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Membrane trafficking Flashcards

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1
Q

Why do eukaryotes need membrane trafficking?

A

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

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2
Q

Secretory/ exocytic pathway

A

ER
golgi
PM/endosome/lysosome

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3
Q

Endocytic pathway

A

cell surface
endosome
golgi/ER/lysosome

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4
Q

How are proteins modified in the ER/golgi?

A

glycosylation
proteolytic cleaving

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5
Q

What happens in the ER lumen?

A

addition of pre-formed oligosaccharide to an aspargine amino acid in a consensus sequence

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6
Q

What happens in the golgi?

A

further sugars are added and structure can be further branched
oligosaccharide group is trimmed

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7
Q

What is the purpose for glycosylation?

A

to assist folding for trafficking and sorting inside the cell
to assist interactions with the ECM and with other proteins/sugars on other cells

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8
Q

Advantages of yeast as a model organism

A

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

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9
Q

Disadvantages of yeast as a model organism

A

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

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10
Q

What was the rationale for the Novick and Scheckman experiment?

A

if proteins couldn’t be secreted the cell would increase density as vesicles accumulate

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11
Q

Novick and Schekman experiment

A

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

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11
Q

Novick and Schekman experiment

A

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

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12
Q

What does alpha factor have to do with the secretory pathway?

A

it is glycosylated and proteolytically cleaved at different stages
this helps us to follow it progress

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13
Q

What are the limitations of Novick and Schekman’s experiment?

A

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

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14
Q

Endocytosis

A

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

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15
Q

What is endocytosis important for?

A

retrieval of molecules that form part of the secretory vesicle for recycling
downregulation of signals
remodelling cell surface lipid and protein composition

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16
Q

Stages in the endocytic pathway

A

plasma membrane → endocytic vesicle → early endosome → late endosome or recycling to the plasma membrane → golgi or vacuole

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17
Q

What is the major function of a lysosome/vacuole in endocytosis?

A

degradation of extracellular material taken up by endocytosis and certain intracellular components by a process termed autophagy

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18
Q

Carboxypeptidase Y

A

normally transported to lysosomes having been trafficked through ER and golgi
glycosylated and proteolytically cleaved at different stages which helps us follow its progress

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19
Q

How are vacuolar mutants classed?

A

depending on what stage at which they appear to block the route to the vacuole

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20
Q

What are the 4 possible destinations from the late golgi?

A

plasma membrane
early endosome
late endosome
vacuole

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21
Q

CPY transport

A

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

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22
Q

What does the transport of CPY require?

A

cytoplasmic factors clathrin
2 adaptors called Gga1 and Gga2

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23
Q

Nuclear pores

A

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

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24
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
25
What amino acids are rich in nuclear transport recognition sites?
Lys Arg Pro
26
What is required for mRNA transport?
ATP hydrolysis as it is inhibited by cooling to 4 degrees
27
What happens in the absence of ATP in protein transport?
the protein binds to the pore complex but remains outside the nucleus
28
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
29
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
30
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
31
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
32
BiP
chaperone in ER that associates with a newly synthesised protein and ensures it fold properly via complex quality conrol mechanisms in the ER
33
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
34
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
35
Anterograde transport
through the secretory pathway
36
Retrograde transport
backwards (opposite of secretory)
37
Essential components for transport vesicle formation
GTPase adaptor proteins coat proteins
38
GEFs
guanine nucleotide exchange factors allow the exchange of GDP for GTP to activate GTPase
39
GAPs
GTPase activating proteins
40
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
41
COPII coated vesicles
formed at the exit sites of the ER and are a paradigm for transport vesicle formation
42
COPII GTPase
called Sar 1 and is a member of the Arf family adaptor is a sec protein and so is the coat protein
43
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
44
What allows recruitment of the adaptor complex?
when Sar1 is converted from GDP into GTP form by GEF
45
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
46
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
47
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
48
Smooth microsomes after centrifugation
have a low density and stop sedimenting and float at low sucrose concentration
49
Rough microsomes after centrifugation
have a high density and stop sedimenting and float at high sucrose concentration
50
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
51
What acts as a GAP for Sar1?
Sec23
52
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
53
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
54
When Sar1 is in its active form
leads to formation of coat
55
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
56
GDP mutants
will sequester GEFs so GTP dependent reactions will be suppressed
57
GTPase mutants
cannot hydrolyse GTP so these normally have dominant negative effects
58
What does expression of Sar1GDP cause?
inhibition of COPII formation
59
COPII
GTPase- Sar1 coat- COPII cargo- newly synthesised
60
COPI
GTPase- Arf1 coat- COPI cargo- retrieved & newly synthesised proteins
61
Clathrin (TGN)
GTPase- Arf1 coat- clathrin cargo- lysosomal proteins and regulated secretory proteins
62
Clathrin (PM)
GTPase- ? coat- clathrin cargo- endocytosed material
63
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
64
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
65
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
66
Rough ER function
protein synthesis
67
Smooth ER
sites of lipid synthesis and has roles in calcium storage
68
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
69
CLEM
correlative light and electron microscopy allows localisation of fluorescently labelled proteins to membrane contact sites
70
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
71
Tethers of membrane contact sites
protein-protein protein-lipid distance is usually around 30nm inhibition of membrane fusion
72
Membrane contact sites provide platforms for:
calcium mobilisation lipid transfer signalling organelle division
73
Sarcoplasmic reticulum
specialised ER for handling Ca2+ transients required for muscle contraction
74
Contact sites in the ER
non-vesicular transfer of lipids unidirectional
75
LTPs
lipid transfer proteins use concentration gradients of lipids to promote lipid transfer
76
Neimann pick disease
affects spleen, liver, lungs, bone marrow, brain happens when sphingomyelin accumulates in lysosomes
77
How do MSCs participate in cell signalling?
ER tubule interacting with a later endosome endosomes can drag sections of the ER
78
Other functions of MSCs
regulating movement of EGFR into MVB also involved in organelle fusion
79
TDP-43
pathologically linked to ALS which regulates ER-mitochondria contacts
80
Hereditary spastic paraplegia
disease associated mutations linked to REEP1 which leads to disruptions of ER mitochondria contacts

Molecular and cell biology (8 decks)

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