3.2-3.4 Molecular traffic in the cell Flashcards
Where does gated transport occur?
It is movement between the cytosol and the nucleus
What is imported into the nucleus?
What is exported from the nucleus?
What is the interior of the nucleus topologically equivalent to?
What are the structural features of the nuclear pore complex?
- 30 plus different protein types with 450 plus individual proteins
- octagonal arrangement
- central aqueous pore
Which molecules can pass through the nuclear pores freely by diffusion?
- Small molecules less than 5000 daltons pass through pores by diffusion
in what form do nuclear proteins pass through nuclear pores?
Nuclear proteins complete synthesis on cytosolic ribosomes and pass through pores in fully folded state
What shows that certain proteins are targetted to the nucleus?
- Nuclear localisation signal sequences
- They are a continuous stretch of amino acids 15-60 residues long
- Either at the N terminal or at a signal patch which has multiple internal sequences
- Recognised by complementary receptors
What is the amino acid composition of nuclear localisation signals?
- Commonly lys rich sequence PKKKRKV targets a protein to the nucleus
- Mutation of Lys to Thr causes cytoplasmic retention
What do nuclear import receptors bind to on the cargo protein?
- Cargo proteins which need to get into the nucleus bind to specific Nuclear import receptors (importins) via Nuclear localisation signals (NLS)
- Nuclear import receptors interact with the nuclear pore complex (NPC) proteins to transfer cargo in/out of the nucleus
What are the steps of cargo being transported into the nucleus?
- Cargo with Nuclear localisation signal binds Nuclear import receptors
- Nuclear import receptors shuttles into the nucleus via the Nuclear Pore Complex (F-G repeats)
- Ran GTP binds to the Nuclear Import receptor to discharge the cargo
- Nuclear import receptor shuttles out of the nucleus via the Nuclear Porte Complex (F-G repeats)
- Ran-GTP is hydrolysed to Ran-GDP in the cytosol
- Nuclear import receptor is free to shuttle more cargo into the nucleus
How does export of proteins and RNA from nucleus differ from import?
- It works like import but in reverse
How is Ran-GTP and Ran-GDP compartmentalised?
- Ran-GEF stimulates Ran-GDP to release its GDP and pick up GTP whihc releases cargo
- When Ran-GTP coming out of the nucleus Ran-GAP binds to it and activates GTPase for hydrolysis
What is nuclear import regulated by?
- NFAT is a transcription factor found in the cytosol (T-cells)
- Nuclear localisation sequence is cryptic by exposed after dephosphorylation of amino acid residues (Ser) by Ca2+ regulated phosphatase (calcineurin)
- Dephosphorylation produces a conformational change
How does regulation of nuclear protein import influence fate decision in embryos?
- Fate decision in embryo determined by nuclear accumulation of specific transcription factors
- Dorsal protein has nuclear localisation only in ventral cells of early Drosophila embryo
- Mutation of Dorsal results in dorsalisation of embryos (no ventral structures)
How many membranes are cross in protein transport to the mitochondria?
Mitochondria have two membranes that need to be crossed for proteins to be targeted correctly
In what form are proteins imported to the mitochondria?
- Mitochondrial proteins are imported as fully synthesised but unfolded polypeptide chains
- mRNA produced in the nucleus exported through NPC, chaperones keep it unfolded
- They have specific targeting/signal sequence
What is the signal sequence for mitochrondial protein transport?
- Amphiphillic signal sequence which forms an alpha helix, non-polar (hydrophobic), polar, and hydrophillic residues on separate sides of the helix
- Hydrophobic region matches hydrophobic groove of receptor
What are the different protein translocators that move proteins through mitochondrial membranes?
- TOM complex = transport through outer membrane
- TIM23 complex = complex for transport through inner membrane
How does the protein use translocators to get through mitochondrial membranes?
- Protein snakes through translocators in unfolded state
- Signal sequence binds to TOM complex receptor
- TIM23 complex aligns with TOM complex and fed through TIM23
- In the matrix signal sequence is cleaved off and then the protein folds
How does mitochrondrial import use energy (ATP)?
- Chaperone proteins (cytoplasmic Hsp70) bind to the precursor peptide
- Their release requirs ATP hydrolysis to push the protein through the TOM complex
- Import via TIM results in mitochondiral Hsp70 binding
- ATP hydrolysis to pull protein through TIM complex
What other protein transport requires translocators?
Transport of proteins into peroxisomes involves translocators
Where might proteins go once passing through the ER lumen?
- Many proteins pass through the ER lumen en route to other destinations
Where does the synthesis of proteins targeted for the ER begin?
