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Does sorting to the lysosome have to go through an endosome?
No, it can go directly from the TGN to the lysosome
What is the main purpose of a lysosome?
To break down large molecules into simpler subunits that can be recycled and reused by the cell
What are some characteristics of a lysosome?
They are membrane-bounded and were discovered by cell fractionation; they vary in size (200 nm - 500 nm) and contain degradative acid hydrolases that only work at low pHs; there are many different types of acid hydrolases that break down different types of molecules; the optimal pH of a lysosome is 5, and this pH is maintained by H+ pumps in the membrane that use ATP to move H+ against its gradient into the lysosome; only need about 250 H+ to reach pH 5
Why don’t the lipases inside lysosomes destroy the lysosome membrane?
Because the lumenal leaflet of the lysosome membrane has many glycoproteins that are extremely glyscosylated attached to it; this thick layer of oligosaccharides is called a glycocalyx; this blocks the lipases from accessing the lipids in the membrane
Why don’t the glycosidases break down the glycocalyx on the inside of the lysosome membrane?
The oligosaccharides in the glycocalyx are not recognized by the glycosidases due to their topology
What are vacuoles?
They are large lysosomes found in plant and fungal cells that can take up 30-90% of the cell; these vacuoles have other functions too such as maintaining turgidity and storing proteins
What can problems with the functioning of the lysosome be caused by?
defect in protein trafficking → acid hydrolases never make it to the lysosome
defect in lysosomal proteins → acid hydrolases have a mutation and don’t function properly
defect in regulation of lysosomal proteins → acid hydrolases present, but regulatory proteins are malfucntioning
How does trafficking of acid hydrolases to the lysosome work?
Controlled by both a pH gradient and protein-protein interactions
The mannose residue on the oligosaccharide of a specific N-linked glycoprotein gets phosphorylated, forming mannose-6-phosphate; there are protein-protein interactions that specify which proteins get this mannose-6-phosphate tag
mannose-6-phosphate is recognized by a M6P receptor in the membrane of the TGN and binds to it
This occurs at an exit site of the TGN where the membrane buds off and forms a vesicle with a double protein coat around it; M6P receptor interacts with coat of adaptins, which binds to clathrin, forming clathrin-coated vesicles
eventually the vesicles lose this coat and dock and fuse with a late endosome, which has a pH of about 5.5, which is lower than that of the TGN; this change in pH causes the protein bound to the M6P receptor to release and its phosphate group gets removed and you have an acid hydrolase (not yet functional because pH is too high)
The M6P receptor gets recycled back to the TGN
The late endosome matures over time into a lysosome
For protein sorting in general, what mechanisms can have an effect on the sorting of a protein?
pH gradient
thickness of membrane gradient
specific amino acid sequences
protein topology
Is there any vesicle trafficking between the late endosome and the lysosome?
No, the late endosome matures into the lysosome
What is a primary lysosome?
A lysosome that has no material in it being degraded
What is a secondary lysosome?
A lysosome that has material in it being degraded
Which proteins coats are associated with which trafficking processes?
COPII vesicles → ER to Golgi
COPI vesicles → retrograde Golgi to Golgi or Golgi to ER
Clathrin-coated vesicles → Trans Golgi Network to late endosome or endocytosis to lysosome
How do clathrin-coated vesicles coming from the TGN form?
the activation of an ARF GTPase on the TGN membrane causes it to interact with kinases that change the lipid composition of the membrane, allowing adaptin proteins to bind to the membrane
the cytosolic tails of the M6P receptors interact with the adaptin proteins
The clathrin proteins then assemble on top of the adaptins
the budding off of the vesicle is mediated by a GTPase called dynamin (NOT a small monomeric GTPase) which forms polymers, and the hydrolysis of GTP into GDP and Pi releases energy that allows the vesicle to pinch off, forming the vesicle
What is the structure of clathrin?
It is composed of 3 heavy chains and 3 light chains in a triskelion shape; when multiple clathrin molecules come together, the arms lock together, and 36 clathrin molecules will come together to form a sphere/basket thing
What are the 3 pathways to degradation in a lysosome?
endocytosis - phagocytosis
endocytosis - pinocytosis
autophagy
What is autophagy and how does it work?
It is the degradation of old organelles or organelles that the cell needs nutrients from
a double membrane begins to form around the organelle, forming a compartment called an autophagosome
the autophagosome is then brought into the lysosome whole and its membrane and contents are broken down
How did we figure out the order of events in autophagy?
A researcher isolated temperature-sensitve yeast mutants that were blocked in different parts of the autophagy pathway and did double-mutant analysis to determine the order of events in the pathway; also, because this is a genetic approach, they were able to identify the genes in which the mutations resided, allowing them to predict the proteins involved and understand how the mechanism works
What is phagocytosis and how does it work?
