Endomembrane System

Question 1

What is the Endomembrane System?

Answer 1

It is a dynamic, co-ordinated and interconnected network of the cell’s organelles and related structures

Question 2

What are the organelles in the endomembrane system?

Answer 2

Endoplasmic Reticulum, nucleus, peroxisomes, lipid bodies, Golgi, Endosomes, lysosomes/vacuoles, secretory granules, plasma membrane

Question 3

What happens with transport vesicles?

Answer 3

Large amounts of material are exchanged (trafficked) between each organelle/structure via the small, membrane-bound transport vesicles

Question 4

How are proteins/material trafficked through the endomembrane system?

Answer 4

They are trafficked via transport vesicles and involve four general steps

Question 5

What are the four steps involved in protein trafficking through the endomembrane system?

Answer 5

1) Cargo-containing vesicle buds off the donor membrane compartment
- -> the vesicle ‘coat’ proteins select which ‘donor’ membrane and soluble (lumenal) ‘cargo’ proteins enter (or not enter) nascent transport vesicle and regulate vesicle formation and budding
2) Once the vesicle buds off, the nascent vesicle is transported through the cytoplasm to the ‘acceptor’ membrane compartment
- -> Vesicle receptor (coat) proteins regulate the intracellular trafficking of the vesicle to proper acceptor membrane, it also involves molecular motors and cytoskeleton highways
3) Vesicle ‘fuses’ with proper acceptor membrane compartment
- -> receptor proteins also regulate vesicle-acceptor membrane fusion
- -> vesicle (donor) membrane & lumenal cargo proteins are incorporated into the acceptor compartment
4) Entire process of budding and fusion is repeated and can occur in the reverse direction
- -> Other receptor proteins regulate the recycling of proteins that ‘escape’ from the acceptor membrane compartment back to the donor membrane compartment

Question 6

What do motor proteins do?

Answer 6

Direct vesicle movement within the cell by linking to vesicle surface and cytoskeleton element

Question 7

What exists within the endomembrane system?

Answer 7

Several different trafficking pathways exist within the endomembrane system that all rely on transport vesicles.

Question 8

What is the biosynthetic pathway?

Answer 8

Materials are transported from the ER to Golgi, to endosomes, and then to either lysosomes and sometimes materials transported, via exosomes, from endosomes to the plasma membrane (pm) and extracellular space

Question 9

What are the two secretory pathways?

Answer 9

Constitutive Secretion and Regulated Secretion

Question 10

What is constitutive secretion?

Answer 10

ER derived materials are continually transported from Golgi to pm and/or ‘released’ (via exocytosis) outside of the cell (i.e extracellular space): ER materials are targetted to plasma membrane

Question 11

How are secretory transport vesicles incorporated?

Answer 11

The secretory transport vesicle membrane components are incorporated into pm, and vesicle luminal ‘cargo’ is released into extracellular space

Question 12

What is exocytosis?

Answer 12

Vesicle trafficking to and fusion with pm, and release of contents, this is how receptors can be delivered.

Question 13

What is regulated secretion?

Answer 13

Occurs only in specialized cells
ER derived materials from Golgi are stored in secretory granules
In respond to a cellular signal, secretory granules fuse with pm and release (via exocytosis) luminal ‘cargo’ into extracellular space
Secretory granule membrane components are incorporated into pm

Question 14

What is the endocytic pathway?

Answer 14

Operates in opposite direction of secretory pathway (materials move into the cell)
Materials from pm (e.g., receptor proteins destined for degradation or bound to a ligand) and/or extracellular space are incorporated into the cell (via endocytosis) and then transported to endosomes and lysosomes

Question 15

How does the amount of secretion vary between cell?

Answer 15

It does vary and some cells have high levels of secretion and others have low levels of secretion
Yeast & plant cells: Cell wall materials
Pancreatic acinar cells: Digestive enzymes
Epithelial cells of the small intestine: Mucus

Question 16

What is special about pancreatic & epithelial cells?

Answer 16

They are highly polarized

Question 17

How are organelles distinct?

