl-CoA than pyruvate
produces.
TCA cycle:
Each acetyl-CoA produces: 3
NADH + 1 FADH
2
+ 1 ATP
NADH and FADH
2
are used by the
elctron transport chain (ETC) for
oxidative phosphorylation.

Electron transport chain
(ETC):
Is composed of 4
complexes
Transfers electrons from
TCA cycle reactions to
terminal electron
acceptors.

O
2
+ 2H
+
H
2
O

Making ATPs Steps 3:
Oxidative phosphorylation
Electron transport chain
Electrons are donated to complex I, pass through complex I
(coupled to transport of H
+
across membrane).
Electrons are carried by ubiquinone (UQ) to complex III; pass
through complex III (coupled to transport of H
+
).
Electrons are carried by cytochrome c to complex IV.
Electrons pass through complex IV and are donated to O
2
this
is coupled to transport of H
+
across membrane
Electron donors: NADPH
Complex II is also part of the TCA cycle: producing FADH
2
.
FADH
2
donates electrons to ubiquinone carrier complex III
complex IV (coupled with transporting H
+
).
Complex II does not transport H
+
.
Electron donors: FADH
2


Coupling proton movement across mitochondrial inner
membrane to electron transport results in chemiosmotic
gradient across inner membrane.

Electrochemical gradient combines both a pH gradient and
voltage difference across inner membrane (proton motive
force).
DpH
H
+
H
+
Chemiosmotic gradient of proton
ATP synthase (F
0
-F
1

ATPase) utilizes the H
+

gradient to synthesize ATP.
H
+
move down the
concentration gradient
and pulled by electrical
gradient across inner
membrane through F
0
F
1

ATP synthase.

The F
0
domain (membrane
integral) form the H
+
channel.

The F
1
domain (peripheral on
the matrix side of the inner
membrane) synthesizes ATP
by rotational catalysis.

ATP synthase utilizes proton motive force
Structure of ATP synthase
Major subunits:
a/b
g,
- c
d

F
0
F
1
1 ATP molecule is produced for every 3 to 4 H
+
moving through ATP
synthase
3 ATP molecules for each 360
o
degree rotation of g subunit
Letters indicated refer to: O = open, L = loose, T = tight conformations of the F
1
subunit
Rotational catalysis by ATP synthase

The ATP and
chemiosmotic
gradient is used in the
transport of other
molecules across the
inner membrane by
antiport transporter
activities.
CHLOROPLASTS: Photosynthesis
Sites of photosynthesis in plants
conversion of photon (light)
energy into chemical energy
(ATP and NADPH).
Sites of conversion of CO
2
to
sugars at expense of ATP and
NADPH (CO
2
fixation also
known as the Calvin-Benson
cycle).

Site of synthesis and assembly of
some chloroplast components:
chloroplast genome, translational
machinery etc.
Major functions of chloroplasts
1) Outer membrane: permeable to small molecules (810 kDa
substrates) diffused through porins.
2) Inner membrane: relatively impermeable, transporting
molecules for exchange to and from the cytoplasm
3) Thylakoid membrane: site of photosynthesis/
photophosphorylation
- H
+
pumps and chloroplast ATP synthase (CF
0
CF
1
ATPase
complex) is located in this membrane
Structure of chloroplast: Membranes

Stroma:
ATP and NADPH production
CO
2
fixation
starch synthesis and storage
chloroplast genome (generally circular DNA)
Thylakoid lumen:
accumulation of H
+

Structure of chloroplast: Stroma and lumen
Light (photons) can be absorbed
by pigments such as chlorophyll
and carotenoids to transfer
excited electrons.

Excitation of electron results in
the displacement of an electron
from a weak electron donor
(chlorophyll at the reactive center
of a photosystem) to an electron
acceptor.
Chlorophyll green/ blue
Repeats 4-5
times
HO
OH
Carotenoids red to orange
Porphyrin Ring
Photosynthesis: Light-dependent reactions
A typical light harvesting complex
(LHC)
Two kinds of light
harvesting complexes
(LHC):
LHCII: Higher light
energy (lower
wavelength) absorption
LHCI: Lower light
energy (higher
wavelength) absorption

Electrons are transported through photosystems and e
-

carriers within the thylakoid membrane.

H
+
are transported from stroma into thylakoid lumen and
coupled to electron movement.
NADP is final electron acceptor

H
+
gradient used by CF ATP synthase (CF
0
CF
1
ATPase)
to synthesize ATP.
Some electrons generated are used to synthesize
NADPH from NADP
Light-dependent reactions: ATP and NADPH
Plants use two photosystems:
Chlorophyll is the primary light absorbing pigment in
both photosystems.
There are thousands of chlorophyll molecules in each
photosystem but only one at the reactive center.
Energy is transferred from absorbing pigments to
reaction center through resonance energy
transfer.
Absorbed photons by antenna pigments cause
electron displacement from chlorophyll at the reactive
center to a primary electron acceptor.
Photosystems
LHCII & PSII
1- PSII operates maximally at 680 nm wavelength (chlorophyll a
P680)
- Uses H
2
O as donor for replacement electrons
- Water-splitting activity associated with PSII: 2H
2
O 4H
+
+ O
2

- H
+
contribute to a proton gradient across thylakoid membrane

2- Electrons are passed from PSII to cytochrome b/f complex by
plastoquinone (PQ) (insoluble electron carrier).

3- Electrons are passed through cytochrome b/f to plastocyanin
(soluble electron carrier). Movement through cytochrome b/f coupled
to H
+
pumping across thylakoid membrane, into the thylakoid lumen.

4- Plastocyanin (PC) is used as electron donor to replace electron
displaced in photosystem I (PSI).
Photosystem II
e
- e
-
e
-
LHCI & PSI
1- PSI operates maximally at 700 nm (chlorophyll a P700).

2- Electrons from plastocyanin (PC) are replaced.

3- Electron flow:
- Linear: The activated electron is transferred to NADP to form
NADPH by ferridoxin NADP reductase.
- Cyclic: The activated electron cycles back through
cytochrome b/f complex to pump more H
+
across thylakoid
membrane. produces a H
+
gradient without NADPH
production.
Photosystem I
Linear electron flow
of photosystem I
e
-
Fd
FNR
Cyclic electron flow of photosystem I
e
-
e
-
e
-
CF ATP synthase: H
+

gradient used by CF ATP
synthase to synthesize
ATP.
ATP synthesis is similarly
organized as in
mitochondria.
4. Glyceraldehyde 3-
phosphate (GAP) is
converted to
carbohydrates.
2. After splitting,
phosphoglycerate (PGA)
is phosphorylated by
expending ATP.
5. Conversion to
ribulose-1,5-
bisphosphate
(RuBP) to begin
the cycle again
3. NADPH is oxidized
to NADP
+
and the
newly added P
i
is
removed from PGA to
form GAP.
1. CO
2
is fixed
by adding onto C2

of RuBP.
Light-independent reactions: Calvin cycle

GAP (glyceraldehyde-3-phosphate) of the Calvin-
Benson cycle is used to synthesize:
starch in the stroma
sugar in the cytoplasm

An antiport mechanism is used to export GAP in
exchange for inorganic phosphate.
Starch amylose (+ amylopectin) Sugar sucrose
D-glucose D-fructose
D-glucose
VS.