2014-07-09 Lecture 4 - Lysosome, Vacuole, Mitochondrion, Chloroplast (Posted)

8/12/2019 2014-07-09 Lecture 4 - Lysosome, Vacuole, Mitochondrion, Chloroplast (Posted)

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LYSOSOME


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Functions of lysosomes

1

2

3

3. Controlled

Uptake ofnutrients

1. Digestive

2. Autophagic


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1. Digestive Optimal pH for function is low (pH 4.6 - 5.0)

H+-ATPase activity (1001000 times cytoplasm acidity)

Glycosylated interior (inner leaflet) protectscompartment from pH damage

Enriched with ~40 types of hydrolytic (degradative)enzymes

2. Controlled uptake regulator Endocytic particles (or bacteria) form endosomes whichare routed to the lysosome for degradation.

Some bacteria target and happily live in endosomes eg. Coxiellaburnetti (Q fever)

3. Autophagic (micro/macro types) Organelle (macro) and ribosome (micro) turnover is

essential to remove damaged or malfunctioning cellcomponents (eg. mitochondria or chloroplasts)


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Digestive

enzymes

Lysosome

Food vacuole

Plasma membrane

Digestion


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Lysosome

Vesicle containing

damaged mitochondrion

Digestion


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Mannose-6-phosphate (M6P)is added onto lysosomalproteins in the cis-Golgi (two step reaction)permits theiridentification later.

M6Pis recognized by the M6P receptor (MPR) in theTGNwhich sorts these proteins away from secretedprotein Patients with I- cell diseaseare deficient in the enzymes that

convert mannose to M6P, or lack proper M6P receptorsresultsin lysosomes filled with undegraded cell structures/molecules

At TGN, lysosomal proteins are packaged into clathrin-coated vesiclesfor transport to the lysosome

Mechanism for sorting lysosomal proteins


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Lysosomal sorting using

clathrin coated vesicles

(CCV)

1 2

3 4

Cyto

TGN


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Endocytosis involves the uptake of proteins andother macromolecules at the plasma membrane.

Bulk materials are taken up by the cell in two

ways: Within the membraneProteins are concentrated

during uptake (receptor mediated endocytosis).

Within the fluid phaseNo increase in theconcentration of the molecules

Pinocytosis (cell drinking)

Phagocytosis (cell eating)

Lysosomes in endocytosis


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Pinosomes are generated by the process of pinocytosis(celldrinking). No pseudopod formationplasma membrane pinching using

receptors and COP like proteins in the coated pits

Pinch sites

Pinososome

Endocytosis: Pinocytosis


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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


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Many of the events in receptor mediated

endocytosis are similar to vesicle transport in the

secretory pathway.


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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 receptorsto internalize

1

Endocytosis step # 1: Formation of coated pits


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Coat is formed from clathrin.

Three heavy chains and 3light chains are assembled

into a triskelion. Triskelions are assembledinto a basket-like structure onthe cytoplasmic face of thevesicle.

Adaptor proteinsconnectthe cytoplasmic side ofreceptors to clathrin.

2

3

1

Endocytosis step # 2: Coat assembly continues

until vesicle is formed and released into cytoplasm


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Clathrin coated vesiclesused for both receptor-mediated endocytosisand for vesicle transport

from TGN to lysosome.

The adaptor proteins forTGN are different fromthose for plasma

membrane.


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Example: Cholesterol uptake

Cholesterol is carried with apo-B

protein as LDL particle.

LDL receptor internalizes LDL.

Familial hyper-cholesterolemialeads to elevated blood

cholesterol:

Mutations to LDLRgene (encodes

the LDL receptor)

Mutations to apoBgene


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Tftransferrin

TfRtransferrin receptor

Example: Iron uptake Iron is released from transferrin

in endosome


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Peroxisomes

Peroxisomes can be formed eitherby de novosynthesis orgrowth/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 fattyacid chains

- Toxic oxygen species formation/breakdown


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Peroxisomes

Urate oxidaseis not found in

humansuric acid can build

up leading to the illness called

gout.

Peroxisomes have 32unique proteins called PEX

that function as:

- Protein import machinery

- Enzymes

- Receptors


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VACUOLE


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Vacuoles are apredominant feature of

plant cells. Lysosomes are

predominately found inanimal cells and rarely inplant cells.

