Endocytosis and Protein Degradation Flashcards
Organelles get damaged and
need to be removed and replaced
Proteins have a
finite lifetimes.
Misfolded proteins will
create havoc if they are not destroyed.
Both good and bad endocytosed materials
need to be degraded.
Major Functions of the ER
- Synthesis of lipids (phospholipid, ceramide, and cholesterol)
- Control of cholesterol homeostasis (cholesterol sensor and synthesis)
- Ca+2 storage (rapid uptake and release)
- Synthesis of proteins on membrane bound
ribosomes - Co-translational folding of proteins and
early post-translational modifications - Quality Control
Molecular chaperone proteins
- Help fold a protein by binding to exposed hydrophobic patches in incompletely folded proteins (hsp70)
- Form large barrel-shaped structures to act as an “isolation chamber” into which misfolded proteins are fed to prevent their aggregation and help it to refold (hsp60)
ER Quality Control
- Provide optimized oxidizing environment
– Folding
– Oligomeric assembly - Folding enzymes
– ERp57- thiol oxidoreductase (allows formation of disulfide bonds)
3 . Molecular Chaperones- ATPases
– BiP- Hsp70 family - Folding Sensors/ Quality Control
– UDP-glucose: glycoprotein glucosyltransferase (UGGT)
Proteasome
(ATP-dependent protease that constitutes 1% of cellular protein)
Dispersed throughout the cytosol and nucleus
Proteosomes also monitor the
monitors the ER (proteins detected to be misfolded are retrotranslocated back into the cytosol for degradation)
Proteasomal degradation (ubiquitin mediated)
ubiqutiation-covalent attachment of ubiquitin (76 aa) to acceptor lysine E-amino group in protein to be degraded
Proteolysis occurs within the
central chamber
Only β-subunits are _____
proteolytically active”
α-subunits regulate
substrate entry into the “death chamber””
Substrate is “spiraled” through the chamber and cleaved by
different activities associated with different b-subunits; ~7-9 amino acid peptides are released;
cleavage does not require
ATP;
ATP required for
unfolding and translocation
Degradation:
mechanisms that provide spatial control
Proteasome-
degrades only proteins
– Polyubiquibitinated proteins (tetra-ubiquitin minimal targeting signal)
Lysosome-
degrades all cellular components
– Targeted via the endocytic pathway
– Monoubiquitinated transmembrane proteins
– Regulated at the Multivesicular Body
• Autophagy
– Direct transport into the lysosomal lumen from cytoplasm
Lysosomes Degrade all
Macromolecules in an Acidic Environment
Transporters in lysosomal membrane allow exit of
amino acids, monosaccharides, nucleotides, and lipids for reutilization within the cell.
Lysosomal storage diseases are
- Metabolic disorders that result from defects in lysosomal function
- Specific enzymes are defective or deficient
examples of lysosomal storage diseases
– Tay-Sachs:
– Gaucher’s disease:
– Niemann-Pick:
– Tay-Sachs:
beta-hexosaminidase, breaks down gangliosides in neurons
Gaucher’s disease:
beta-glucosidase, breaks down glucosylceramide in monocytes and leukocytes
Niemann-Pick:
(1) sphingomyelinase, breaks down sphingomyelin in macrophages and
(2) cholesterol transporter, moves cholesterol from the lysosome to the cytosol.
Two major routes fro small volume endocytosis
- clathrin coated vesicles
2. calveolae
Clathrin Coated Vesicles function to
Selectively sort cargo at the cell membrane, trans-Golgi network and endosomal compartments for multiple membrane traffic pathways.
In clathrin coated vesicles, after a vesicle buds into the cytoplasm, the
the coat rapidly disassembles allowing the clathrin to recycle while the vesicle gets transported to a variety of locations.
In clathrin coated vesicles, Adaptor molecules are responsible for
self assembly and recruitment.
