Membrane trafficking Flashcards
Why do eukaryotes need membrane trafficking?
compartmentalisation allows more complexity
enzymes can modify subsets of proteins in certain environments
sequential modifications require exposure to distinct sets of enzymes
important in retrieval of proteins back to resident compartment
Secretory/ exocytic pathway
ER
golgi
PM/endosome/lysosome
Endocytic pathway
cell surface
endosome
golgi/ER/lysosome
How are proteins modified in the ER/golgi?
glycosylation
proteolytic cleaving
What happens in the ER lumen?
addition of pre-formed oligosaccharide to an aspargine amino acid in a consensus sequence
What happens in the golgi?
further sugars are added and structure can be further branched
oligosaccharide group is trimmed
What is the purpose for glycosylation?
to assist folding for trafficking and sorting inside the cell
to assist interactions with the ECM and with other proteins/sugars on other cells
Advantages of yeast as a model organism
amenable for genetic studies as they can grow as haploid or diploid
entire genome sequence is fully known and annotated
cheap and easy to grow large quantities
limited gene diversity + fundamental pathways conserved
Disadvantages of yeast as a model organism
limited cell to cell contact so uninformative about multicellularity
small so high resolution imaging of intracellular compartments difficult
cell wall which can preclude some studies
What was the rationale for the Novick and Scheckman experiment?
if proteins couldn’t be secreted the cell would increase density as vesicles accumulate
Novick and Schekman experiment
cells were analyses for their ability to secrete enzymes at permissive and restrictive temps
defined secretory mutants as those that failed to export active invertase and acid phosphatase
23 genes identified to be required to ensure protein transport from ER to membrane
Novick and Schekman experiment
cells were analyses for their ability to secrete enzymes at permissive and restrictive temps
defined secretory mutants as those that failed to export active invertase and acid phosphatase
23 genes identified to be required to ensure protein transport from ER to membrane
What does alpha factor have to do with the secretory pathway?
it is glycosylated and proteolytically cleaved at different stages
this helps us to follow it progress
What are the limitations of Novick and Schekman’s experiment?
only considered secretion to plasma membrane
defects in transport to endosome or vacuole would not be identified
redundantly functioning genes would not be identified as there isn’t much of this in yeast
Endocytosis
process through which the plasma membrane invaginates into the cell resulting in the production of a vesicle that is then able to fuse with endosomes and enter the endo-lysosomal system
What is endocytosis important for?
retrieval of molecules that form part of the secretory vesicle for recycling
downregulation of signals
remodelling cell surface lipid and protein composition
Stages in the endocytic pathway
plasma membrane → endocytic vesicle → early endosome → late endosome or recycling to the plasma membrane → golgi or vacuole
What is the major function of a lysosome/vacuole in endocytosis?
degradation of extracellular material taken up by endocytosis and certain intracellular components by a process termed autophagy
Carboxypeptidase Y
normally transported to lysosomes having been trafficked through ER and golgi
glycosylated and proteolytically cleaved at different stages which helps us follow its progress
How are vacuolar mutants classed?
depending on what stage at which they appear to block the route to the vacuole
What are the 4 possible destinations from the late golgi?
plasma membrane
early endosome
late endosome
vacuole
CPY transport
synthesised in prepro form and transported through to ER to the golgi where it is recognised by Vsp10 receptor
dissociates from Vsp10 at late endosome and trasnported to vacuoles where it is cleaved to generate mature form
Vsp10 retrieved to late golgi through specific aromatic signal in its protein sequence
What does the transport of CPY require?
cytoplasmic factors clathrin
2 adaptors called Gga1 and Gga2
Nuclear pores
consists of multiple copies of 30 different nucleoporins
each complex is made of 8 subunits with a central plug
contains DNA, nuclear pore proteins and tight junctions
Examples of substances moved across the nuclear envelope by pore complexes
transporting histones from the cytoplasm for packaging of new DNA
ribosomal units formed in the nucleus being transported into the cytoplasm
What amino acids are rich in nuclear transport recognition sites?
Lys
Arg
Pro
What is required for mRNA transport?
ATP hydrolysis
as it is inhibited by cooling to 4 degrees
What happens in the absence of ATP in protein transport?
the protein binds to the pore complex but remains outside the nucleus
Why must ribosomes be so closely associated with the ER membrane?
newly made proteins can be translocated into organelles co or post translationally
the majority are co-translationally translocated
How is a soluble protein destined for secretion synthesised co-translationally?
Signal sequence on growing polypeptide chain → cleavage of signal peptide → (through signal peptidase) NH2 → mature polypeptide chain in the ER lumen
How is the ER lumen adapted for protein synthesis and modification?
ER lumen is rich in chaperones and protein disulphide isomerases which allows the formation of sulfide linkages within proteins
How are membrane proteins inserted into the ER?
signal sequence that starts the transfer is recognised by sec61, the translocator → fed through but encounters a stop transfer sequence, usually a hydrophobic part of the protein -> means it is anchored to the ER → so the rest of the protein is exposed to the cytoplasm
BiP
chaperone in ER that associates with a newly synthesised protein and ensures it fold properly via complex quality conrol mechanisms in the ER
What is the unfolded protein response?
translation is closed down and the synthesis of chaperones is upregulated due to quality control becoming swamped
cell can recover but apoptotic pathways will be activated if overwhelmed
How do mitochondria, chloroplasts and bacteria move proteins post-translationally?