Vacuoles are membranebound compartments thathave similar functions aslysosomes. 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/


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1. Defensive Toxic chemical repository

cyanide containing

glycosides

glucosinolates2. Vacuoles are storage

units

Stores solutes and

macromolecules such asions, sugars, amino acids,

proteins, and

polysaccharides, organic

acids

VacuoleGolgi

ER

chloroplast

mitochondria

Functions of vacuoles in plant cells


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3. Intracellular

digestion

pH ~ 25

Organic acids assist

in maintaining low pH

The vacuolar

membrane isreferred to as the

tonoplast

Specific proteins:

Tonoplast intrinsic

proteins (TIP)

Vacuole

Turgor pressure

[Solute]

H2O

H2O

Plant cells have high

osmotic pressure


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The Mitochondrion:Aerobic Respiration


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Aerobic respiration

Aerobic respiration is a biological/metabolic process

where molecular oxygen (O2) is used to generateenergy 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 CO2and water (H2O) by

the tri-carboxylic acid (Krebs) cycle (TCA).

The process generates electrons (e-) and NADHused

to drive the electron transport chain and create a proton

gradient used to synthesize ATP.

C6H12O6+ O2

6 CO2+ 6 H2O + energy


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Aerobic respiration

Glucose

O2present

Pyruvate

Glycolysis

Fermentation

Lactate

NADH

NAD+

NAD+

NADH

O

Plasma membrane

NAD+

2ATP

O2absentPyruvate

Acetyl-CoA

TCA

Cycle

3NADH, 2FADH2

Electron transport chain

36ATP Cytoplasm

NAD+

NADHCO2

5 e- pairs

OH

OH

OH

OH

2CO2+ H2O

O

O

O-

(Ethanol)

CH2OH

HC

CH2OPO32-

OH

Glycerol 3-P

H3C COO-

Fatty acid

Fermentation is useful for aerobic respiration replenishes NAD+ supply


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Mitochondria can self-replicate by fission.

Functions:

1. Synthesis of ATPvia the oxidation ofpyruvate: most ATP is produced byoxidative phosphorylation.

- The more energy a cell needs themore mitochondria (skeletal muscle)

- Mitochondria are localized near siteswhere ATP requirement is greatest

Example: near baso-lateral surface

of gut epithelium, where Na

+

/K

+

ATPase activity is highest

2. -oxidationof fatty acids (peroxisomesalso participate in this).

3. Apoptosis(programmed cell death).

Controlled

Cell death3

-oxidation

TCA

cycle

Acetyl-CoA

Acyl-CoA

2

1

ATP

Mitochondria


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Mitochondrial outer membrane is distinct from the inner

membrane.

The outer membrane is permeable to small molecules.

Enriched with porinsthat form non-specific channels in

outer membrane (permeable to molecules < 10 kDa)

Outer membrane

Intermembrane space lumen

Cyto side

Intermembrane sideMatrix

H+

ADP + Pi ATP

MITOCHONDRIAL PORIN

Structure of mitochondria: Outer membrane


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Mitochondria inner membrane:

Contains high abundance of cardiolipin

make membraneless permeable to protons.

Contains high abundance of protein (approx. 75% of mass):

electron transport chain (ETC) complexes and ATP

synthases.

Is highly convolutedjoins to double layered membrane

sheets called as cristae.

Is highly impermeable.

Both the outer and inner membranes have distinct protein importsystem.

Structure of mitochondria: Inner membrane


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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


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Mitochondrial DNA (mtDNA),RNA, ribosomes:

Self-replicating and maternallyinherited.

Can be circular or linear

Protein: very high abundance

Pyruvate and fatty acidoxidations

TCA/Krebs cycle Mostly encoded by nuclear

genome.

Structure of mitochondria: Matrix


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NADH

NADH FADH2

ATP ATP

ATP

CYTOPLASM

Glycolysis

Electrons

carried by NADH

Glucose PyruvatePyruvate

OxidationCitric Acid

Cycle

Oxidative

Phosphorylation

(electron transport

and chemiosmosis)

Mitochondrion

Substrate-level

phosphorylationSubstrate-level

phosphorylation

Oxidative

phosphorylation

ATP production

Major energy reserves in cells:


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Major energy reserves in cells:

polysaccharides and fats

Starch granulesin potato tuber cells

Glycogen granulesin muscle

tissueGlycogen

Glucosemonomer

Starch

Cellulose

Hydrogen bonds

Cellulosemolecules

Cellulose microfibrilsin a plant cell wall

Fatty acids

Glycerol

Fat (triglyceride)

Making ATPs Step 1:


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Glycolysis:break-down of glucoseto pyruvate

Each glucose produces: 2 NADH + 2

ATP

Lipolysis:

Triglyceridesfatty acids

Glycerol-3-phosphate shuttle:

triglyceridesglycerolglycerol-3-

phosphate

(In mitochondria: glycerol-3-phosphate

dihydroxyacetone-3-phosphate to

make FADH2)

Making ATPsStep 1:

Breaking down energy sources in the cytoplasm

Making ATPs Steps 2:


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Making ATPsSteps 2:Acetyl-CoA production and TCA cycle

Fatty acids and pyruvate are

transported into mitochondria viapermeases.