On free ribosomes in the cytosol
When is translation completed for proteins targetted to the ER?
- After the ribosome binds to the ER
- Co-translational translocation is where the mitochondrion and nuclear proteins have translation occur first and then the fully translated protein gets translocated
What are the steps for protein targetted to the ER?
- The signal sequence on the mRNA is a recognition sequence for a protein called the signal recognition particle
- The binding pocket of signal recognition particle bind to the signal sequence and causes signal recognition particle to attach to large ribosomal subunit and pause translation
- Part of signal reocngition particle engages with receptor in ER membrane allowing signal sequence to be fed through
- Signal recognition particle gets released and protein is fed through the translocator
How does the Sec61 translator structured (protein translocator into ER)?
- Conducting channel through which the polypeptide is fed through
- It has a hinge structure
- When signal peptide fed through the pore it displaces the plug and opens the hinge, sequence fed through in a loop
How does the insertion of an integral protein into the ER differ from usual protein translocation?
- Insertion of transmembrane protein has a similar signal (start tranfer) sequence but additional hydrophobic “stop transfer” sequence causes the polypeptide chain to stop in the translocator
- Translation of the -COOH terminal continues into the cytosol (ribosome detaches)
How is the Stop transfer seqeunce of integral proteins of the ER membrane resistant to peptidase?
It is enclosed by lipids so peptidase can’t get to it
How do transmembrane proteins in the ER membrane have different orientations?
Where does glycosylation of proteins take place?
- Glycosylation of proteins takes place in rough ER as proteins enter lumen
- Transfer of precursor oligosaccharide (glucose, mannose, N-acetylglucosamine) to nitrogen atom on Asn by transferase enzyme associated with translocator
What is the role of dolichol in the glycolyslation of proteins?
- Glycolipid dolichol sits adjacent to the translocator
- Cuts the phosphate bond and transfer oligosaccharide complex on to Asparagine
How are oligosaccharides used as tags to mark the state of protein folding?
- Chaperone proteins can recognise unfolded proteins because of terminal glucoses
- Glucosidase removes two terminal glucoses (glucose trimming)
- Terminal glucose comes in contact with calnexin at glucose binding sites and protein is folded
- If protein folding incomplete it goes through process again
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Where do misfolded proteins go?
- Misfolded proteins are exported into the cytosol and degraded
- Up to 80% need to leave ER. Modification of mannose (time dependent) may mediate recognition by translocator protein which shunts them out the cytosol where they are ubiquinated
Within the vesicle transport pathways, what are the main groups of pathways?
What are the three different types of coats that vesicles have?
- Clathrin for the endocytic pathway
- COP2 for biosynthetic (secretory pathway)
- COP1 for recycling/retrieval pathway
How are clathrin coated vesicles formed?
- Receptors bind to the cargo
- Adaptor proteins (adaptins) bind both clathrin triskelions and cargo receptors
- Binding of receptros/adaptins by clathrin concentrates cargo in the pre-vesicle
What regulates the pinching off and uncoating of coated vesicles?
- Cytosolic proteins regulate the pinching off and uncoating of coated vesicles
- Dynamin is a GTPase that destabilises the bud neck so that the lipid bilayers flow together
What proteins guide the targetting of vesicles to specific location?
- Rab proteins (small GTPase) guide vesicle targetting and SNAREs mediate membrane fusion
- It sits at the surface and recognises specific effector molecules
- When rab binds to the effector it bends over and brings vesicles closer to the target membrane
- Complementary sets of vesicle snare (v-snares) and target snares (t-snares) determine the selectivity of transport-vescile docking
What mediates transport from the ER to the Golgi apparatus?
- Vesicular tubular clusters mediate transport from the ER to the golgi apparatus
- If proteins end up in the golgi when they shouldn’t be, the coding sequence KDEL allows it to bind to specific adaptins to transport back to the ER
In what way are oligosaccharide chains processed by the Golgi apparatus?
- Oligosaccharide processing in the Golgi is highly ordered
- Product of each reaction becomes substrate for the next reaction
What two models does transport through the Golgi apparatus occur by?
Where do hydrolytic enzymes from Golgi and materials to be digested converge?
Hydrolytic enzymes from Golgi and materials to be digested converge at early endosome
What are lysosomes?
- They are the principal sites of intracellular digestion, degrading proteins and lipids.
- Membrane bound vesciles containing soluble hydrolytic enzymes (40 different types)
- Membrane bound to prevent random digestion of the cell
What is the pH like of the lysosome?
