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Lecture 3:

Cell Biology interactive media ”video” or ”interactive”1

Cell biology 2017 version 13/1 2017 Note – endosome vs lysosome handout

Text book Alberts et al.: Chapter 12 - 14(Topics covered by the lecture)

A lot of reading! Focus on principles

Protein trafficking between cell compartments

Mitochondrion

NucleusER

Golgi

Cytosol

Lysosome

Ribosomes

Ribosome populations

Free cytosolic

Attached to the endoplasmic

reticulum

Protein

Various address tags(without a tag cytosol)

N C

>90 % of all membranes are part of organelles within the cytoplasm 2

Glucose

Pyruvat

ATP (2)

NADH (2)

1. 2.

3.

Translation Glycolysis

Signal transduction

1.2.

3.

The cytosol

Nucleus

Viscous solution high concentration of proteins (~400 mg/ml)

Key processes/components of

the cytosol

3The mitochondrion - the power plant of the cell

1 m

0.5 m

The invaginations are denoted cristae increased surface area

• Reproduce by dividing in two (binary fission)

+

• The mitochondrion is a double membrane-enclosed organelle that specialize in ATP regeneration (>100 per cell)

• From Greek, mitos, thread, + chondros, granule

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Metabolic pathways of the mitochondrion

1.

2.

Intermediary metabolism

Respiration (electron transport chain) and ATP synthesis

Krebscycle

1.2.

Pyruvate

Acetyl CoA Acetyl CoA

Fatty acid

Anim. 02.5-citric_acid_cycle.mov (1.5 min)

Anim. 14.3-electron_transport & 14.4-ATP_synthase

H+ gradient and utilization of its energy for ATP production.

NADH

The origin of the mitochondrion and its genome

• Most of the mitochondrial proteins are encoded in thenucleus and have to be imported from the cytosol

• Mitochondria have circular DNA and bacteria-like ribosomes

• Mitochondria are only inherited from the mother

37 genes

22 tRNA genes

2 rRNA genes

13 mRNA encoding genes

Aerobicbacteria ”Founding”

eukaryote

+

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Targeting proteins to the mitochondrion

Translocation of mitochondrial proteins through this channel requires proteins to be kept unfolded

Protein translocation across themitochondrial membranes is mediated by proteins that form a channel spanning both membranes

Folded protein Unfolded protein

Successful passageNo passage

Chaperone, keepingthe protein unfoldedin cytosol

Signal sequence

N

Anim. 12.3-protein_import7

Endoplasmic reticulum – ”network within cytoplasm”

1. Protein sorting and modification (Rough ER )(starting point of the “secretory pathway” of protein synthesis)

2. Lipid synthesis (Smooth ER)

Ca2+4. storage (Smooth ER)

3. Detoxifications (Smooth ER, eg. P450)

1.

L+i+p+i+d = Lipid

4.

3.2.

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Protein targeting to the endoplasmic reticulum

mRNA

Ribosome

tRNA

Signal-recognitionparticle (SRP)

ER lumen

SRP receptor

Protein translocator

Pause in translation during localization step

Cytosolic ribosome

ER associated ribosome

ER signal sequence, stretch of hydrophobic a.a.

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Anim. 06.6-translation-I

Co-translational protein translocation

Protein is translocated into the lumen of theER co-translationally

Signal sequence is cleaved bya peptidase after completion of translation/ translocation

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Integration of a transmembrane protein into ER

Translocation is initiated but stops at a hydrophobic ~15 aa

sequence termed stop-transfer signal

Translation completethe stop-transfersignal sequence integrates into

the ER membrane

However,translation continues

N

C

11

12

Note the opening of the protein translocater, which allows lateral diffusion within the ER-membrane of both the ER-signal sequence and trans-membrane domain

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Synthesis of multi-pass transmembrane proteinsER signal sequence (N-terminus)=

Stop-transfer sequence

Re-start-transfer sequence

C-

Translocation stop and re-start several times, which results in a multi-pass transmembrane protein

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SRP

Anim. 12.6-protein_translocation.mov

the initial ”start transfer signal” followed by a signal peptidase recognition site

ER entry: the first step to various destinations14

ER

Post officeOut of the cell (secretion)

Plasma membrane

LysosomeGolgi

Secretory pathway

ER

January 2017

Proteins are glycosylated during passageof the secretory pathway

Post-translational modification by attachment of oligo-saccharides

N-linked oligo-saccharides are attached via the amide groupof asparagine in ER

O-linked oligo-saccharides are attached to hydroxyl group of serine or threonine in Golgi

N

H

O

ER

Cytosol

Extracellular

Golgi15

“Glycocalyx – a carbohydratezone on the cell surface”

Vesicular trafficking post ER

ER

Post officeOut of the cell

Plasma membrane

LysosomeGolgi

Transport from ER to Golgi, within Golgi, and from Golgi to either lysosomes or cell surface is carried out by transport vesicles (liposomes made of phospholipids)

Secretory pathway

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Video 13.2-biosy_secret_path

The architecture of the Golgi apparatus

ER

Transport vesicles

3-10 Golgi cisterna(containing differentsets of processingenzymes)

Cis-faceTrans-face

Downstream target

compartments

NucleusProteins that keep theGolgi cisterna together

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Principle of vesicular transport

Donor compartment

Budding of vesicle fromdonor compartment

Docking and fusion ofa vesicle with its target compartment

Target compartment

1.

