Protein trafficking (J.B) Flashcards
Why is compartmentalization needed?
It is important as it allows for specialization.
What is the difference between the cytosol and cytoplasm?
Cytosol –> the intra-cellular fluid
Cytoplasm –> Total content within the cell membrane other than the contents of the nucleus
What is the structure of the nucleus and whats its function?
- Function –> protect the genome
- Structure –> Nucleus is continuous with ER –> structurally held together with intermediate filament based basket/lamina –> nuclear pores govern the movement of molecules in and out –> lamina proteins facilitate the breakdown of the nuclear envelope (important for cell division).
Briefly outline the activation of lamins and the breakdown of the nuclear envelope.
- Phosphorylation of lamins
- Results in the breaks down nuclear envelope
- Dephosphorylation of lamins
- Results in the fusion of the nuclear envelope
This shows how the envelope breaks down and reforms quickly.
Summary of the nucleolus.
Nucleolus
- Largest structure in the nucleus of eukaryotic cells
- Not membrane-bound
- The primary site of ribosomes synthesis and assembly
- High-density region is due to the aggregate of macromolecules used for ribosomes synthesis.
- Assembled ribosomes subunits are transported to the cytoplasm
- They are often multiple nucleolus – with varying sizes.
Summary of the endoplasmic reticulum.
ER
- Linked to the nucleus
- double membrane structure
- Unique structure that varies between cells
- Half of the total membrane in the eukaryotic cell is the ER
- Forms net-like labyrinth of tubes and sacs extending throughout the cytosol.
- Very dynamic structure
- Can be RER (rough ER) with ribosomes on the outside –> used for protein synthesis.
- Or it can be SER (smooth ER) –> used in lipid synthesis and also acts as a calcium storage and a site of carbohydrate metabolism.
Summary of Golgi.
Golgi –> Known as the postal sorting office
- Located in the middle of the trafficking pathway.
- Smooth tubular ordered stacks of flattened cisternae
- It sorts proteins, packages them into membrane-bound vesicles –> which are then sent to the appropriate destination.
Summary of the lysosome.
Lysosome
- Spherical organelle –> enclosed by a lipid bilayer
- Contains digestive enzymes
- Low pH inside
- Acts as a waste disposal centre –> break down protein, nucleic acids, carbohydrates, lipid, etc.
Summary of endosomes.
Endosomes
- Spherical –> also enclosed by a lipid bilayer
- Endosomes provide an environment for material to be sorted before it reaches the degradative lysosome.
- Three main types:
1. Early (sorting)
2. Recycling (return)
3. Late (target of degradation)
Summary of peroxisomes.
Peroxisomes –> key site for redox reactions
- Spherical organelle –> enclosed by a lipid bilayer
- Plays an important role in oxidative reactions with O2
- Take organic substrates and oxidizes them to produce H2O2 –> which is then used to process toxic substances (Ethanol)
Summary of plastids.
Plastids –> Mitochondria or Chloroplasts
- Important for ATP generation
- Some are involved in the storage of synthesis role –> varies depending on cell type.
Explain the endosymbiotic origin of organelles.
According to the endosymbiotic origin –> Symbiotic relationships between organisms is the driving force for evolution.
Endosymbiotic origin
- An initial ancestral prokaryotic cell with DNA
- Invaginations/infolding of P.M to form ER and nuclear envelope
- Consequently –> the cell engulfed another prokaryote –> symbiotic relationship to form mitochondria and chloroplasts
- Evidence for the engulfing process –> double membrane, own genome, 70s ribosomes.
What are the three models of protein trafficking?
- Gated transport –> proteins and RNA molecules between cytosol and nucleus –> through nuclear pore complexes in the nuclear envelope.
- Transmembrane transport –> form cytosol across the membrane into different spaces –> transmembrane protein translocators directly transport specific proteins across a membrane from the cytosol into a space that is topologically distinct –> protein usually has to unfold to be transported.
- Vesicular transport –> vesicles transport between compartments –> vesicles and fragments become loaded with a cargo of molecules derived from the lumen of one compartment as they bud and pinch off from its membrane –> discharge their cargo into a second compartment by fusing with the membrane (topologically the same).
What is the destination number one for protein trafficking?
Nucleus
- Nuclear membrane –> major site of protein import/export via nuclear pore complex (regulate the passage of proteins)
- Nucleus takes up a varying amount of space in the cell
- Function –> storage/protection of genome + creation of ribosomes and tRNAs + transcription.
- Double membrane (outer (continuous with ER) and inner) each membrane containing different proteins
What is the nuclear pore complex (NPC)?
