Lecture 3:

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

Cell biology 2014 (Revised 23/1 -14) Note handout concerning endosomes/lysosomes

Alberts et al5th edition

Chapter 12699-702713-714723-739743-745

Chapter 14813-819

Chapter 13749-751754-800809

A lot of reading!Focus on general principles and topics highlighted in the lecture synopsis

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 NADH

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

3

The mitochondrion - the power plant of the cell

1 mm

0.5 mm

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

4

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

Generation of a H+ gradient and utilization of its energy for ATP production (Alberts et al Fig. 14-10)

NADH

The origin of the mitochondrion and its genome

• Most of the mitochondrial proteins are encoded in the nucleus 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

+

6

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_import 7

Endoplasmic reticulum – ”network within the cell”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 = LipidC

a2+

4.

3.2.

8

Protein targeting to the endoplasmic reticulum

mRNA

Ribosome

tRNA

Signal-recognition particle (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.

9

Anim. 06.6-translation-I

Co-translational protein translocation

Protein is translocated into the lumen of theER co-translationally

Signal sequence is cleaved by a peptidase after completion

of translation/ translocation

10

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-transfer signal sequence

integrates intothe 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 domains

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

13

SRP

Anim. 12.6-protein_translocation.mov

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

Entry into ER is in most cases only

the first step to a final destination

ER

Post officeOut of the cell (secretion)

Plasma membrane

LysosomeGolgi

Secretory pathway

14

ER

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

NH

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

16Video 13.2-biosy_secret_path

The architecture of the Golgi apparatus

ER

Transport vesicles

3-10 Golgi cisterna(containing different sets of processing enzymes)

Cis-faceTrans-face

Downstream target

compartments

Nucleus Proteins that keep theGolgi cisterna together

17

Principle of vesicular transport

Donor compartment

Budding of vesicle fromdonor compartment

Docking and fusion of a vesicle with its target compartment

Target compartment

1.

2.

3.

Vesicle transportThe cytoskeleton isused often used asrailway tracks

18

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

19

Different coating proteins in vesicular trafficking

Cargo

Sorting receptor

Adaptin Lysosome

Golgi

ER

Endocytosisat the plasmamembrane

Clathrin

COPI

COPII

20

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.

21

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 proteinRab effector(tethering protein)

22

Fusion of a vesicle with its target compartmentv-SNARE

t-SNARESNARE 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 fusion 23

Protein trafficking in the vesicular pathway

Endoplasmic reticulum

Golgi

Plasmamembrane Lysosome

Retrograde transport

Anterograde transport

Clathrin

COPI

COPII

24

Retrieval of ER proteins

(KDEL receptor)

Protein trafficking: post-Golgi

Constitutive Regulated(e.g. insulin)

AB

C

A

B

C

Primary lysosome

Secondary lysosome

Endosome

Exocytosis Lysosomal pathway

25Anim. 13.1-clathrin

The term lysosome defines a function: lys: digest some: 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 and endogenous macromolecules and structures

PATP ADP +H+

H+H+

H+pH 5

pH 7.2

0.2-0.5 mm

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

• The inside of the lysosome is acidic

26

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

+

27 Hydrolases: proteases, nucleases, phosphatases etc etc.

Uptake of material from the exteriorPhagocytosis (“cell eating”) – specific uptake of large (0.5 – 2 mm) particles, primary by immune cellsReceptor-mediated endocytosis - specific uptake of moleculesNon -specific endocytosis, pinocytosis (“cell drinking”) - anything small in the extracellular fluid is taken up indiscriminately

1.

2.3.

1.

2. 3.

28

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)

x

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%)

30

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

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