Secretory/Endocytic Pathways Flashcards
Give an overview of the secretory pathway?
Refers to proteins that traffic to the ER, Golgi, plasma membrane and vesicles that travel between them
Named ‘secretory’ as it is the pathway through which cells secrete proteins into the extracellular environment
Not all proteins are secreted - transmembrane domain, endosomes, lysosomes
Some are directed towards organelles
Describe the initial secretory pathway?
- Secretory proteins move from the RER lumen through the Golgi and then to the cell surface
Incorporated into small (~50 nm) transport vesicles - Fuse with the cis-Golgi from the ER
- Proteins to be secreted move by cisternal migration to the Trans-Golgi
These proteins are sorted in the trans-Golgi network (TGN) into transport vesicles
What are the next potential options in the secretory pathway after the TGN?
At the TGN, proteins can be continuously secreted
Example: serum proteins in hepatocytes
OR regulated secretion also occurs - TGN sorts proteins into secretory vesicles that are stored awaiting a stimulus
Hormones (e.g. insulin) - KATP channels
Rise in the cytosolic Ca2+ triggers fusion of the secretory-vesicle with the plasma membrane and release of the vesicle contents by exocytosis
OR some proteins are also delivered to lysosomes for degradation
OR Membrane proteins are delivered to the cell surface or other organelles to become part of their repertoire of membrane proteins
How can we study the secretory pathway?
Vesicular stomatitis virus G protein (VSG-G) is a membrane glycoprotein
VSV-G transfected cells rapidly synthesise VSV-G as they would there own secretory proteins
There are 3 experiments
What are the 3 experiments to study the secretory pathway?
Experiment 1
Temperature sensitive version of VSV-G allows researchers to switch its transport on and off
40°C - misfolded and ER retained
32°C - correctly folded
By varying the incubation temperature of cells from 40°C to 32°C - allows VSV-G movement through the secretory pathway to be followed
Experiment 2
We can also follow the modifications made to VSV-G
Experiment 3
Cells pulsed with radiolabelled AAs at 40°C (to prevent correct folding of VSV-G)
Cells returned to 32°C for to permit folding and ER exit
VSV-G proteins extracted and assayed for Endo-D cleavage
Describe the vesicular budding/fusion pathway?
Assembly of a protein coat drives vesicle formation and cargo selection
Driven by polymerization of soluble protein complexes on the membrane
Protein coat provides curvature and acts as a filter allowing other proteins to be added.
Can disassemble to leave a completed transport vesicle
Sorting, Budding, uncoating, tethering/docking and fusion
What are the types of mechanisms of membrane curvature?
Types of phospholipids differ across the bilayer
- Lipid asymmetry (however, most are protein mediated)
- Large curved proteins scaffold onto and bend the membrane
- Insertion of amphipathic α-helices in leaflet of the bilayer
- Oligomerization of monomers on the membrane
- High surface concentration of membrane-binding proteins leads to crowding and steric pressure that bends the bilayer
What are coat proteins?
Vesicles are named after their major coat proteins
Formed by reversible polymerization
COPII vesicles: ER to Golgi
COPI vesicles: Cis-Golgi back to the ER
What is the role of GTPases?
They control the assembly of different vesicle coats
The coats of vesicles contain small GTP binding proteins that regulate coat assembly
ARF1: COPI and clathrin
SAR1: COPII vesicles
These switch between GDP and GTP bound forms
Control the initiation of coat assembly
Give an example of ER to Golgi COPII transport - Sar1?
COPII coat - ternary complex between Sar1, Sec23 & Sec24
Together mediate membrane deformation and vesicle fission
- Sar1 membrane binding - recognised by sec12 and inserts through the hydrophobic N-terminus
Using GTP exchange to give a conformational change in Sar1 - COPII coat assembly - Sar 1 binds sec23/sec24 complex (recruited by a signal)
- Sec23 promotes GTP hydrolysis of Sar1
- Release of Sar1-GDP allows coat disassembly = uncoated vesicle formed containing our cargo
What is used for releasing the vesicle?
Dynamin - polymerises around the neck of the vesicle
Uses energy from the hydrolysis of GTP to drive a conformational change that stretches the neck of the vesicle until it pinches off - fission event
Experimental evidence
Cells incubated with GTP—Ƴ-S a non-GTP hydrolysable derivative of GTP
Image shows a long-necked clathrin bud that cannot pinch off in the absence of GTP hydrolysis
Describe vesicle uncoating?
Clathrin coated pits also uncoat
Depolymerisation is driven by HSP70
Allows clathrin triskelion’s to be re-used for another round of vesicle formation
Permits the exposure of v-Snares for docking onto target membranes - allows specific targeting
What is involved in tethering, docking and fusion?