2.

3.

Vesicle transportThe cytoskeleton isused often used asrailway tracks

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Vesicle formation in donor compartment

Cargo (i.e., the protein to be transported)

Sorting receptor

Coat protein

Bud formation1.

Vesicle formation2.

Vesicle pinching off3.

Lumen of donor compartment

Constricting protein complex

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Different coating proteins in vesicular trafficking

Cargo

Sortingreceptor

Adaptin Lysosome

Golgi

ER

Endocytosisat the plasmamembrane

Clathrin

COPI

COPII

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Coat:

Vesicle docking and fusion with target compartment

Lumen of target compartment

Uncoating of vesicle subsequent to ”pinching off”1.

Vesicle tethering with target compartment (specificity Rab’s)2.

Vesicle docking and fusion with target compartment (SNAREs)3.

1.

2.3.

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Tethering of vesicles to the correct target compartment

Rab protein on vesicle docks with Rab effectoron target compartment

Compartment X

Compartment Y

Different Rab proteins – different target compartments

Rab protein

Rab effector(tethering protein)

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Fusion of a vesicle with its target compartmentv-SNARE

t-SNARE

SNARE proteins on vesicle and targetcompartment interacts

1.

Conformational changes of SNAREs bring the membranes closer together…..

2.

…..until they are in physical contact3.

1. 2. 3.

4.

4. This leads to exclusion of H2O membrane fusion23

Protein trafficking in the vesicular pathway

Endoplasmicreticulum

Golgi

Plasmamembrane Lysosome

Retrogradetransport

Anterogradetransport

Clathrin

COPI

COPII

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Retrieval of ER proteins

(KDEL receptor)

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Protein trafficking: post-Golgi

Constitutive Regulated(e.g. insulin)

A

B

C

A

B

C

Primary lysosome

Secondary lysosome

Endosome

Exocytosis Lysosomal pathway

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Anim. 13.1-clathrin

Video 13.4-endosome_fusion

The term lysosome defines a function:lys: digestsoma: body

Lysosomes develop from endosomes by fusion with vesicles carrying lysosomal enzymes

The lysosome – the digestive system of the cell

• The lysosome is responsible for degradation of exogenous andendogenous macromolecules and structures

PATP ADP +H+

H+H+

H+pH 5

pH 7.2

0.2-0.5 m

• Vesicles (~ 300/cell) filled with ~ 40 acid hydrolases that has capacity to degrade more or less anything

• The inside of the lysosome is acidic

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The pH regulates the activity of hydrolytic enzymes

+

Lysosome contains many types of hydrolytic enzymes These are only active in an acidic environment

Hydrolases are inactive in ER and

Golgi (pH ~7) Hydrolases are active in the acidic lumen of

the lysosome

Degradation of endocytosed

material

+

27Hydrolases: proteases, nucleases, phosphatases etc etc.

Uptake of material from the exteriorPhagocytosis (“cell eating”) – specific uptake of large (0.5 – 2 m) particles, primary by immune cells

Receptor-mediated endocytosis - specific uptake of molecules

Non -specific endocytosis, pinocytosis (“cell drinking”) - anything small in the extracellular fluid is taken up indiscriminately

1.

2.

3.

1.

2. 3.

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video 13.5 phagocytosis .mov

Three routes to the lysosome

Primary lysosome Secondary lysosome

Endosome AutophagosomePhagosome1. 2. 3.

4. 5. 29

Autophagy

Phagocytosis

ER

1.

2.

3.

4. 4.

4.5.

Endocytosis

Anim. 13.3-receptor_endocytosis (Note: vesicle fusion with endosome)

Summary: cellular organelles and trafficking

Mitochondrion (22%)

Nucleus (6%)

Cytosol(54%)

Lysosome (1%)

Ribosomes

ER (12%)

Golgi (3%)

>10-fold more internal membranes than plasma membrane

% = volume of a liver cellEndosome (1%)

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3 types of protein transportA. Gated (nuclear pores)B. Across membranes**(translocation channels)C. Vesicle (budding and fusion)

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