Nuclear pore complex –> form of gated transport
- 1000’s per nucleus with 30+ types of nucleoporins
- They transport 500 macromolecules per second in and out –> how? –> unknown.
- Allows for bidirectional import and export
- Allows proteins to move out into the cytoplasm –> mRNA, tRNA and ribosome
- Allows proteins to move into the nucleus –> DNA + DNA polymerases, histones, lamins, transcription factors.
Briefly outline ribosome synthesis.
Ribosomes are proteins that are made in the cytoplasm –> transported back into the nucleus where they are assembled with the help of ribosomal RNA (rRNA) –> subunits are transported back into the cytoplasm.
What factors determine the method of transport into the nucleus.
Size determines the method of transport in and out of the nucleus.
- Molecules that are smaller than 50,000 daltons diffuse freely through NPC –> ions
- Molecules that larger than 60,00 daltons are too large –> to fit through the disordered mesh –> can’t move through by passive diffusion.
Note –> The pore is aq. so folded proteins can enter and exit –> no changed of state needed when substances are transported.
Describe the structure of the NPC.
Many techniques used to obtain structure –> recently Cryo-electron tomography has been used to obtain images to determine the structure.
The structure consists of…
- Cytoplasmic fibrils
- Central framework
- Nuclear basket
What is a nuclear localization signal?
- Before a protein gets transported into the nucleus –> it requires a signal that tells it can be transported through the NPC –> located almost anywhere in the amino acid sequence and are thought to form loops or patches on the protein surface (precise location is not important).
- This signal is called the nuclear localisation signal (NLS) –> Acts as a molecular postcode that tells the protein to go to the nucleus.
- Made of basic amino acids –> sequence is found in the amino acid sequence and is not added to the protein post-translationally.
How was this figured out?
- A protein composed of a core and exposed tail –> the tail was removed via enzymatic digestion –> the core no longer enters the nucleus –> supports the theory that there is an NLS. –> Note: 1 NLS signal is sufficient for transport –> however more = faster rate of transport.
How is nuclear import/export achieved? Name of the process?
Nuclear import and export use the Ran Cycle –> cycle is orchestrated by a small GTPase called Ran.
- The process uses a choreographed set on interactions between cargo proteins and import/export chaperons (molecules that aid in movement)
- Recognition of molecules is done using NLS signal
What are the key players in nuclear import/export?
- Ran –> RanGTP and RanGDP (different phosphorylated forms)
- Karyopherins (Importins/exportins) –> consist of an alpha and beta subunit.
- Cargo –> proteins being transported –> contain NLS
- Nucleotide exchange factors –> RanGEF and RanGAP –> factors that stimulate change between GTP and GDP.
- Helper proteins.
How are RanGTP and RanGDP interconverted? Where is each molecule mostly found (inside or outside the nucleus)?
RanGEF –> catalyzes the conversion of RanGDP to RanGTP.
RanGAP –> catalyzes the conversion of RanGTP to RanGDP.
- RanGEF –> found in the nucleus in the nucleus –> results in higher concentrations of RanGTP in nucleus.
- RanGAP –> found in the cytoplasm –> results in higher concentrations of RanGDP in the cytoplasm.
Explain the process of import into the nucleus using the NPC.
- Cargo (with NLS signal) binds to the importin –> adaptor protein may be used to bridge import receptor and NLS.
- Importing transports the cargo through the NPC into the nucleus –> F-G repeats
- Inside the nucleus, RanGTP binds to the importing (Beta subunit) –> makes the beta-importin dissociate from the cargo + alpha importin
- Alpha Importin + cargo is taken up by CAS nuclear export factors catalyzed by Nup50 –> releases the cargo in the nucleus
- Importing alpha/beta taken through NPC to the cytosol –> takes RanGTP with them.
- Outside RanGTP can get hydrolyzed back to RanGDP by RanGAP which releases the importin –> both are ready to restart the cycle.
Explain the process of export out of the nucleus using the NPC.
Nuclear export occurs by a similar mechanism, except that Ran-GTP in the nucleus promotes cargo binding to the export receptor, rather than promoting cargo dissociation.
Once the export receptor moves through the pore to the cytosol, it encounters Ran-GAP, which induces the receptor to hydrolyze its GTP to GDP. As a result, the export receptor releases both its cargo and Ran-GDP in the cytosol.
Free export receptors are then returned to the nucleus to complete the cycle
What is the net result of the import/export cycle using the NPC?
- RanGTP concentrated in the nucleus (RanGEF)
- NLS-cargo taken to the nucleus
- RanGTP and importin all recycled out for future import.
