ER-GOLGI TRAFFICKING AND ENDOCYTOSIS Flashcards
Cotranslation / translocation steps
- The N’ end of the peptide emerging from the ribosome contains a signal sequence targeting it to the RER
- Signal recognition particle (SRP) binds to the signal sequence and stops translocation
- SRP binds to an SRP receptor associated with a closed channel (translocon) on the RER membrane, mediating GTP binding
-
GTP binds the SRP & SRP receptor and hydrolysis opens translocon and kicks off the SRP to be recycled
- Translation restarts w/o SRP, translating the peptide through the open translocon
- Signal peptidase cleaves off the signal peptide
- For a non-integral soluble protein, the entire peptide is formed inside the ER lumen
- Ribosome falls off mRNA and RER, then dissociates into 40S and 60S
- Translocon closes, peptide folds
What proteins are translocated through the ER membrane and into the ER lumen/matrix?
What proteins are inserted directly into the ER membrane?
soluble matrix proteins
integral proteins
What’s the first modification that occurs, either within the ER or Golgi?
What does it help with?
Glycosylation: enzymatic addition carbohydrates/glycans
- protein folding/targeting
- protein stability
- cell-to-cell contact via lectins (carbohydrate-binding proteins)
- antigens (e.g. ABO blood typing)
Non-enzymatic mechanism of adding carbohydrates
Glycation
O-linked glycosylation
Addition of individual carbohydrates to -OH groups on Ser or Thr by glycosyl transferases
Ex) Addition of carbohydrates to RBCs to generate ABO blood groups
N-linked glycosylation
-
Oligosaccharyl transferase adds glycans to proteins as a preformed oligosaccharide complex to asparagine (Asn) in an Asn-X-(Ser/Thr) consensus sequence
- Occurs in the ER while the peptide is translocated through the ER membrane
- Glycosidases further process the complex
- Final product signals that the glycosylated protein is ready for transport from the ER to the Golgi, where it may be further processed
The oligosaccharide precursor of N-linked glycosylation is initially synthesized where?
Where is it finalized?
Initially built upon a dolichol phosphate in the cytosol on the outersurface of the ER.
Later finalized in the ER lumen
Activated nucleotide sugar donors, ___ & ___, add the sugars to dolichol phosphate sequentially until the oligosaccharide precursor for N-linked glycosylation is complete
UDP-sugar or GDP-sugar
Tunicamycin
inhibitory analog of UDP-gluNAc, which is the first nucleotide-sugar substrate in formation of the oligosaccharide precursor of N-linked glycosylation.
–> stops N-linked glycosylation
–> prevents folding, stability, targeting, and secretion
What enzyme transfers the oligosaccharide precursor complex from the dolichol phosphate to the Asn residue of the consensus sequence?
Oligosaccharyl transferase
What kind of proteins tend to have disulfide bonds?
Secreted proteins
&
Proteins on the extracellular leaflet of the plasma membrane
Disulfide bonds aid in
protein folding by providing strong covalent structure & stability
You woul dnever find disulfide bonds in proteins synthesized from free ribosomes because
Disulfide bonds form in the ER lumen only under oxidative conditions, and as the protein is translocated through the ER membrane.
PDI
Protein disulfide isomerase transfers oxidized disulfide bonds from its Cys to reduce dprotein substrates –> forms and rearranges disulfide bonds
What does A-1AT do?
Inhibits trypsin and elastase to prevent excessive elastase (from neutrophils) damaging the lungs.
Alpha-1 antitrypsin (A-1AT) deficiency
-
Point mutation in the A-1AT protein prevents proper folding in the ER of hepatocytes
- –> Crystalline aggregates can’t be secreted and build up, damaging the liver and impairing secretion of other liver proteins
- Liver diseases (jaundice, cirrhosis) in children
- –> Excessive elastase activity damages lung
- Familial emphysema in adults
- –> Crystalline aggregates can’t be secreted and build up, damaging the liver and impairing secretion of other liver proteins
A-1AT deficiency is treated by
Immunizations to prevent lung infection
Avoid smoking and lung irritants
A-1AT replacement therapy
Bronchodilators
Inhaled steroids to open airways
O2 administration
Liver transplant (most common)
After translcoation into the ER and processing in the ER, proteins are ultimately transported to function in what 3 possible places?
Golgi
Endosomes/lysosomes
Plasma membrane
Anterograde vs Retrograde transport
-
Anterograde: vesicular movement from
- ER to the cis-Golgi
- Trans-golgi to endosomes/lysosomes or plasma membrane
-
Retrograde: vesicles return proteins to previous compartments in the process
- Ex) cis-Golgi to the ER, trans to medial
Cisternal maturation
New cis-Golgi forms when ER-to-golgi anterograde vesicles bud and fuse with it –> moves previous cis-Golgi up to the medial Golgi position –> moves previous medial Golgi up to trans-Golgi
- Proteins get processed and/or sorted into vesicles
- Cisternae mature through retrograde vesicles transporting enzymes
- Ex) When ER vesicle fuses to form a new cis-Golgi, retrograde vesicles transport needed ER enzymes back tothe ER
What anterograde vesicles move from the ER to the cis-Golgi?