Answer 17

They are organized in a distinct way
Basal end of cell: nucleus, rough ER (RER)
Apical end (duct/lumen): secretory granules containing digestive/enzymes mucus

Question 18

What did the Autoradiography and pulse chase labeling experiments demonstrate?

Answer 18

How proteins move through the secretory pathway - proteins are associated with organelles and move via ‘membrane-bound intermediate (not the cytoplasm)
RER –> outside of cell to be secreted

Question 19

What is the pulse chase labeling experiment process?

Answer 19

Pancreatic tissue is briefly incubated ('pulse') with radioactive amino acids labeled amino acids are incorporated into newly synthesized proteins
Tissue washed and incubated ('chase') for varying lengths of time with non-radioactive amino acids 
Protein synthesis continues and radio-labeled proteins traffic through cell
Tissue fixed (killed) and exposed to X-ray film (autoradiography)

Question 20

What did the pulse chase labeling experience prove?

Answer 20

Brief ‘chase’ (3 min) = rough ER (site of protein synthesis)
Intermediated ‘chase’ (20 mins) = Golgi (site of protein modification)
Long ‘chase’ (120 mins) = Secretory Granules (including those fused with the pm)
The results helped define secretory pathway and organization and coordination of protein trafficking in endomembrane system
ER –> Golgi –> PM through secretory granule

Question 21

What have similar experiments shown with respect to non-secreted proteins?

Answer 21

Similar experiments with non-secreted proteins (in non polarized cells) revealed trafficking also occurs between other organelles of endomembrane system (ER to Golgi to Lysosomes)

Question 22

How is autofluorescence used?

Answer 22

Using standard molecular biology techniques, a gene encoding of auto-fluorescent protein (GFP, RFF) is linked to a gene of interest
Recombinant gene fusion is introduced via cloning into a selected organism/tissue/cell
Intracellular localization and trafficking of the expressed fluorescent fusion protein is visualized in the living specimen using fluorescence microscopy

Question 23

What do modern ‘labeling’ experiments study?

Answer 23

Protein transport through the secretory pathway involves live-cell imaging (fluorescence microscopy) using auto-fluorescent proteins

Question 24

What is the modern ‘labeling’ experiment steps?

Answer 24

Temperature sensitive viral glycoproteins fused to GFP and introduced (transfected) into mammalian-cultured cell
Mutation in VSVG in reversible- allows for turning on/off intracellular transport
–> 40 C (restrictive temperature)- nascent (newly synthesized) VSVG protein is mis-folded and remains in ER due to quality control processes: prevent mis folded from leaving ER
–> 32 C (permissive temperature) - VSVG protein properly folds and transported from ER

Question 25

What is the result of the VSVG-GFP fluorescence microscopy experiments?

Answer 25

40 C restrictive temperature = rough ER (filament like network(site of protein synthesis))
32 C permissive temperature (40 min) = Golgi (near nucleus(site of protein modification))
Longer times (180 min) = cell surface (plasma membrane)
These results are consistent with the pulse-chase labelling studies

Question 26

What is sub cellular fractionation using centrifugion?

Answer 26

Techniques used to separate and purify specific organelles on the basis of their varying sizes/and or densities
It allows for the study of organelle’s (component’s) structure and function

Question 27

What is an example of sub cellular fractionation using centrifugion?

Answer 27

Isolation of rough endoplasmic reticulum for cell-free assays for studying protein synthesis (translation) and co-translocation or vesicle trafficking

Question 28

What is the homogenization part of sub cellular fractionation using centrifugation?

Answer 28

Cell/tissue disrupted by gentle homogenization (ensures organelles remain intact)

Question 29

What is the homogenate part of sub cellular fractionation using centrifugation?

Answer 29

Filtered (removes unbroken cells and large fragments) and subjected to differential centrifugation
Separates intact organelles/cellular components of different size/density with increasing higher centrifugation steps
If it is more dense, the particle comes down faster
600g x 10 min: isolated nuclei in pellet

Question 30

What is and what happens to the resulting supernatant of the homogenate sub cellular fractionation?