Two main ways nuclear transport is regulated?
- Cells control transport by regulating nuclear localization and export signals—turning them on or off, often by phosphorylation of amino acids close to the signal sequences.
- Transcription regulators are bound to inhibitory cytosolic proteins that either anchor them in the cytosol (cytoskeleton or specific organelles) or mask their nuclear localization signals so that they cannot interact with nuclear import receptors –> stimulus releases the gene regulatory protein from its cytosolic anchor or mask, and it is then transported into the nucleus
Characteristics of Karyopherins (importins/exportins)?
- Large proteins that are going to be imported have NLS made of basic amino acids (Arginine and lysine) –> this is used to bind to the karyopherin.
- Karyopherin –> soluble proteins (can also bind to an adaptor –> protein that binds and changes the shape of karyopherin) –> giving flexibility to what can be imported/exported.
How exactly are things transported through the nuclear pore complex?
In the unstructured domains (mesh-core work) of the NPC + the fibrils –> you find F-G repeats (G -> Glycine/F–>Phenylalanine)
Receptor-cargo complexes move along the transport path by repeatedly binding, dissociating, and then re-binding to adjacent FG-repeat sequences to move across.
Alpha subunit binds to the NLS signal whereas, the beta subunit binds to the alpha subunit and to the F-G repeats.
There are four different models (no detail needed)
- Selective Phase/Hydrogel model (most popular)
- Virtual gate/polymer brush model
- Forest model
- Reduction of dimensionality model
Briefly outline how the Selective phase model works (NPC)?
NPC
- Mesh functions as a 3D sieve
- Mesh size determined by hydrophobic clusters within FG- Nups (hydrogel)
Role of the sorting signal?
- Specific sorting signals that direct their transport from the cytosol into the nucleus, the ER, mitochondria, plastids, or peroxisomes.
- Some proteins do not have a sorting signal and consequently remain in the cytosol as permanent residents
Characteristics of signal sequences.
- Sorting signals involved in transmembrane transport reside in a stretch of amino acid sequence, typically 15–60 residues long.
- Where do you find signal sequences? –> 1.Signal sequences are often found at the N-terminus of the polypeptide chain –> 2. Internal stretches of amino acids (remain part of the protein) –> 3. multiple internal amino acid sequences that form a specific three-dimensional arrangement of the atom (form signal patch).
- Complementary sorting receptors that guide proteins to their appropriate destination –> unload chargo.
- Many cases specialized signal peptidases remove the signal sequence from the finished protein once the sorting process is complete
Can most organelles be constructed De Novo?
Organelles duplicate by increasing in size and then dividing.
However…. information required to construct an organelle does not reside exclusively in the DNA that specifies the organelle’s proteins.
Information in the form of at least one distinct protein that preexists in the organelle membrane is also required, and this information is passed from parent cell to daughter cells in the form of the organelle itself
What do molecules need to be exported from the nucleus?
Molecules need nuclear export signals, as well as there has to be complementary nuclear export receptors, or exportins.
Why does unloading only occur on the nucleus?
Because the Ran-GDP in the cytosol does not bind to import (or export) receptors, unloading occurs only on the nuclear side of the NPC. In this way, the nuclear localization of Ran-GTP creates the directionality of the import process.
What can happen when a protein has just recently been synthesized on a ribosome?
A new protein…
- Newly translated protein leaves the ribosome –> most proteins lie in the cytosol whereas others are sorted into organelles or onto the cell surface.
How is the cell sorting possible? –> Protein trafficking via the ER.
Where can proteins be produced?
- Proteins can be produced in the cytoplasm/nucleus
- Proteins can be produced in the ER –> transported to different destinations –>
- Proteins can be created by plastids (mitochondria/chloroplasts)
Hence….
Proteins can either be created by free ribosomes (destined for cytosol) whereas, everything else has something to do with the ER (except for protein that go back into the nucleus)
Functions of the ER?
ER/RER Functions (notes mainly focussed on protein trafficking –> less on lipids)
- Most membrane proteins are assembled in the ER membrane.
- ER makes all secreted proteins (out of the cell)
- Makes almost all proteins destined for the lumen of ER, Golgi and lysosomes
- Proteins made in the ER are also folded and modified there (glycosylation)
- A major site of protein quality control
- Cellular lipids also made in the ER
The main idea to take away –> ER is responsible for proteins in the secretory pathway destined for extracellular space, cell membrane, Golgi, lysosomes, peroxisomes, etc.. Basically not what remains in the cytosol/nucleus.
What is a hallmark that can be used to identify a signal peptide? (Amino acid sequence)
Usually N-terminus –> followed by hydrophobic residues –> followed by polar residues.
