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



























