6 - endomembrane system Flashcards
1
Q
what is the endomembrane system made of?
A
- dynamic, coordinated, and interconnected network of the cell’s organelles and related structures
- except mitochondria and chloroplasts
- ER
- ER derived organelles such as nucleus, peroxisomes, and lipid bodies
- golgi
- endosomes
- lysosomes/vacuoles
- secretory granules
- plasma membrane
2
Q
how does the endomembrane system function?
A
- large amount of material are exchanged between each organelle via small membrane bound transport vesicles
- several distinct trafficking pathways exist within the endomembrane system
- all rely on transport vesicles
3
Q
what is the biosynthetic pathway?
A
- materials are transported from the ER to the golgi, to endosomes, and then to lysosomes
- sometimes material goes from endosomes to the plasma membrane and extracellular space via exosomes
4
Q
why is the secretory pathway studied?
A
- amount of secretion varies between different cell types
- yeast and plant cells secrete cell wall materials
- pancreatic acinar cells which secrete digestive enzymes
- epithelial cells of the small intestine which secrete mucus
- pancreatic and intestine epithelial cells are highly polarized
- organelles are organized in a distinct way
- basal end of cell has the nucleus and rough ER
- central region has the golgi and lysosomes
- apical end has the secretory granules containing digestive enzymes and mucus
5
Q
how does the modern labelling experiment for studying the secretory pathway work?
A
- uses live cell imaging via fluomic with autofluo proteins
- experiment: temp sensitive viral glycoprotein (VSVG) fused to GFP and introduced to mammalian cell
- mutation in VSVG is reversible
- allows for turning on and off intracellular transport
- 40 degrees is restrictive temp
- nascent VSVG protein is misfolded and remains in ER
- 32 degrees is permissive temp
- VSVG protein properly folds and transported from ER
- after longer times, the VSVG goes to the pm
- consistent with results from pulse chase labelling
- jennifer lippincott schwartz
6
Q
how are the components and underlying molecular mechanisms of the EMS characterized?
A
- subcellular fractionation using centrifugation
- cell free systems (jim rothman)
- mutant phenotype analysis (randy schekman)
7
Q
what is subcellular fractionation?
A
- techniques used to separate and purify specific organelles on the basis of their varying sizes or densities
- allows for the study of organelle’s structure and functions
- e.g. isolation of rough ER for cell free assays for studying translation and cotranslational or vesicle trafficking
8
Q
how does subcellular fractionation work?
A
- isolation of organelles by centrifugation
- homogenization: cell tissue disrupted by gentle homogenization, ensures organelles remain intact
- homogenate is filtered and subjected to differential centrifugation
- unbroken cells and fragments are removed
- separates intact organelles of different sizes with increasing higher centrifugation speeds
- nuclei isolated in the pellet
- supernatant: liquid at top of the tube
- mitochondria, lysosomes, etc.
- microsomes: fragments of the ER network that fuse and reform into small spherical vesicles
- individual organelles in each pellet fraction can be further purified
- pellet fraction: mixture of organelles
9
Q
what is the cell free systems approach?
A
- characterization of the activities of specific endomembrane protein components in vitro
- components are purified from different organelle/ER microsomal fractions
- isolated proteins are incubated with liposomes
- liposome: artificial spherical vesicle that has a phospholipid bilayer surrounding aqueous centre
- liposomes are mixed with purified proteins
- allows for study of proteins in vitro in its natural membrane lipid environment
- allows for processes underlying protein trafficking in endomembrane system to be reconstituted in vitro
10
Q
what is the mutant phenotype analysis approach?
A
- approaches to identify genes and proteins and steps involved in protein trafficking in the endomembrane system by screening for mutant phenotypes
- vesicle trafficking is evolutionarily conserved
- yeast has a secretory pathway that is essential and can only be studied as with conditional mutants
- secretory mutants: collection of temperature sensitive mutants that secrete proteins at permissive temps but not at higher nonpermissive temps
- in higher eukaryotes, RNA interference (RNAi) mutants have visible defects in endomembrane organelle morphology
- e.g. drosophilia RNAi mutant cultured cells
- golgi seen in wild type and RNAi mutant cells using GFP tagged golgi protein as a marker for the golgi
- RNAi of genes encoding other proteins involved in golgi morphology leads to unique classes of mutant phenotypes
- class i mutant: mislocalization of golgi marker to the ER
- disruption in ER to golgi vesicle trafficking
- class i mutant: golgi fragmentation
- defect in golgi fission/fusion
- mutant genes are cloned and the encoded proteins are characterized
- explains molecular mechanisms in protein trafficking in the EMS
11
Q
what are sec yeast mutants?
