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Cell Biology > Protein Trafficking > Flashcards

Protein Trafficking Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

Describe the mechanisms of how proteins move between organelles

A

Gated transport- between cytosol and nucleus
Transmembrane transport- from cytosol across membrane to different spaces
Vesicular transport- vesicles between compartments

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2
Q

Architecture and organisation of nucleus

A

Double membrane each with different proteins in them
Outer membrane continuous with ER and studded with ribosomes
Perinuclear space is continuous with ER lumen

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3
Q

Architecture of NPC

A

High rate of macromolecules in and out
Bidirectional
Nucleus to cytoplasm and cytoplasm to nucleus
Molecules smaller than 5000 dalton (D) diffuse freely
Molecules >60000 D kept out by disordered mesh
Aqueous pores so folded proteins can enter and exit
Proteins need to be carried across which requires a signal (NLS)
NPC has cytoplasmic fibrils, central framework within membrane and a nuclear basket

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4
Q

Key studies that led to discovery of active nuclear transport

A

Purified nuclear protein accumulates in nucleus of frog oocytes after being injected into cytoplasm - John Gurdon (shows active process)
Used protein nucleoplasmin to identify a domain in the protein that acts as a signal (shows specifically trafficked)
Exposed tail and protected core, if you remove the tail core fails to enter nucleus, more signposts means greater likelihood of coming into contact with a receptor and a signal occurring
NLS is sequence dependent, you can search genomes to find it

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5
Q

Roles of Ran, GEF and GAP in nuclear transport

A

RanGTP and RanGDP are 2 distinct shape conformations
RanGDP in cytoplasm and RanGTP in nucleus
Cargo protein NLS binds to importin alpha/beta
Passes through npc
In nucleus, RanGTP binds to importin beta, dissociating it from NLS and importin alpha
Importin alpha taken up by CAS nuclear export factor
Importin alpha/beta taken back through npc to cytosol, taking RanGTP with them
Ran shuttled back into nucleus by NTF2
In nucleus, ranGEF catalyses conversion of Ran GDP to Ran GTP
RanGAP- GTPase activating protein, in cytosol so RanGDP in cytosol
RanGEF- binds to chromatin so localised in nucleus, Guanine exchange factor so RanGTP in nucleus

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6
Q

Potential models for transport through NPC

A

Large proteins have an NLS made up of basic amino acids
Cargo protein NLS binds to importins/exportins
Soluble proteins so gives flexibility to what can be imported
Doesnt have to be a direct interaction, cargo protein can bind to a secondary intermediate with an NLS
Importins/exportins bind to F-G repeats in unstructured domains of NPC nucleoporins
Alpha and beta chain importins interaction controls interaction with cargo and NPC
Alpha chain binds to NLS
Beta chain outer surface binds to FG repeats
Beta chain binds alpha chain with cargo
Beta can bind RanGTP but RanGTP displaces alpha and NLS as it partly covers loop in receptor required for NLS binding

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7
Q

Structure and function of ER

A

Continuum with nuclear membrane
Function focused on proteins: membrane protein assembly, makes most secreted proteins and those destined for lumen of ER, golgi and lysosomes, site of protein folding and modification, quality control, cellular lipids made in ER membrane

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8
Q

RER:

A

Ribosomes
More RER means higher capacity of cells to secrete proteins, membrane bound ribosomes synthesise secretory proteins

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9
Q

SER

A

Makes and metabolises fat e.g. those involved in steroid hormone synthesis
Major Ca2+ store, some regions specialised for storage e.g. sarcoplasmic reticulum
Males lipoprotein lipid carriers and detoxifies lipid soluble drugs, poisons and metabolites to make them water soluble for excretion in liver

