Introduction to membrane trafficking Flashcards
Describe secretory pathway - image
Aim to cell surface
Er
Golgi
Describe secretory pathway general plan
Er
Pre golgi intermediates - intermediate compartments, Vtcs
Golgi apparatus - intro Golgi transport
Post Golgi carriers - several kinds - secretory vesicles
Cell surface, cell exterior
INSIDE OUT
Describe endocytic pathway - image
Outside in
4 MAJOR ORGAnelles = endosome, recycling endosome, late endosome, lysosome
Targeted to lysosome for degradation
Describe endocytic pathway - general plan
Cell surface - cell exterior
Early endosome - alternate pathways too
Late endosome
Lysosome
Describe what George palade did - general overview
1950s-60s
Using ‘em and membrane fractionation - defines the secretory pathway
Er —> Golgi
Golgi —> secretory granule
Secretory granule —> cell surface
What is autoradiography
Pulse chase technique
autoradiography - sample labelled
Sample labelled with isotopes while alive - tritium tagged aas
Tritium usually bc emits low energy beta particle that moves only a very short distance - so hit something close to where protein is
autoradiography- sample prep
Prepared normally for microscopy and sectioned - Epon-embedded for em
Photo sensitive silver - absorbs light
Silver emulsion placed over sample
Sample stored in dark at low temp - to allow radioactivity to expose emulsion
autoradiography - sample treated
Sample treated with developer to produce metallic silver from exposed silver emulsion - grains of metallic silver individually visible by em
= creates silver lights head where proteins are = can see, silver grains
What is pulse chase - explain
Triniated leucine - small # mins, protein visible = pulse then chase = take pics at diff times and see proteins
What did autoradiography And em do
Provided first direct evidence that proteins to be secreted appeared first in er then in Golgi
Describe Palade exp - step 1
Autoradiography pulse chase - messy bc would label every protein, so used pancreatic cells, bc many protein digestions that makes it neater
Labelled with 3h leucine for 3 mins
Silver grains show over er
(Fix then silver then put in freezer in dark - several weeks, get many silver) - JUST PULSE
Describe Palade exp - step 2
LABELled for 3 mins with 3h leucine
Then incubated for 7 mins further - chase
Label now over Golgi apparatus
= move from er to Golgi
Describe Palade exp - step 3
Label like usually
Incubate for 37 mins - longer = chase further
Label now over vacuoles
Describe Palade exp - step 4
Incubate 117 mins = 2hrs
Label now over zymogen granules (ready to fuse with cell surface when eat)
= er —> Golgi —> cells surface
Describe Palade exp conclusions
Proposed transport vesicles shuttled proteins between compartments - but molecular mechanisms of trafficking between compartments unknown
1980s
the use of yeast genetics (Schekman group) and in vitro systems (Rothman group) identified many of the molecules involved in transport through the secretory pathway and provided a first picture of how carrier vesicles transported proteins from one compartment to another
Name 2 major organelles of secretory pathway
Er
Golgi
Describe er - secretory pathway
Newly synthesized proteins inserted
Folding
N linked glycosylation
Quality control
Describe Golgi apparatus - secretory pathway
N linked oligosaccharides modified
Sorting to various destinations occur
Describe destinations of secretory pathway
Multiple
Includes cell surface directly, secretory granules, endosomes
Describe set up of secretory pathway = where things are in cell
Golgi near nucleus and mtoc - move to neg end mt
Name categories of protein transport between compartments
Transmembrane transport
Gated transport
Vesicular transport
Direct connections
Protein detaches and diffuses through cytoplasm
Describe transmembrane transport
Cytoplasm —> er
Cytoplasm —> miso
Cytoplasm —> peroxiosme
Cytoplasm —> chloroplast
Describe gated transport
Nuclear pores
Describe vesicular transport
Between organelles of secretory pathway and endocytic pathway
Vesicles leaves first organelles then fuses with second organelle
Describe direct connections transport
Also possible but less common
Kiss and run in endocytic pathway
Connections between Golgi stacks in secretory pathway
Describe protein detaches and diffuses through cytoplasm transport
Only possible for peripheral proteins on outside of compartment
Name the 4 steps in vesicular transport
Sorting of cargo, budding and separation from source membrane (coat proteins, also separation= seperate step)
Transfer to destination
Storage - synaptic vesicles
Recognition of target membrane and fusion
Do you need a signal for recognition of target membrane and fusion
Hum sometimes
Zymogen granules
Synaptic vesicles
Describe orientation of proteins during secretory pathway
End that faces cytoplasm will still face cytoplasm
Orientation of membrane stays teh same
What are ways that cargo can be sorted, budded and separated form source membrane
Coated vesicles
Tubular transport intermediate
Describe coated vesicle
Binds cytoplasmic domain of integral membrane protein and buds off into vesicle
Coat lost after buds off
Describe tubular transport intermediate
Some from Golgi or endosomes - lipid rafts
Motor proteins = pulls out the porteins
No coats at all
Some uncoated - phagosomes
Name and describe locations of coats - all main 3
Clathrin = on vesicles, Golgi, cell surface, endosomes
Cop1 = coats Golgi apparatus
Cop2 = coats er
Name all coat proteins
Clathrin/adaptin
Cop1
Cop2
Caveolin
Retromer
Describe clathrin/adaptin
Cell surface
Golgi
Endosomes
Tyrosine, dileucine signals recognized - arf1 (on Golgi endosomes)
Describe cop1
Golgi
Pre Golgi
Dilysine signals
Golgi to er trafficking
Arf1
Describe cop2
Exit from er
Di acidic, diphenyalanine motifs - sar1
Describe caveolin
Cell surface
Cholesterol rich raft domains
Describe retromer
Endosomes
Involved in recycling of cargo receptors to Golgi
How are proteins transferred to destination
Motor proteins - if have to go far
Name and describe all motor proteins used for transfer to destination
Dyenin - to neg end of my - cell centre in fibroblasts = towards Golgi
Kinesins = most to + end mt = cell periphery = away from golgi
Myosins - actin filaments usually used to travel short distances
Describe recognition of target membrane and fusion
2 step process = tethers bind - docking factors then snares bind - drive active fusion
Tethering proteins - docking factors + rabs = may be responsible for initial recognition in targeting membrane
V/t snares = responsible for fusion
Since many diff organelle specific snares = may have a role in recognition also