This clustering of both hydrophobic and polar a.a. is indicative of a signal peptide
Explain the step for co-translational translocation?
Steps
- mRNA bound to ribosome –> free in the cytosol
- Starts translation
- Eventually, the signal peptide is revealed
- Translation Stop when signal peptide is revealed
- SRP binds to the signal peptide
- SRP delivers the ribosome to the ER membrane to the Sec translocon –> signal sequence is cleaved (As the signal sequence is hydrophobic it moves into the membrane –> doesn’t associate with aq. environment.)
- Translation continues and the protein enters the ER lumen via the Sec translocon –> energy for translocation is derived from translation.
- New unfolded protein is now in the ER lumen.
Explain the process of post-translational using a ratchet system.
- Protein delivered to Sec translocon
- Protein Bip ADP grabs the protein (Sec63 catalyzes the conversion of Bip ATP to ADP –> In ADP form it can bind to protein –> energy –> every Bip protein must be converted to its ADP form)
- Protein moves in again inside (random movement) –> Bip grabs it again –> acts as a ratchet –> prevents the protein from moving backwards.
- This process continues until the entire protein is inside the ER lumen.
Extra notes:
- This process is entirely random –> protein simply has an affinity to enter –> randomly moves in and Bip simply grabs it when it does.
- This occurs in yeast and higher eukaryotes.
- Does not use SRP or receptor.
Characteristics of Sec translocon?
Sec Translocon - General (Name Sec is derived from the origin of discovery –> yeast)
- Used in both Co- and post-translational translocation
- Passive pore –> associating partner provides energy
- Depending on energy partner –> the channel functions differently
- Two forms the Sec translocon can take –> open and closed
- Water filled pore
- There is a helical plug that closes the translocon when it is not used –> gated –> as soon as SRP brings ribosome the plug is removed and signal peptide can pass –> polypeptide can then be fed through the cores hydrophobic lining.
- The core can move sideways (lateral gate) to allow cleaved signal sequence and membrane proteins to be integrated into the membrane.
How are membrane proteins formed using the Sec translocon?
Three cases:
- N-terminal signal sequence initiates translocation, just as for a soluble protein, but an additional hydrophobic segment in the polypeptide chain stops the transfer –> known as a stop-transfer signal –> anchors the protein in the membrane –> lateral gating mechanism transfers the stop-transfer sequence into the bilayer –> N-terminal in lumen –> C-terminal in cytosol.
- Internal signal sequence –> like before SRP binds to this internal sequence (recognises hydrophobic region) –> ER signal sequence then serves as a start-transfer signal that initiates the protein’s translocation –> start-transfer sequences can bind to the translocation apparatus in either of two orientations –> determines whether C-terminal or N-terminal is moved into lumen –> depends on the distribution of nearby charged amino acids
How are multi-pass transmembrane proteins integrated into the membrane?
- Internal signal sequence serves as a start-transfer signal in these proteins to initiate translocation –> continues until the translocator encounters a stop-transfer sequence (double pass stop transfer release protein)
- More complex multipass proteins, in which many hydrophobic α helices span the bilayer, a second start-transfer sequence reinitiates translocation further down the polypeptide chain until the next stop-transfer sequence causes polypeptide release, and so on for subsequent start-transfer and stop-transfer sequences
What happens to proteins that have been misfolded in the ER?
Only correctly folded and assembled proteins leave the ER –> 80% are misfolded –> Chaperones facilitate the translocation out into the cytosol –> where they are broken down
Steps:
- Misfolded protein is recognised
- Chaperones move misfolded protein into the cytosol
- Proteasome –> breakdown protein into subunits –> get transported into lysosome –> breaks it down even further
Note –> during cell stress –> there is an increase in misfolds –> this is signalled to the nucleus to stop mRNA production except for proteins that are involved in protein folding + chaperones + proteins involved in the removal of misfolds.
Characteristics and structure of the Golgi apparatus?
- Membrane proteins in ER membrane/lumen are sorted by the Golgi
- It has intrinsic directionality –> proteins/lipids are transported in one direction through golgi.
- Very dynamic organelle
- Number of stacks in structure vary
- Cis end (near ER) and Trans end (near to Plasma membrane) –> vesicles from ER fuse with the Cis-side and then proteins exit to the cell surface/other organelles via trans side
Briefly explain N-linked glycosylation.
N-Linked glycosylation
- Precursor molecule (dolichol) is added in the ER
- In the Golgi, the precursor can be modified by enzymes (glycosyltransferases and glycosidases) to form high mannose, hybrid and complex glycans.