What is the associated GTPase?
COPII vesicles
Sar1
What are the retrograde vesicles?
What is their GTPase?
COPI
ARF
Clathrin-coated vesicles transport from where to where?
What’s their coat protein?
Associated proteins
Their GTPase?
- TGN -> endosomes or lysosomes
- Plasma membrane -> endosomes, lysosomes, other cell-specific structures (endocytosis)
Coat protein: clathrin
Associate adaptor protein: AP1, AP2, AP3
GTPase: ARF
Describe ER to cis-Golgi anterograde transport for a soluble cargo protein
- Soluble cargo binds to an ER membrane-bound receptor
- COPII coat protein binds to the receptor and causes formation of a curved COPII vesicle
- Vesicle pinches off and de-coats, exposing v-SNAREs
- The v-SNARE binds to the t-SNAREs on the target membrane, the cis-Golgi –> vesicle fusion
- Soluble cargo is now bound to its receptor, bound in the cis-Golgi membrane
How would the ER to cis-golgi anterograde transport be different for an integral membrane cargo protein (as opposed to a soluble one)?
- COPII coat protein binds to the cytosolic domain of the integral membrane cargo
- (instead of binding to the receptor for soluble cargo)
- The end product places the integral cargo in the cis-Golgi membrane
- (instead of bound to a receptor)
Sar1 and ARF are GTPases - what are their 2 important funcitons?
vesicle formation
&
de-coating
Describe the GTPase function of Sar1
-
GTP-bound Sar1 inserts its hydrophobic tail into the ER membrane to interact with COPII coat proteins
- –> binds coat protein to the vesicle membrane, thus recruiting cargo, and promoting curvature for vesicle formation
- Hydrolysis –>GDP-bound Sar1 retracts its tail –> de-coating
After the ER, the preformed N-linked oligosaccharide complex goes to the cis-Golgi by anterograde vesicle transport.
What kind of processing could happen here?
High mannose glycosylation in the cis-Golgi
Complex glycosylation in the trans-Golgi
Modifications of the high mannose structure can target the protein to the lysosome.
From the TGN, where can proteins be targeted?
- Previous trans-Golgi (retrograde transport)
- Lysosomes
- Late endosomes
- Constitutive or regulated secretion
Remember: The transport vesicles <u>usually</u> have a clathrin coat protein (exceptions), adapter protein, and ARF
What’s the function of the AP?
It binds a cytosolic domain sorting signal on the cargo or cargo receptors AND the coat protein
–> gets cargo into the correct vesicle
Transport from TGN to trans-Golgi would use..
COPI (retrograde)
ARF
clathrin is not the coat protein
Transport from TGN to the lysosome would use
what AP?
what GTPase?
what signal?
Maybe clathrin
AP3
ARF
Cargo signal: Mannose6-phosphate
Transport from the TGN to the endosome involves
what coat protein?
what ap?
what GTPase?
What about from the plasma membrane to the endosome (receptor-mediated endocytosis)?
- TGN to endosome
- Clathrin, AP1, ARF
- Receptor-mediated endocytosis
- AP2 & ARF
Secretion/exocytosis can be __ or __
constitutive or regulated
Regulated secretion involves aggregation
Child with coarse facial features, craniofacial abnormalities, psychomotor retardation, and regression of developmental psychomotor milestones
I-cell disease: defect in lysosomal transport
In transporting to the lysosome,
- M6P-containing lysosomal proteins are packaged into AP3 vesicles in the TGN –> bud off
- Vesicle de-coats, recycling the clathrin and AP3
- Uncoated vesicle fuses w/ lysosome (or late endosome that matures into lysosome)
What happens to the receptor?
- It is recycled back to the TGN or goes to the plasma mambrane
- Goes to plasma membrane in case a hydrolase is accidentally constitutively secreted –> receptor can bind it in the ECM and bring it back into the late endosome/lysosome
If lysosomal proteins aren’t tagged with the M6P signal, like in I-cell disease, what happens?
Constitutive secretion of ALL lysosomal proteins (not just the hydrolases) and none are ever returned to the lysosome via receptor-mediated endocytosis
–> lysosomal storage disease
Creation of the M6P sortign signal
- GlcNAc phosphotransferase uses UDP-GlcNAc as a substrate to add GluNAc-phosphate to one of the Mannose sugars.
- Phosphodiesterase removes Glu-NAc, leaving the Mannose 6-phosphate sorting signal
I-cell disease / Mucolipidosis II
- Defective GlcNAc phosphotransferase –> can’t make M6P so all proteins destined for the lysosome are mis-targeted and secreted from the cell, causing a buildup of inclusion bodies (carbs & lipids) in the lysosome
*
ML-I
ML-II
ML-IIII
ML-I: sialidase deficiency
ML-II/ I-cell disease: defective GlcNAc phosphotransferase
ML-IIII/Pseudo-Hurler polydystrophy: less severe deficiency of GlcNAc phosphotransferase
What would you seein a patient with pseudo-Hurler polydystrophy or I-cell disease?