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10
Q

Describe co-translational translocation

A

Uses signal recognition particle (SRP) and SRP receptor
SRPs move between ER membrane and cytosol and bind to any Er signal sequence
SRP binds to this and the ribosome to pause translation
Signal peptide recognised by SRP in cytosol and translocator in ER membrane
Polypeptide transloacted through pore in translocator
Signal peptidase cleaves off signal peptide
Mature protein released into ER lumen
Translocator closes

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11
Q

Describe post translational translocation

A

Doesnt use SRP or receptor
BiP provides direction
BiP bound to ATP coverts to ADP
Successive binding pulls polypeptide through

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12
Q

How does the Sec translocon work

A

Used in co and post translational translocation
Aqueous pore
Closed by helical plug when unused so ‘gated’
Polypeptide is fed unfolded through cores hydrophobic lining
Core can open to let cleaved signal sequences out and integrate membrane proteins

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13
Q

Transmembrane proteins

A

Have stop transfer sequence and start
Translocator protein contains hydrophobic stop and start transfer sequence binding sites
Start transfer sequence cleaved off, stop remains in membrane
Stop transfer sequence stops the transfer of the peptide through the translocon and then exits laterally
Ribosome translates the rest of the protein

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14
Q

Multipass membrane protein

A

Contain multiple hydrophobic stop and start transfer regions
By looking at hydrophilic and phobic regions of the peptide we can predict the topology as hydrophobic regions will be inside the membrane

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15
Q

Function of Golgi apparatus

A

Membrane proteins from ER and ER lumen sorted here
Accepts proteins and lipids from ER
Post translational modifications
Covalently modifies, labels, sorts and sends off
Oligosaccharide modifications, further additions to N-linked glycosylation added to many proteins
Has directionality, cis face is receiving end and trans face is exit end
Fused vesicular tubules with lipids and proteins from er enter
Proteins and lipids exit to cell surface or another organelle
Dynamic

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16
Q

Structure of Golgi

A

Enzymes compartmentalised in Golgi cisternae
Allows you to regulate activity
Enzyme complexes for further N and O modification
Number of stacks varies depending on cell type

17
Q

What post-translational modifications occur in the golgi

A

Modifying N-linked glycosylation
Already added in ER then modified in golgi, made more complex by exposure to golgi enzymes
Also O-linked glycoslyation
Sugars added to -OH group of Ser or Thr side chains
Glycosyl transferases add the sugar nucleotides
Example of heavily O-linked glycosylation are mucins, core proteins of mucus
Proteins move through golgi by vesicular transport model and cisternal maturation model
Some proteins go back to ER if they are ER resident proteins or for membrane replenishment
Prevents depletion of lipids

18
Q

Structure of the lysosome

A

Vary in size and number
Single compartment
Full of hydrolytic enzymes
Often appears crystalline because it is so packed
Low internal pH protects the cell if lysosome breaks as most enzymes wont work
PH 4.5-5, cytosol s 7.2
H+ ATPase acidifies and drives metabolite transport

19
Q

Function of lysosome

A

Hold enzymes in active state, fuse with target cell, a hybrid forms, enzymes get to work
Delivery of proteins to mature at lysosome
Repairing membranes
Debris, phagocytosed microorganisms
Autolysis (cell destruction in injured or dying cell)
Apoptosis

20
Q

Alternative routes to the lysosome

A

M6P tag allows material to enter lysosome (salmonella hijacks this by stopping recycling of M6P receptors)
Endocytosis
Autophagy
-allows orderly degradation and recycling of Elul are components
-targeted parts of cytoplasm isolated from cell within a double membrane vesicle (autophagosome)
-fuses with lysosome so contents can be degraded and recycled
-helps people live longer as it stops damage accumulating
Phagocytosis

21
Q

What are lysosomal storage disorders

A

Arise from mutations in genes that encode enzymes e.g. enzymes that label enzymes for delivery to lysosome, failure processing in ER can inactivate a whole class of enzymes
Defects in proteins that help endosomes and autophagosomes fuse with a lysosome
Defects in processing enzymes can cause undigested material to accumulate
Can be treated with enzyme therapy, substrate reduction therapy to stop damaging product accumulating, gene therapy, bone marrow to replace set of healthy stem cells