- Products that are formed are extremely variable
Briefly explain O-linked glycosylation.
O-linked glycosylation.
- sugars added to -OH groups of Ser or Thr side chains
- glycosyl transferases add the sugar nucleotides
What are the two models of transport through the Golgi?
- Vesicular transport model
- Small vesicles constantly pinch and deliver molecules to the next compartment –> i.e. Cis Golgi –> vesicle –> medial Golgi. - Cisternal maturation model
- Substances from ER arrive at the Cis Golgi and mature through the different compartments
Likely a mixture of both.
What is one of the main post-Golgi destinations?
Lysosome –> simple compartment that contains many hydrolytic enzymes + low pH 4.5-5 –> used to break down molecules to simpler forms.
Enzymes
- 40+ acid hydrolases: proteases, nucleases, glycosidases
- lipases, phospholipases, phosphatases, sulfatases… often appear crystalline – because they are so packed!
pH
- H+ ATPase: acidifies and drives metabolite transport
Outline the process of autophagy.
Autophagy allows the orderly degradation and recycling of cellular components.
- Targeted cytoplasmic constituents isolated from the rest of the cell within a double-membraned vesicle known as an autophagosome.
- Autophagosome fuses with a lysosome and the contents are degraded and recycled.
- Three different forms of autophagy are commonly described: macroautophagy, microautophagy, and chaperone-mediated autophagy
What are the different trafficking destinations?
Outline the general process that leads to vesicle formation.
What are the three types of vesicle coats?
- COPII covers vesicles emanating from the ER (to Golgi).
- COPI surround vesicles originating from Golgi
- Clathrin surrounds those from the plasma membrane (Also involved in other parts)
- Clathrin, COPI, and COPII drive the formation of transport vesicles by polymerizing on cellular membranes.
- Each protein polymerizes into a polyhedral lattice with triangular & pentagonal faces (COPII), hexagonal & pentagonal faces (COPI and Clathrin).
Explain the process of vesicle formation using via COPII.
COPII –> ER to Golgi
- Sec12p is a GEF –> exchanges GDP to GTP in Sar1p (a switch that initiates the process)
- Sar1p is activated when in the GTP form –> Sar1p inserts helix to embed in ER Membrane
Note: interaction between sec12p and Sar1p is like the Ran cycle
- Activated Sar1p interacts diffuses in the plane of the membrane acquires Sec23/24p to form core inner coat of COPII coat.
- Sec23/24p collided and sample different membrane proteins –> Molecules that are destined for transport are picked up
- Acquires outer coat scaffold complex Sec13/31p
- Results in Budding/pinching off
- GTP Hydrolysis by Sar1p –> releases coat to form a naked vesicle –> has exposed snare proteins that allow the vesicle to dock and fuse with the target membrane.
Explain the process of vesicle formation using via COPI.
Retrograde pathway
- Activation of ARF1 GTPase –> from GDP to GTP form
- Recuirtment cargo
- Recruitment of coat complex
- Pinching and budding
- Hydrolysis of ARF1 GTPase –> release COPI coat –> form a naked vesicle.
Explain the role of dynamin in vesicle formation.
Pinching the vesicle –> most energetically demanding
Molecular strangulation: Dynamin
- Dimeric GTPase recruited to the membrane
- GTP hydrolysis leads to a power stroke and increased constriction
- Fission of the membrane where the membrane stress is the largest
- Disassembly of the oligomer and recycling of dynamin
Where those dynamin play an important role in humans?
Neuron synapse –> constant formation of vesicles containing neurotransmitters.
Outline the process doe SNARE complex formation.
What is the function of Rab GTPase in SNARE complex formation?
- Guide vesicle targeting lots of different forms each specific for different cargos
- Active Rab-GTP binds Rab effectors for movement or tethering vesicles to membranes.
Includes: motor proteins (e.g. myosin 5, walks) or tethering proteins or SNAREs-coupling tethering to fusion
What is the basal lamina?
- The thin mesh of ECM molecules, important in tissue repair and development
- Contains multi-adhesive matrix proteins, e.g. Laminins
- Type IV collagen (principal structural component)
- Perlecan (proteoglycan), bind diverse partners, crosslink components
Explain how Rab proteins work?
Two states:
In their GDP-bound state, they are inactive and bound to another protein (Rab-GDP dissociation inhibitor, or GDI) –> keeps them soluble in the cytosol.
In their GTP-bound state, they are active and tightly associated with the membrane of an organelle or transport vesicle.
Rab-GEFs activate Rab proteins on both transport vesicle and target membranes
