Cell Bio 3 Flashcards
(35 cards)
Where do proteins go after the ER
proteins go through the non-secretory pathway or through the secretory pathway.
Golgi Complex
Consists of flattened, dislike cirsternae with no ribosomes
Vesicles at cisterna tips fuse or pinch off
Three types of cirsternae (cis, medial and trans) and two flanked networks of tubules: CGN faces RER and TGN is opposite to RER.
Processing and sorting of proteins (secreted, membrane, lysosomal)
Transport Vesicles: types + function
Coats promote budding of vesicles
SNARE proteins promote fusion of vesicles with target membranes
COPII vesicles -> RER to Golgi (anterograde)
COPI vesicles -> cis-Golgi to RER (retrograde transport)
Clatherin vesicles->TGN/OM to late endosomes
Contents of the Vesicles
- Membrane cargo protein: traverses the GA membrane and it has portion that faces into the lumen of ER and into the cytosol. Transmembrane protein we are getting from the ER to the membrane
- Soluble cargo proteins: free floating proteins in the ER they bind to receptors in the vesicle
- GTP binding proteins: function to help promote the coat protiens to bind to certain parts of the ER membrane to help form that budding to create the vesicle
Snare: once the vesicle has formed and mvoed towards its target destination the snare proteins interact with one another, we have the v-snare on teh vesicle and t-snare on the target membrane. They interact wiht one another, twisting to result in the membrane fusin to one another.
GTP binding proteins control assembly and disassembly of COPII coat proteins
Membrane associated GTP binding proteins promote association of COPII coat proteins on ER membrane they will bind to areas where you might have a cargo protein receptor.
Localized recruitment of coat proteins to a small portion of the ER membrane that causes a physical conformational change of the membrane to form a bud, it buds oof the membrane and then it forms a complete vesicle.
Once COPII vesicles are released from donor membrane, hydrolysis of GTP occurs which triggers disassembly of coat proteins.
RER to cis-Golgi Transport
Anterograde Transport
COPII coat proteins assemble onto the budding vesicle: GTP, DXE sorting signal on cargo membrane proteins.
Vesicle moves towards the GA, hydrolysis of GTP causes the coat protein to fall off, snare proteins interact causing the fusion of the membrane and vesicle.
ATP hydrolysis is required for dissociation of SNARE complexes: naked vesicle exposes the snare proteins, they twist around and bring the membrane vesiclesin close proximity to the membrane of the golgi
RER to cis-Golgi Transport
Anterograde Transport
COPII coat proteins assemble onto the budding vesicle: GTP, DXE sorting signal on cargo membrane proteins.
Vesicle moves towards the GA, hydrolysis of GTP causes the coat protein to fall off, snare proteins interact causing the fusion of the membrane and vesicle.
ATP hydrolysis is required for dissociation of SNARE complexes: naked vesicle exposes the snare proteins, they twist around and bring the membrane vesiclesin close proximity to the membrane of the golgi
Cystic Fibrosis
Recessive genetic disease
Abnormal transport of chloride and Na+ across epithelium leading to thick viscous secretions
After lung, liver, pancreas and intestine
Caused by a mutation in the gene for the protein cystic fibrosis transmembrane conductance regulator
delta508 is most common mutation in CFTR
Affects the ability of CFTR to bind COPII coat proteins
The CFTR protein is stuck in the ER, it is a transmembrane proteins and has pushed faces into the cytosol, mutation has the proteprty where it alters the conformation of the protein in such a way that is causes the DEX sorting signal to be obscured, so it can’t be recognized by the COPII coat proteins.
cis-Golgi to RER transport: KDEL
Golgi reroutes soluble ER resident enzymes back to the ER
Such protiens bear a c-terminal KDEL sequence
KDEL receptor is sensitive to pH. when they end up in GA, the pH is slightly more acidic, the KDEL peptide will bind to KDEL receprtor, the KDEL receptor has a slighltly higher affinity for the KDEL sequence at lower pH.
KDEL receptor contains c-terminal KKXX sequence (faces cytosol) which binds COPI coat proteins.
When you get a binding it causes a conformational change on the receptor, there is a protion that faces into the cytosol, recruitment and binding of COPI proteins, movement towards the ER and shedding of the COPI proteins followed by the fusion of COPI vesicles with the ER membrane. Releasing the ER residue protein back into the lumen and the KDEL receptor will not have an affinity because the pH is slightly higher in the lumen of the ER.
Luminal proteins including chaperons (BiP) and lectins have KDEL sequence
Protein Glycosylation in Golgi
Golgi sub-compartments differ
Glycosidases (removing sugars) and glycosyltransferases (adding sugars):
- cis-golgi: mannosidases
- medial-golgi: GlcNac-transferase, mannosidase, fucosyiltransferase, sialyltransferase.
- Function of the golgi is to modify proteins by adding or subtracting sugars.
- The golgi contains (depending on cis or trans) different types of enzymes.
- Unerstand that sugars need to be put and taken off within the golgi.
trans-golgi network
1-COPI vesicles (retrograde transport)
2-AP (adapter protein) coated vesicles
3-Clatherin coated vesicles
Other proteins that end up going to the lysosome can directly via vesicles that are coated with AP, they can form and the vesicles can fuse with the lysosome. Proteins will get encapsulated with the vesicles coated in clatherin.