22
Q

How do vesicles form

A

Coat protein assembly at membrane causes bilayer to begin to bend
Coat proteins may also select cargo to be packaged into the vesicle
More coat protein binding results in the formation of a sphere of membrane
Once vesicle pinches off, coat detaches and vesicle is transported to destination
Helps to concentrate proteins into a specialised patch on membrane for designated transport

23
Q

Roles of COPI, COPII and Cathrin in vesicle trafficking

A

COPII covers vesicles coming from ER
COPI surrounds vesicles from golgi
Clathrin surrounds vesicles from plasma membrane

24
Q

How does COPII work

A

Sec12p is a GEF that exchanges GDP to GTP in Sar1p
Sar1p is activated by GTP so inserts helix to imbed in ER membrane
Activated Sar1p acquires Sec23/24p to form the core inner coat of COPII coat
Acquires outer coat scaffold complex sec13/31p
Pinching off
GTP hydrolysis by Sar1p releases the coat
Highly selective process
Exit signals involved
Only proteins correctly folded and assembled leave the ER

25
Q

How does COPI work

A

Movement from golgi to ER
Coat formed from 7 polypeptides, all one coat not inner and outer like COPII
Arf1 GTPase similar to Sar1
GAP and GEF

26
Q

Clathrin

A

Arf GTPase initiates assembly recruiting co-factors
Adaptor proteins give specificity
Dynamin pinches off vesicles
Clathrin coated vesicles composed of triskellions composed of 3 Clathrin heavy chains interacting at their C termini
Each heavy chain has a light chain tightly bound
Heavy chain provides structural backbone of lattice and light chain regulates formation and disassembly of lattice
Vesicle uncoating mediated by Hsc70 and auxilin
Movement from Pm to endosomes, from trans golgi network to endosome, TGN to pm , TGN to lysosome, each destination has different adaptor protein to provide specificity

27
Q

Molecular strangulation with dynamin

A

Dimeric GTPase recruited to membrane
GTP hydrolysis creates power stroke that increases constriction of vesicle
Fission of membrane where membrane stress is the largest
Disassembly of the oligomer and recycling of dynamin
Non-functional at high temperatures, no vesicle regeneration at synapse so causes paralysis

28
Q

How does vesicle fusion with target occur

A

Fusing membranes energetically unfavourable
Thought to go through intermediates

29
Q

SNARE hypothesis for vesicle fusion

A

Series of molecules thought to play a role in vesicle fusion
Hypothesis: each type of transport vesicle carries a specific V-SNARE that binds to a t-SNARE on the target membrane making a stable 4 helix bundle
1 helix contributed by V-snare and 3 by oligomeric t-snare
Promote fusion by overcoming energy barrier
Help ensure specificity of membrane fusion
Different complexes form at different steps of intracellular transport
Highly specific interaction
Clinical relevance: many neurotoxins inhibit snare complex formation e.g. tetanus

30
Q

Rab GTPases in vesicle fusion

A

Guide vesicle targeting as lots of different forms each specific for different cargos
Active Rab-GTP binds Rab effectors for tethering vesicles to membranes or movement
Includes it or proteins, tethering proteins or SNARES coupling the ring to fusion

31
Q

Secretion of proteins to outside e.g. ECM

A

Some of key proteins secreted are components of ECM and cell-cell adhesion complexes
ECM made up of collagen, proteoglycans and laminins
Basal lamina is a thin mesh of ECM molecules that are important in tissue repair and development, contains collagen, perlecan and multi-adhesive matrix proteins
Tensile strength of connective tissue provided by collagen
These molecular components come together to make up tissues such as epithelium
Cells moving through ECM have to secret factors that disrupt the ecm

Cell Biology (6 decks)

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