- Regulated by pH (proton pump)
- Low pH activates hydrolases, proteases, nucleases
- Enzymes active at low pH
What are the pathways that deliver materials for destruction to the lysosomes?
- phagocytosis
- endocytosis
- autophagy
How does autophagy occur?
How does the endosome progressively mature into the lysosome?
- Vesicles from the golgi containing lysosoma enzymes will fuse with the endosome adding lysosomal enzymes
- Maturation gives endolysosome as lysosome fuse with late endosome
- Not distinct structures but progressive maturation process
Where are oligosaccharide chains processed?
How are vesicles containing hydrolytic enzymes targeted to the early/late endosome?
- Lysosomal proteins are tagged with Asn-linked N-linked oligosaccharides for protein folding
- Nascent protein gets fed into ER lumen and oligosaccharides get added to asn
- terminal glucose gets cleaved off until one remains only which binds to calnexin
- Exit from ER and end up at golgi
Where are lysosomal proteins modified?
- in the golgi network
- A signal patch (amino acid sequence) determines the addition of a mannose-6-phosphate tag
- Enzymes act on the N linked oligosaccharide and M6P is added in the early cisternae of the Golgi and modified as it progresses through the Golgi so that is is recognised by a specific receptors in the trans Golgi
What is the process by with the Mannose 6 phosphate tag is added on the lysosomal protein?
- A specific amino acid sequence (signal patch) in the lysosomal hydrolase enzyme is a binding site for glucosamine transferase enzyme
- N-acetyl glucosamine is added and then removed to leave a phosphate group on the mannose
What recognises the Mannose 6 phosphate tag added to lysosomal protein?
- A mannose-6-phosphate receptor recognises lysosomal proteins in the trans Golgi network
- The addition of Mannose-6-PO4 causes binding of the lysosomal proteins to M6P receptors in the trans golgi memrbane
- adaptins and clathrins form the vesicle
Where does the mannose 6 phosphate receptor move to when transporting lysosomal protein?
- Modified hydrolase binds to receptor and transferred by clathrin coated vesicle to early endosome
- receptor is re-cycled back to GOlgi (pH dependent release of cargo)
What happens in hurler’s disease?
- Enzyme (alpha-L-iduronidase) required for breakdown of glycosaminoglycan (GAG) chains is defective
- Person with this mutation cannot breakdown GAG and end up in the lysosome which keeps growing
What is pinocytosis?
- Smaller molecules and fluids can be taken up by pinocytosis (non specific uptake, not involving receptors)
- it is clathrin dependent
How is cholesterol imported?
- Cells use receptor linked endocytosis to import selected extracellular macromolecules
- Cells need cholesterol - uptake via endocytosis using clathrin coated pits/vesicles
- LDL = low density lipoprotein = lipid and protein (blood carrier for cholesterol)
What does defective LDL receptors lead to in cholesterol import?
- Defective LDL receptors lead to defective cholesterol transport and atherosclerosis
- LDL still binds to the receptor, vesicles still forms but no interaction means vesicles empty
- Accumulation of cholesterol in blood and failure to uptake into cells
What is the role of the early endosome?
- They are the main sorting station for the endocytic pathway
- clathrin shed from vescles and naked vesicles target the endosome
- receptors cycle back to cell surface and take these molecules into the endosome
- Early endosome constantly changing structure, more LDL taken in it matures into late endosome
How do multivesicular bodies form on the pathway to late endosomes?
- Multivesicular bodies form on the pathway to late endosomes
- Membrane buds inwards and forms multivesicular bodies in order to get rid of transmembrane proteins
What are the two ways that secretion to the outside of the cell can occur?
- Constitutive pathways is the default (continuous/basal pathway)
- Regulated pathway requires signals
What are the steps of the constitutive pathway of secretion?
How does I cell disease affect the constitutive pathway of secretion?
I-Cell disease results in failure to put tags on enzymes and therefore they are not regulated but exit cell continuously via the constitutive pathway.
How does the cargo concentration of secretory vesicles change?
- Contents of secretory vesicles become concentrated by vesicle fusion and membrane recycling
- Proteins targeted to secretory vesicles are concentrated in the vesicles (more effective when released quickly after signal)
- Vesicles fuse with others to form larger vesicles which are remodelled and small parts of the membrane are pinched off and returned to golgi
What happens to the membrane of secretory vesicles after contents are released into the extracellular space?
- membrane of secretory vesicles is rapidly retrieved
- If membrane was not recycles the cell would get uncontrollably large
- Need a recycle mechanism called kiss and touch