How could you differentiate?
- Cells filled with dense inclusion bodies
- High level of lysosomal enzymes in patient’s serum and body fluids
Pseudo-hurler polydystrophy ocurs later in life and the patient surives past the first decade
How are I-cell diseae and psuedo-Hurler polydystrophy different from other lysosomal storge disorders?
Both result from a targeting defect, not a single enzyme defect –> ALL lysosomal enzymes are affected
In both phagocytosis/pinocytosis and receptor-mediated endocytosis. endosomes are formed and ..
acidified and enriched with acid hydrolases –> becomes late endosomes, and ultimately lysosomes
Phagocytosis involves evagination of the plasma membranae to engulf a foreign particle and internalize it in…
membrane-enclosed phagosomes
Autophagy
removal of old and worn-out organelles into autophagosomes for digestion
- Cell may digest its organelles as a stress response
- Plays a role in immunity and infection, destroying intracellular pathogens
- Can result from inflammation and is increase in neural injury/degeneration
Steps of receptor-mediated endocytosis for soluble cargo protein (or the receptor itself)
- Integral receptor binds a cargo protein
- Either the receptor or the cargo binds to AP2
- AP2 interacts with Clathrin
- ARF decoats clathrin, creating an uncoated vesicle (Early endosome)
- Early endosome gains other acid hyrolases & ATP-dependent proton pump via vesicular trafficking from TGN –> late endosome
- Late endosome becomes a lysosome as pump keeps lowering pH
- Ligand-receptor complexes dissociated and the receptors are recycled to the PM or degraded in the lysosome
The LDL particle transports cholesterol. Its hydrophobic core contains cholesteryl-esters.
What on its outer surface serves as the ligand for RME?
How does it get digested by lysosomes?
ApoB
The LDL receptor on a cell surface binds ApoB for RME.
In the acidic endosome or lysosome where the pH is lower, the receptor becomes positively charged, decreasing its affinity for ApoB –> LDL particle is released into lysosome for digestion
The LDL receptor is bound in _____
At neutral pH, it will bind ApoB on the LDL particle, causing internalization of the ligand-receptor complex into a ____
the vesicle de-coats, creating an early endosome that acidifies into a late endosome.
The acidic pH promotes release of LDL into the lysosme, while the LDL receptor is ___
The LDL receptor is bound in a clathrin-coated pit to AP2
At neutral pH, it will bind ApoB on the LDL particle, causing internalization of the ligand-receptor complex into a clathrin-coated endocytic vesicle
The vesicle de-coats, creating an early endosome that acidifies into a late endosome. The acidic pH promotes release of LDL into the lysosome, while the LDL receptor is forms into another vesicle that recycles back to the PM
Familialhypercholesteremia occurs due a
mutation in the LDL receptor (absence, defective, premature degradation, inability to sort) or in the ApoB ligand –> LDL builds up in blood –> plaques
Homozygous is worse than heterozygous
Fe3+ + apotransferrin = ferrotransferrin
Describe iron transport
- Ferrotransferrin binds to the transferrin receptor on cell surface
- Receptor-mediated endocyotsis
- Vesicle uncoats and acidification of the late endosome releases the iron from ferrotransferrin
- Apotransferrin binds transferrin receptor in the acidic pH of the late endosome
- Apotransferrin-transferrin is recycled back to the PM, where neutral pH causes them to dissociate and apotransferrin is released
What happens when a receptor on the plasma membrane or in the Golgi needs to be degraded, NOT recycled?
The membrane and proteins invaginate to become vesicular bodies within the late endosome. Because the vesicle is completely within the lysosomal matrix, it can be degraded by acid hydrolases.
Formation of vesicular bodies
- Hrs proteins and proteins required for degradation get monoubiquitinated
- Monoubiquitinated Hrs interacts with ESCRT to induce invagination of endosomal multivesicular bodies that are then degraded
- ESCRT disassembled and recycles
this process is ued in the budding of HIV particles
HIV budding
- HIV Gag gets monoubiquitinated to serve as a scaffold for ESCRT assembly on the plasma membrane–> evagination into the extracellula rmatrix
Gag is part of the HIv envelope
Describe the role of acid hydrolases for the lysosomal degradation of cellular macromolecules.
Proteases, glycosidases, nucleases, lipases, sulfatases and phosphatases in lysosomes are collectively referred to as acid hydrolases, in that they possess maximal activity in an acidic environment.
Inactive in the cytosolic ph
In receptor-mediated endocytosis, does the clathrin coat directly bind the cargo/receptor to be internalized?
NO
Instead, the integral receptor (or cargo) is bound through interactions between its cytosolic domain and the AP2 protein. AP2 interacts with Clathrin
apotransferrin has a high affinity for the transferrin receptor under what kind of pH?
low pH, like in the lysosome when it binds the transferrin receptor to be recycled back to the PM together
Back at the neutral pH of the extracellular matrix, apotransferrin has a low affinity for the transferrin receptor, and apotransferrin is released.