A
- accumulate normally secreted proteins at points in the endomembrane pathway blocked by mutation or have defects in organelle morphology or distribution
- five major classes of sec yeast mutants (A to E)
- class A: accumulation of secretory proteins in cytosol
- defect in protein cotranslational translocation
- class B: accumulation in ER
- defect in ER vesicle formation
- double mutants highlight the order of steps in the pathway
- B + D = B mutants
- ER vesicle budding occurs before golgi vesicle budding
- mutant sec genes cloned and the encoded wild type proteins are characterized
12
Q
what is equilibrium density gradient centrifugation?
A
- separates intact organelles on the basis of density
- supernatant from differential centrifugation is gently resuspended in sucralose solution
- organelle fraction layered on top of sucrose gradient
- centrifugation results in individual organelles going to their corresponding equilibrium density
- different layers of gradient are removed and purified organelle fractions are identified based on electron microscopy or organelle marker proteins
- determine composition of the isolated organelles using proteo/lipidomics or use in cell free/in vitro import and vesicle trafficking assays
13
Q
what are the two types of secretion?
A
- constitutive secretion
- regulated secretion
14
Q
what is constitutive secretion?
A
- ER derived materials are continually transported from the golgi to the plasma membrane or released via exocytosis outside of the cell
- secretory transport vesicle membrane components are added to the pm and the lumen cargo in the vesicle is released into the extracellular space
15
Q
what is regulated secretion?
A
- occurs only in specialized cells
- ER derived materials from golgi are stored in secretory granules
- in response to a cellular signal, secretory granules fuse with pm and release lumen cargo outside of the cell
- e.g. release of neurotransmitters by nerve cells in the gap junction
- secretory granule membrane components are added to the pm
16
Q
what is the endocytic pathway?
A
- operates in opposite direction of secretory pathway
- materials move into the cell
- materials from pm or outside the cell are incorporated into the cell then transported to endosome and lysosomes
- e.g. receptor proteins that have to be degraded or are bound to a ligand
- exocytosis: vesicle trafficking to and fusion with pm, and release of contents
17
Q
how does trafficking work?
A
- vesicle with cargo buds off from membrane that donates itself
- - vesicle coat proteins select which donor membrane and soluble/lumen cargo proteins enter the newly formed/nascent transport vesicle
- - also regulate vesicle formation and budding - nascent vesicle is transported through the cytoplasm to the recipient membrane
- - vesicle receptor coat proteins regulate intracellular trafficking of vesicle to proper acceptor membrane
- - also involves molecular motors and cytoskeleton highways
- - - motor proteins direct vesicle movement within cell by connecting to vesicle surface and cytoskeleton element - vesicle fuses with proper acceptor membrane compartment
- - receptor proteins also regulate the fusion of the vesicle with the target membrane
- - the donor membrane and lumen cargo protein are incorporated into the acceptor/target compartment - entire process of budding and fusion is repeated and can occur in the reverse direction
- - other receptor proteins regulate recycling of proteins that escape to acceptor membrane compartment by bringing them back to the donor membrane compartment
18
Q
how does pulse chase labelling work?
A
- demonstrates how proteins move through the secretory pathway
- proteins are associated with organelles and move via membrane bound intermediates and not the cytoplasm
- experiment: pancreatic tissue is briefly incubated (pulse) with radioactive amino acids
- labeled amino acids are added to the newly synthesized proteins
- tissue is washed and incubated (chase) for varying lengths of time with non radioactive amino acids
- protein synthesis continues and radiolabelled proteins traffic through cell
- tissue is fixed and exposed to xray film
- results helped define secretory pathway and organization and coordination of protein trafficking in EMS
- brief chase has proteins in the rough ER (site of protein synthesis)
- intermediate chase has proteins in the golgi (site of protein modification)
- long chase has proteins in secretory granules (including those fused with pm)
- similar experiments with non secreted proteins in non polarized cells revealed trafficking also occurs between other organelles of EMS
19
Q
what is live cell imaging?
A
- using standard molecular bio techniques, a gene that produces an autofluorescent protein is linked to the gene of interest
- recombinant gene fusion is introduced via cloning into a selected organism
- intracellular localization and trafficking of the expressed fluorescent fusion protein is visualized in the living specimen using fluorescence microscopy