Oxidation:

Pyruvateacetyl-CoA

Fatty acidsacetyl-CoA (-oxidation)

Fatty acids produce much more

acetyl-CoA than pyruvate

produces.

TCA cycle:

Each acetyl-CoA produces: 3

NADH + 1 FADH2+ 1 ATP

NADH and FADH2are used by the

elctron transport chain (ETC) for

oxidative phosphorylation.

Making ATPs Steps 3:


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Electron transport chain(ETC):

Is composed of 4

complexes

Transfers electrons from

TCA cycle reactions to

terminal electron

acceptors.

O2

+ 2H+H2

O

Making ATPsSteps 3:

Oxidative phosphorylation


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Electron transport chain


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Electron donors: FADH


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Complex II is also part of the TCA cycle: producing FADH2.

FADH2donates electrons to ubiquinone carriercomplex IIIcomplex IV (coupled with transporting H+).

Complex II does not transport H+.

Electron donors: FADH2


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ATP synthase utilizes proton motive force


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ATP synthase (F0-F1

ATPase) utilizes the H+

gradient to synthesize ATP.

H+move down theconcentration gradientand pulled by electrical

gradient across innermembrane through F0F1ATP synthase.

The F0domain (membrane

integral) form the H+

channel.

The F1domain (peripheral onthe matrix side of the innermembrane) synthesizes ATP

by rotational catalysis.

ATP synthase utilizes proton motive force


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Rotational catalysis by ATP synthase


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1 ATP molecule is produced for every 3 to 4 H+moving through ATP

synthase

3 ATP molecules for each 360odegree rotation of g subunit

Letters indicated refer to: O= open, L= loose, T= tight conformations of the F1subunit

Rotational catalysis by ATP synthase


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Major functions of chloroplasts


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Sites of photosynthesis in plants

conversion of photon (light)energy into chemical energy

(ATP and NADPH).

Sites of conversion of CO2to

sugars at expense of ATP andNADPH (CO2fixation 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

Structure of chloroplast: Membranes


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1) Outer membrane:permeable to small molecules (810 kDasubstrates) diffused through porins.

2) Inner membrane:relatively impermeable, transportingmolecules for exchange to and from the cytoplasm

3) Thylakoid membrane:site of photosynthesis/photophosphorylation

- H+pumps and chloroplast ATP synthase (CF0

CF1

ATPasecomplex) is located in this membrane

Structure of chloroplast: Membranes

Structure of chloroplast: Stroma and lumen


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Stroma:

ATP and NADPH production CO2fixation

starch synthesis and storage

chloroplast genome (generally circular DNA)

Thylakoid lumen: accumulation of H+

Structure of chloroplast: Stroma and lumen


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A typical light harvesting complex

(LHC)

Two kinds of lightharvesting complexes(LHC):

LHCII: Higher lightenergy (lowerwavelength) absorption

LHCI: Lower light

energy (higherwavelength) absorption

Light dependent reactions: ATP and NADPH


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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 (CF0CF1ATPase)

to synthesize ATP.

Some electrons generated are used to synthesize

NADPH from NADP

Light-dependent reactions: ATP and NADPH


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LHCII & PSII

Photosystem II


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1- PSII operates maximally at 680 nm wavelength (chlorophyll a

P680)- Uses H2O as donorfor replacement electrons

- Water-splitting activity associated with PSII: 2H2O4H++ O2

- H+contribute to a proton gradient across thylakoid membrane

2- Electrons are passed from PSII to cytochrome b/f complex byplastoquinone(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


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e- e-

e-


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LHCI & PSI

Photosystem I


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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 formNADPH 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 NADPHproduction.

Photosystem I


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Linear electron flowof photosystem I

e-

Fd

FNR


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Cyclic electron flow of photosystem I

e-

e-

e-

CF ATP synthase: H+


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CF ATP synthase: H+

gradientused by CF ATP

synthase to synthesize

ATP. ATP synthesis is similarly

organized as in

mitochondria.

Light-independent reactions: Calvin cycle


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4. Glyceraldehyde 3-

phosphate (GAP) is

converted to

carbohydrates.

2. After splitting,

phosphoglycerate (PGA)

is phosphorylated by

expending ATP.

5. Conversion toribulose-1,5-

bisphosphate

(RuBP) to begin

the cycle again

3. NADPH is oxidizedto NADP+ and the

newly added Piis

removed from PGA to

form GAP.

1. CO2is fixed

by adding onto C2

of RuBP.

Light independent reactions: Calvin cycle


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2014-07-09 Lecture 4 - Lysosome, Vacuole, Mitochondrion, Chloroplast (Posted)