Introduction to membrane trafficking Flashcards by Misha Gandhi

Describe secretory pathway - image

Aim to cell surface
Er
Golgi

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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

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Describe endocytic pathway - image

Outside in
4 MAJOR ORGAnelles = endosome, recycling endosome, late endosome, lysosome
Targeted to lysosome for degradation

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Describe endocytic pathway - general plan

Cell surface - cell exterior
Early endosome - alternate pathways too
Late endosome
Lysosome

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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

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What is autoradiography

Pulse chase technique

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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

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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

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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

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What is pulse chase - explain

Triniated leucine - small # mins, protein visible = pulse then chase = take pics at diff times and see proteins

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What did autoradiography And em do

Provided first direct evidence that proteins to be secreted appeared first in er then in Golgi

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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

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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

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Describe Palade exp - step 3

Label like usually
Incubate for 37 mins - longer = chase further
Label now over vacuoles

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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

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Describe Palade exp conclusions

Proposed transport vesicles shuttled proteins between compartments - but molecular mechanisms of trafficking between compartments unknown

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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

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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