Membrane Trafficking Flashcards
What are the two main cell trafficking routes?
- secretory pathway
- endocytic pathway
How is cell trafficking studied?
- Pulse- chase
- incubate in florescent or media or allow accumulation
- Chase is then using normal media or removing inhibition for varying periods
What are the topologies of the smooth and rough ER?
- Smooth is tubular
- Rough is sheet-like consisting of high density of tubules
What occurs in the rough ER?
- lipid biosynthesis
- protein folding environment
- ribosomes
- Oxidising for bisulphide bridge formation
- Glycosylation, helps folding and chapreon binding
- Folding quality control
What occurs in the smooth ER?
- Lipid biosynthesis
- ER export
What is the ERGIC?
- ER Golgi intermediate
- COP II vesicles fuse
- Create vesicular tubular clusters (VTCs)
- First round of recycling
- Pulled to golgi with MTs and dyneins
What modifications occurs in the golgi?
- Gradual maturation of cisternae from cis, medial trans
- Protein modification: cleavage, addition or extension of glycans, sugar phosphorylation
How does export occur at the trans golgi network?
- Tubes and vesicles being converted to vesicles
- Point of sorting between proteins
- Either bulk, directed transport or secretory vesicles which release on signal
How are proteins designated for the ER transfered?
- First part of translation is secretion signal sequence
- Recognized by signal recognition particle (SRP) which halts translation
- Binds to SRP receptor and Sec61 translocon in ER membrane
- Transfered across ER membrane during translation
- Signal cleaved by signal peptidase
How specific is the ER signal sequence?
- Random combinations work but less efficient
- Requires hydrophobic region
How does te Sec61 translocon prevent leaking ions between ER and cytosol?
- Has a plug, only opens when attached to SRP
How are different proteins inserted to the ER?
- Soluble proteins: Signal peptide
- Single pass membrane protein: signal anchors type I, II
- Multi pass proteins: signal anchors
How is the topology of the protein determined by ER translocon?
- Cleavable vs uncleavable signal sequence/anchor
- Positive charged side stays on cytosolic side
How are COPII vesicles made?
- Sec13/31 structural
- Sec23/23 cargo selective adaptor
- In vivo starts with GDP-GTP exchange in small GTPase Sar1
- Cytosolic in D form
- Recruits Sec23/24, which recruits Sec13/31
- Sar1 T to D activity acts as timer for uncoating
How are COPI vesicles made and what signals do they use?
- In ERGIC or golgi
- Specific ER sequences
- soluble K/HDEL
- Type I KKXX
- Type II RR
- Arf1 recruits coatomer (structural and recognition)
What is the role of Erd2 receptor?
- recognizes signals for COPI
- Affinity depends on pH, low pH in golgi means it attaches to cargo and release in ER
How do maturing golgi compartments alter composition?
- Sterol and sphingolipid content increases with maturation
- Allows proteins to sort into prefered environment
- COPI helps transfer enzymes back
How does sorting in the TGN work?
- Secretion default
- Regulated secretory vesicles sorted by kin recognition, association of multiprotein complex
- Clathrin coating targets endosomes
What are different types of endocytosis?
- Phagocytosis: large particles
- Pinocytosis: membrane ruffling to take up fluid
- caveolae: flask shape internative cargo
- Clathrin coat
What are the stages of endosome to lysosome and how do they occur?
- EE receives cargo from plasma membrane and TGN
- Stops accepting plasma membrane cargo, accumulates LE proteins
- Acidified due to proton pumps
- Vesicles bud within LE
- Fuse with lysosome
What kind of fusion events occur between the LE and lysosome?
- Kiss and run, minor exchanges
- Hybrid organelle formation
- Then matures back to lysosome
How does clathrin mediated vesicle budding occur?
- Cargo selective adaptor protein complex 2 (AP2) and clathrin
- AP2 complex recognises cytoplasmic tails
- Due to specificity clathrin coated pits have much higher concentration of product making them efficient
- GTPase dynamin forms collar and pinches vesicle
- Shedding coat is ATP dependent, mediated by auxilin and chapeon hsc70
How are extracellular nutrients taken up by constitutive receptors?
- Receptors bind to nutrients then use specific cytoplasmic tails to attach to AP-2
- Once in EE acidity reduces affinity allowing receptor to be recycled
- Receptors without cargo also internalized
How are extracellular nutrients taken up by ligand triggered receptors?
- Only internalize when bound
- Becomes ubiquinated when binding, targetting it for lysosome
- In EE ESCRT(0 to III) prevents recycling
- Then creates intralumen vesicle away from ESCRT to make cargo accessible to lysosome
- Thereby ESCRT proteins avoid degredation
- Ex. epidermal growth factor (EGF) recognized by Epsin which binds to AP2
How are proteins targeted from the TGN to endosome?
- Ex. lysosomal hydrolase have signal patch
- Becomes phophorylated in golgi to display mannose-6-phosphate (M6P)
- M6P binds to clathrin adaptors, AP1 and GGAs
- Low pH dissociate from protein and recycled using retromer coat
What protein coats transport what pathway?
- sec23/24, sec13/31, Sar1 in ER to ERGIC
- Coatomer, Arf1 in golgi and ERGIC to ER
- Clathrin, AP1, Arf1 in TGN to endosome
- AP4 in TGN to autophagosome
- Retromer or AP5 in endosome to TGN
- Clathrin, AP-2 dynamin in plasma membrane to EE
How many Phosphoinositides (PIPs) can be made and what is their role?
- 7
- Membrane identity markers, easily converted
- Provides coincidence detection by join binding with cargo to AP2
How does vesicle transport work?
- Mediated by motor proteins and rearrangement of cytoskeleton
How are tethers recruited to the right membrane?
- Different GTPases localize to different membranes based on membrane GEFs
- Recruit diverse Rab effectors, creating a microdomain of specific lipid protein composition
- GEFs recruit tether
What two extra proteins are needed for vesicle fusion?
- ATPase NSF helps uncoil snares
- a-SNAP helps NSF attach
What is the SNARE hypothesis?
- SNAREs are SNAP receptors
- For every synaptobrevin there is a syntaxin which are fusogenic
How do SNAREs drive membrane fusion?
- Energy release from binding
- Multiple bind to create trans-SNARE complex
- Deform membranes
How are SNARE’s recycled?
- NSF breakup of SNARE complexes
- Retrograde transport of inactive and active SNAREs