4 and 5 secretory vesicles- constituitive and regulated (unknown coat proteins)
Clatherin
Clatherin plays and important role the formation of many different types of coats of vesicle.
Clatherin has three heavy and light chains that form a triskelion, they interweave with one another spontaneously to form a cage structure, they form the vertices in the pentagons. The Triskelion proteins form together and bind onto together to force it into a curved structures. They are very functional in the formation vesicles.
TGN to lysosome transport: M6P sorting signal
- Mannose-6-phosphate (M6P) is carbohydrate
- M6P targets soluble proteins to lysosomes
- Targeting requires an M6P receptor
- M6P is added to lysosomal enzyme in the cis-golgi
Adding the M6P enzyme
- The protein in the lumen of the ER oligosaccarotransferase adds a branched sugar structure and then that protein moves via anterograde COPII vesicles to the cis-golgi.
- protein is recognized by GlcNAc phosphotransferase it has two important sites a recognition site and a catalytic site
- it will recognizes the AA sequence within a lysosomal protein, it will bind to it and bring it in close proximity to a catalytic site that binds a phosphorylated form of mannose sugars on the branched sugar chain, the enzyme sticks on the phosphate.
- an extras sugar on the GlcNac needs to be cleaved off from the phosphate
- this protein can be recognized by a mannose-6-phosphate receptor
TGN to lysosomes transport: vesicles
-receptor binding occurs at low pH in the trans-golgi and when it encounters a lysosomal protein that has a mannose-6-phosphate sugar attached to it, it recognizes it. (6.5)
M6P will bind to phosphorylated mannose ant the attached suagr structure bound to the lysosomal enzyme
- that causes the recruitment of AP1, adaptor protein coat proteins in addition to the clathrin triskerlions they assemble and form the clathrin coated vesicles
- it moves towards the late endosome the coat proteins are removed and the uncoates transport vesicle fuse with the help of snare protein fuse eith the late endosome.
- At low pH M6P detaches from receptor in the GA (pH 5-5.5)
- the late endosome has a lower pH than the trans-golgi it facilitates the dissociation of the phosphate from the M6P receptor
- M6P receptors return to Golgi or to PM
- M6P receptor pinches off in a vesicle that can later go back and fuse with the golgi and reintroduce that receptor back to the trans-golgi or go back to the plasma membrane.
Lysosomal storage disease
- absence of one or more lysosomal enzymes resulting in the accumulation of undegraded material in lysosome.
- inclusion-cell (I-cell) disease is one of the most severe types of LSD
- absence of GlNAc phosphotransferase (no MP6) signal
- lysosomal enzymes are secreted rather than being sorted to lysosomes
- Undigested glycolipids normally degrdaded by lysosomal enzymes accumulate in lysosomes
- Clinical onset at birth
- fatal outcome
How can cells internalize the extracellular materials?
Phagocytosis
Pinocytosis
Receptor Mediated endocytosis
Receptor-mediated endocytosis: a method of selective internalization of specific extracellular molecules( ligands)
LDL
transferrin
Hormones
Receptor-mediated endocytosis: a method of selective internalization of specific extracellular molecules( ligands)
LDL
transferrin
Hormones
Low Density Lipoprotein
- Lipids are transported in large well-defined water-soluble complexes/particles called lipoproteins
- LDL contain approx. 88% cholesteryl eaters and mediates cholesterol transport.
- LDL receptors on plasma membrane (localized in clathrin-coated pits)
- Amphipathic shell: composed of a phospholipid monolayer and apolipoprotein
Apolar core-hydrophobic, mostly
Clathrin coated/AP coated vesicles
Two layer coat: clatherin and adaptor protein
AP complexes recognize sorting signals of cargo proteins or receptors.
Clathrin-coated vesicles pinch off using dynamin and GTP hydrolysis.
- Binding of LDL particle to its receptor leads to the recruitment of coat proteins
- Coat like structure causes the invagination of the vesicle (caltherin pit)
pH-dependent binding of LDL particles to the LDL receptor
LDL-Receptor has three domains:
- Short C-terminal cytosolic segment with sorting signal has an NPXY sorting signal
- long N-terminal exoplasmic segment with a ligand binding domain and beta propeller domain
- At normal pH the ligand-binding arm binds tightly to ApoB
- At acidic pH 5-5.5 histidine residues in the beta-propeller domain become protonated and bind with high affinity to the negatively charged residues in the ligand-binding arm
Targeting LDL and LDL receptors to clatherin/AP2-coated pits
ApoB mediates binding to LDL receptor.
Specific sorting signal (NPXY) in the ctyplasmic domain of receptors binds to the AP2 complex
pH 7 causes the LDL particle to bind to the ligand binding domain of the LDL receptor resulting in a change in conformation of the cytoplasmic domain activating the NPYX sorting signal causing the recruitment of AP2 coat proteins and clathrin causing the invagination of the plasma membrane
Acidification of endosomes and lysosomes
- Vclass proton pumps transport H+ across membranes via ATP dependent mechanism
- Cl- channels are also present on lysosomal and endosomal membranes
- Anions passively follow the pumped proton resulting in acidification of lumen
- pH of lysosomes and endosomes decrease dramatically