SNARE proteins
SNARE proteins: “SNAP” (Soluble NSF Attachment Protein) Receptor” (~60 members)
Two categories
Vesicle or v-SNAREs - incorporated into the membranes of transport vesicles during budding
Target or t-SNAREs - associated with target membranes
Snare proteins guide vesicles to their target membrane for fusion
How can SNARE proteins be classed?
As R-SNAREs and Q-SNAREs
R-SNAREs act as v-SNAREs and contribute an arginine (R) residue
Synaptobrevin: R-SNARE
Q-SNAREs act as t-SNAREs and contribute a glutamine (Q) residue in the assembled core SNARE complex
Syntaxin and SNAP-25: Q-SNAREs
Describe sorting at the Golgi?
As cargo proteins move from the cis to the trans-Golgi they are subject to modifications
Retrograde trafficking of these enzymes ensures their high levels in the cis-Golgi
Example - Glycosyl transferases
Means that only fully processed cargo reaches the TGN
TGN: Major sorting station for proteins
Clathrin and Adaptor proteins mediate transport from the TGN
What are adaptor proteins?
AP1-5 are clathrin-associated adaptor protein complexes
Recognize tyrosine-based and dileucine-based signals
AP1 - TGN and endosome trafficking protein
AP2 - key mediator of clathrin mediated endocytosis, recognises the specific cargo on the proteins of the plasma membrane - undergo endocytosis into intracellular compartments
AP3 - TGN and endosome trafficking protein
AP4 - TGN trafficking protein
AP5 - endosome/lysosome delivery
GGA1-3 (Golgi-localized, γ-ear containing, ADP-ribosylation factor binding) regulate the trafficking of cargo molecules from the trans-Golgi network to the endosome/lysosome system
What is the role of adaptor proteins?
They all bind cargo and bring in the coat subunit to form the vesicle
They have specific localisations within cells - depending on where they need to be trafficked to
Dictated by motifs - targeting sequences
Describe clathrin?
Plays a major role in the formation of coated vesicles
Forms a triskelion composed of three clathrin heavy chains and three light chains
Performs critical roles in shaping rounded vesicles in the cytoplasm for intracellular trafficking
Adaptor molecules are responsible for its self-assembly and recruitment
Describe targeting sequences?
Targeting sequences on cargo make specific contacts with coat proteins
Vesicle coat selects cargo for delivery through direct binding to sorting signals
COPII transport, cargo protein associates with Sec24
Sec24 recognises di-acidic motifs at the ER (COPII)
AP µ subunits recognise tyrosine and di-leucine motifs
What can cargo traficking be dictated by?
Protein modifications
Mannose-6-phosphate (M6P) in the cis-Golgi
Generated by a two step reaction from the Man8(GlcNAc)2 N-linked chains
Addition of MP6 stops cargo proteins from undergoing any further processing reactions that permit their entry into the secretory pathway
Recognised by the MP6 receptor in clathrin/AP1 vesicles
MP6 receptor binds mannose phosphate at pH 6.5 (Golgi)
Does not bind at pH ≤ 6
Lysosomal enzymes are released at lysosomal pH (5.0-5.5)
Late endosome phosphatases also remove the phosphate from MP6 and release it from MP6R
What is endocytosis?
Uptake from extracellular fluid into the cytoplasm of a cell
There are three major pathways:
Phagocytosis
Pinocytosis
Receptor mediated endocytosis
Describe the three endocytic pathways?
Phagocytosis
Cell uses its plasma membrane to engulf large particles
Gives rise to an internal compartment: the phagosome
Pinocytosis
Fluid endocytosis and bulk-phase pinocytosis
Small particles in extracellular fluid are taken up through ruffling of the cell membrane (uses actin?)
Results in a suspension of the particles within a small vesicle that eventually fuses with the lysosome
Involves a considerable investment of ATP
Receptor mediated endocytosis
Motif in the cargo is recognised by the endocytic machinery
Dictates delivery of a cargo or receptor to a specific cellular compartment
Examples - Transferrin receptor, Low-density lipoprotein receptor and Epidermal growth factor receptor
Describe the examples - Transferrin receptor, Low-density lipoprotein receptor and Epidermal growth factor receptor - of receptor mediated endocytosis?
Transferrin receptor - recycling
Carrier protein for transferrin
Required to import iron into the cell
Surface expression is regulated in response to intracellular iron concentration
Imports iron by internalizing the transferrin-iron complex through receptor-mediated endocytosis
LDL degradation
LDL receptors in the liver remove unneeded cholesterol - by binding cholesterol esters and uses endosomes acidic pH to recycle the LDL receptors
Increased expression of LDL receptors: more LDL cholesterol is removed from the body
EGFR degradation
EGFR - produces a signal, and internalise the receptor - it dimerises and is sent to the endosome/lysosome in order to turn off the signal
This is required for:
1. Signalling platforms in endosomes
2. Degradation to switch off EGFR signalling