Answer 30

The resulting supernatant (liquid at top of centrifuge tube) is subjected to 15K g x 5 min: mitochondria, lysosomes, etc
Supernatant subjected to 100K g x 1 hr: pm and ER microbes
Each individual organelles in each pellet fraction (mixture of organelles) can be further purified

Question 31

What is equilibrium density-gradient centrifugation?

Answer 31

Separates intact organelles/cellular components on the basis of density:
15K g x 5 min pellet from differential centrifugation step(containing mitochondria, lysosomes, etc) is gently resuspended in sucrose solution
Organelle fraction layered on top of sucrose gradient (increasing density from top to bottom)

Question 32

What does the centrifugation in equilibrium density do?

Answer 32

Centrifugation results in individual organelles migrating to their corresponding equilibrium density
Different layers of gradient removed and purified organelle fractions identified based on electron microscopy or organelle marker proteins/enzymes
Less Dense is at the top of tube

Question 33

What does equilibrium density-gradient centrifugation do?

Answer 33

Determines the composition of the isolated organelles using proteo/lipidomics and/or use in cell free (in vitro) import and vesicle trafficking assays

Question 34

What is the cell free systems?

Answer 34

Characterization of the activities of specific endomembrane protein components in vitro -
components are purified from different organelle/ER microsomal fractions
Isolated proteins incubated with liposomes, which are artificial spherical vesicles consisting of a phospholipid bilayer surrounding aqueous centre

Question 35

In the cell free systems, what does the liposomes mixed with the purified proteins allow?

Answer 35

The study of protein(s) in vitro in its ‘natural’ membrane lipid environment
e.g., protein(s) involved in the formation of transport vesicles cause liposome membrane ‘budding’
Allows for processes underlying protein/vesicle trafficking(and steps) in endomembrane system to be reconstituted in vitro

Question 36

What is the Mutant Phenotype Analysis?

Answer 36

Approaches to identify genes/proteins and steps involved in protein/vesicle trafficking in the endomembrane system by screening for mutant phenotypes
Vesicle trafficking/endomembrane organization is evolutionary conserved

Question 37

How is yeast related to mutant phenotype analysis?

Answer 37

Secretory pathway is essential and can only be studied as with conditional mutants

Question 38

What are the secretory (Sec) mutants?

Answer 38

Collection of temperature-sensitive mutants that secrete proteins at permissive temperatures, but do not at higher, non permissive temperature
Essential, if they are knocked out, the organism becomes lethal

Question 39

What are the Sec yeast mutants?

Answer 39

Accumulate normally secreted proteins at points in the endomembrane pathway blocked by mutation and/or possess defects in organelle morphology and/or distribution
Mutant SEC genes cloned and encoded (wild-type protein) are characterized

Question 40

What are the classes of sec yeast mutants?

Answer 40

A-E

Question 41

What is the Class A of Sec yeast mutants?

Answer 41

Accumulation of secretory proteins in cytosol (defect in protein co-translation/translocation)

Question 42

What is Class B of Sec yeast mutants?

Answer 42

Accumulation in ER (defect in ER vesicle formation)
Sec12 mutant has large, expanded ER due to disruption in vesicle budding from the ER
Did not form proper vesicle, so remains in ER

Question 43

What is RNA interference?

Answer 43

In higher eukaryotes, RNA interference mutants possess visible defects in endomembrane organelle morphology and/or distribution

Question 44

What is the Drosophila RNAi mutant cultured cells?

Answer 44

Golgi visualized in wild-type and RNAi mutant cells using GFP-tagged Golgi protein as a marker for the golgi
RNAi of genes (mRNA) encoding other proteins involved Golgi morphology/distribution leads to several unique classes of mutant phenotypes

Question 45

What are the RNAi mutant classifications?

Answer 45

Class I mutant: mis-localization of Golgi marker to ER (disruption in ER-to Golgi vesicle trafficking)
Class II mutant: Golgi fragmentation (defect in Golgi fission/fusion)

Question 46

What happens to mutant genes?

Answer 46

They are cloned and the encoded (wild-type) proteins are characterized by elucidating molecular mechanisms in protein/vesicle trafficking in the endomembrane system