N terminal sequence is recognised by the TOM complex → the protein translocates through TOM and TIM23 → translocates through TIM23 into matrix of mitochondria → signal is cleaved off
Anterograde transport
through the secretory pathway
Retrograde transport
backwards (opposite of secretory)
Essential components for transport vesicle formation
GTPase
adaptor proteins
coat proteins
GEFs
guanine nucleotide exchange factors
allow the exchange of GDP for GTP to activate GTPase
GAPs
GTPase activating proteins
Ran GTPase
if cargo needs to enter nucleus it binds to nuclear receptors
when cargo dissociates and exits nucleus it encouters GAP
GAP hydrolyses GTP to GDP so cargo dissociates from receptors, so they can pick up more cargo
COPII coated vesicles
formed at the exit sites of the ER and are a paradigm for transport vesicle formation
COPII GTPase
called Sar 1 and is a member of the Arf family
adaptor is a sec protein and so is the coat protein
What is the role of Sec24 in vesicular transport?
component of Sec24 recognises signal on cargo receptor which allows it to be recruited into a COPII vesicle
What allows recruitment of the adaptor complex?
when Sar1 is converted from GDP into GTP form by GEF
How are ER resident proteins excluded from the bud during COPII vesicle formation?
done by having a high SA:V ratio of the bud
so they don’t become trapped
What is the purpose of COPII vesicle coating?
adaptor complex allows coat to be recruited
acts as a physical structure to stabilise the buds to make sure they form correctly
form a structural scaffold
What occurs after the bud is formed in vesicular transport?
pinches off to create a coated vesicle crowded with receptors and cargo
this helps with exclusion of ER proteins to prevent their escape
Smooth microsomes after centrifugation
have a low density and stop sedimenting and float at low sucrose concentration
Rough microsomes after centrifugation
have a high density and stop sedimenting and float at high sucrose concentration
How can ER be isolates from cells and used for reconsitution?
homogenization to break and vesiculate ER to create rough and smooth microsomes
put in tube with gradient of increasing sucrose concentration
centrifuge to seperate rough and smooth microsomes
What acts as a GAP for Sar1?
Sec23
When is secretion of vesicles blocked?
when coated vesicles cannot fuse with the target and deliver cargo
if they have a bulky coat it must be removed after the vesicle buds
When is GAP activity enhanced?
following recruitment of the Sec13/31 coat
so the forming of the coat is coupled to this
leads to inactivation of GTPase Sar1
causes coat to dissassemble
When Sar1 is in its active form
leads to formation of coat
When Sar1 is in its inactive form
leads to dissassembly of coat
this cycling is necessary as they can’t function properly if activated all the time
GDP mutants
will sequester GEFs so GTP dependent reactions will be suppressed
GTPase mutants
cannot hydrolyse GTP so these normally have dominant negative effects
What does expression of Sar1GDP cause?
inhibition of COPII formation
COPII
GTPase- Sar1
coat- COPII
cargo- newly synthesised
COPI
GTPase- Arf1
coat- COPI
cargo- retrieved & newly synthesised proteins
Clathrin (TGN)
GTPase- Arf1
coat- clathrin
cargo- lysosomal proteins and regulated secretory proteins
Clathrin (PM)
GTPase- ?
coat- clathrin
cargo- endocytosed material
Trans goli network
major sorting station where a decision is made as to whether something will go straight to the mebrane or to the endosomal pathway
Adaptor proteins
recognise and select cargo ensuring specificity
link coat to the membrane
recognise motifs in the cytoplasmic domains of membrane proteins
needed for all coated vesicles
AP2
major clathrin adaptor
recognises a variety of peptide motifs and apendages
the beta 2 subunit is able to bind clathrin and the smaller subunits recognise the signals in transmembrane proteins
Rough ER function
protein synthesis
Smooth ER
sites of lipid synthesis and has roles in calcium storage
How do advances in microscopy allow us to understand structure and function of membranes?
electron microscopy- size and exclusion of ribosomes, visualisation of protein tethers
live cell imaging- dynamics
CLEM
correlative light and electron microscopy allows localisation of fluorescently labelled proteins to membrane contact sites
Structure of membrane contact sites
ribosomes are excluded from contact sites
membranes are very close at 10-80nm
ER contacts can be short or long lived
Tethers of membrane contact sites
protein-protein
protein-lipid
distance is usually around 30nm
inhibition of membrane fusion
Membrane contact sites provide platforms for:
calcium mobilisation
lipid transfer
signalling
organelle division
Sarcoplasmic reticulum
specialised ER for handling Ca2+ transients required for muscle contraction
Contact sites in the ER
non-vesicular transfer of lipids
unidirectional
LTPs
lipid transfer proteins
use concentration gradients of lipids to promote lipid transfer
Neimann pick disease
affects spleen, liver, lungs, bone marrow, brain
happens when sphingomyelin accumulates in lysosomes
How do MSCs participate in cell signalling?
ER tubule interacting with a later endosome
endosomes can drag sections of the ER
Other functions of MSCs
regulating movement of EGFR into MVB
also involved in organelle fusion
TDP-43
pathologically linked to ALS which regulates ER-mitochondria contacts
Hereditary spastic paraplegia
disease associated mutations linked to REEP1 which leads to disruptions of ER mitochondria contacts