Calveolae arises from
invaginations on the surface of the cell o Look like “little caves,” hence their name
3 major coat proteins for calveolae:
- Calveolin 1 – located everywhere
- Calveolin 2 – seem to work with calveolin 1
- Calveolin 3 – specific to skeletal and cardiac muscle
Calveolin 1 – located
everywhere
Calveolin 2 –
seem to work with calveolin 1
Calveolin 3 –
specific to skeletal and cardiac muscle
Mutations in Calveolin 3 cause
muscle diseases such as Limb Girdle Disease and Rippling Muscle Disease
Cholera toxin gets into the cell via _____ exclusively.
calveolae
Dynamin pinches off both
clathrin coated vesicles and calveolae.
The ER provides an optimized oxidizing environment for
folding and oligomeric assembly.
The ER has folding enzymes associated with it such as ______
ERp57
Erp57: is ____ and allows formation of
thiol oxidoreductase
disulfide bonds with the new protein to hold it in place with calnexin
Calnexin has a _____ pocket
o ERp57 holds the protein in place so that it presents its
glucose
glucose molecule to the calnexin pocket:
If the protein has been folded correctly, ______
If it has been folded incorrectly, it will_____
the glucose will fit in the pocket.
not fit in the calnexin pocket
If incorrect, it will be sent to the
lysosome for destruction or else given a chance
to refold correctly.
The ER has ATPase molecular chaperones-
ATPases such as the BiP- Hsp70 family
Finally, the ER has Folding Sensors:
- UDP-glucose
2. Glycoprotein glucosyltransferase (UGGT)
two types of molecular chaperones.
- Hsp70
2. Hsp60
Hsp70:
Binds to exposed hydrophobic patches of incompletely folded proteins to prevent aggregation.
Hsp60
Forms a large, barrel shaped structure that acts as an isolation chamber. Misfolded proteins are
fed into it to prevent aggregation and to promote proper refolding.
Proteasome Structure:
- 2 mega-Daltons in weight – very large
- 26S proteasome made up of 3 macro-subunits o 19S cap on each end – regulatory region
- Base of cap contains 6 “AAA-ATPases”
- Lid unit of cap contains poly-ubiquitin recognition molecules o Central 20S cylinder – the actual active site of the proteasome
- The proteasome is ATP-powered
Beta subunits are ______
proteolytically active
Ubiquitination:
Occurs in three steps:
- E1 ubiquitin – activating enzyme
- E2- ubiquitin – conjugating enzyme which takes activated ubiquitin and transfers it to E3.
- E3- ubiquitin – protein ligating enzyme.
β1
– caspase-like: cleaves after acidic amino acids
β2 –
trypsin-like: cleaves after basic amino acids
β3 –
cleaves after hydrophobic amino acids
E3s confer______to ubiquitination.
specificity and regulation
E1 ubiquitin –
activating enzyme
E2- ubiquitin –
conjugating enzyme which takes activated ubiquitin and transfers it to E3.
E3- ubiquitin –
protein ligating enzyme.
E3 binds directly to
substrate and covalently attaches the ubiquitin to the substrate.
Once a single ubiquitin is covalently attached to a protein, additional ubiquitins are linked preferentially to
Lys48 of ubiquitin to form a poly-ubiquitin chain.
Ubiquitin is
highly conserved
The lysosome degrades all cellular components
- Digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria
- Targeted via the endocytic pathway
- Mono-ubiquitinated transmembrane proteins
- Regulated at the Multivesicular Body
The environment of the lysosome is.
acidic (pH 4.5).
The lysosome maintains this pH differential by
pumping protons (H+ ions) from the cytosol across the membrane via proton pumps and chloride ion channels
The lysosomal membrane protects the cytosol, and therefore the rest of the cell, from the
degradative enzymes within the lysosome.
The cell is additionally protected from any lysosomal acid hydrolases that leak into the cytosol because
these enzymes are pH-sensitive and do not function as well in the alkaline environment of the cytosol.
Transporters in lysosomal membrane allow exit of
amino acids, monosaccharides, nucleotides, and lipids for reutilization within the cell.
two major routes of endocytosis:
1) phagocytosis and
2) pinocytosis or small vesicle formation.
Caveolae
(little cavities) are small endocytic vesicles that form
without coat proteins.