Phagocytosis is “cell-eating” and it is a type of endocytosis; it is important for unicellular organisms and our immune system; phagocytosis can be receptor-mediated or not depending on the cell type; for our immune system, receptors on the plasma membrane recognize antibodies on the bacterium and then extend pseudopodia around it and engulf it, forming a phagocytic vacuole that eventually matures into a lysosome; for unicellular organisms, food is swept onto the surface of the cell and into the food gullet, which is then brought into the cell via phagocytosis
How does endocytosis and exocytosis help regulate the size of the plasma membrane?
They balance each other out to make sure the membrane is not gaining or losing too many lipids
What does endocytosis do for a cell?
Recycling of membrane and proteins → brought back into the cell
“Ingestion” → for feeding or defense
Regulation → signaling; either bringing signaling molecule in or can bring receptor back into cell to stop signaling
Trafficking
Harm → route of entry for some viruses
How does clathrin-independent pinocytosis work?
It depends upon the formation of invaginations of the plasma membrane called caveolae; this captures liquid and soluble molecules in the environment and the caveolae pinches off and brings that material into the cell
How does receptor-mediated clathrin-dependent pinocytosis work?
depends on the formation of clathrin-coated pits; the target proteins associate with receptors on the extracellular side of the plasma membrane, and then clathrin begins to gather on the cytosolic side of the plasma membrane and as the membrane invaginates inward the clathrin coats the budding compartment until it pinches off, forming a clathrin-coated vesicle
What are some examples of cells that have many many clathrin-coated pits (undergoing a lot of receptor-mediated endocytosis)?
Fibroblasts, hepatocytes, macrophages
How does the presence of external cholesterol or statin drugs affect the synthesis of cholesterol in a cell?
The presence of external cholesterol or statin drugs inhibits the synthesis of cholesterol in a cell by blocking the activity of HMG-CoA reductase, an enzyme in the synthesis pathway
What are important steps in cholesterol synthesis?
This part of the pathway occurs on the cytosolic leaflet of the smooth ER
One of the precursor molecules, Acetyl CoA is converted into HMG-CoA
HMG-CoA is then converted into Mevalonate by HMG-CoA reductase → this is the rate-limiting step of the pathway
Does cholesterol float around freely in the blood?
No, it is packaged in LDL or HDL
In what form does synthesized cholesterol leave the cell?
It leaves packaged in chylomicron or VLDL, and these particles then get processed in the blood to form LDL or HDL
What is the structure of LDL?
It is a spherical ball; it has a polar outer monolayer of phospholipids and a core of cholesteryl esters (esterified cholesterol); there is a band of apolipoprotein around the center of the sphere
What is cholesteryl ester?
It is the storage form of cholesterol; it has an ester bond at the -OH group that attaches a long fatty acid tail
What are the general trends of properties of cholesterol packaging molecules?
Density: chylomicron → VLDL → LDL → HDL
Diameter: HDL → LDL → VLDL → chylomicron
# of proteins: chylomicron → VLDL → LDL → HDL
What did Brown and Goldstein’s experiment show?
They were looking at how cells uptake LDL particles and discovered the actual mechanism of receptor-mediated (clathrin-dependent) endocytosis; it also showed that hyperlipidemia can be caused by any number of defects, not just one
How did Brown and Goldstein’s experiment work?
They tracked the LDL particles using radioactively labeled LDL (used 125I); set up Pulse-Chase experiment; they brought all the cells in the cultures down to 4 °C to get them in an inactive state to reduce endocytosis; then added the 125I (Pulse) and after a while washed away the excess (Chase); then they began warming the cells for a period of 45 minutes; at different time points during warming, they took samples and processed them; they measured how much 125I was bound to the plasma membrane at any given time (heparin-releasable LDL); they did this by making LDL release from the PM by mixing cells with heparin (negative charge of heparin disrupts electrostatic interaction between LDL and the PM receptors); they also measured how much 125I was internalized by the cell (heparin-resistant LDL); they found that this LDL was being degraded over time; they measured degradation by seeing if the apolipoproteins on the LDL were still intact or not; they measured this by adding TCA to cell extracts, which polypeptides are non-soluble in, but amino acids are, and they found that over time the amount of TCA-soluble material was increasing, so the apolipoproteins were being degraded
How did Brown and Goldstein figure out how LDL enters a cell?
They labeled LDL particles with ferritin, which binds free iron ions, making the particles electron dense, so they deflect electron beams, showing up as black spots on an EM; they saw that it entered the cell in coated pits
How does LDL enter a cell?
It enters via clathrin-dependent endocytosis; LDL recpetors on the surface of the PM move around in the plane of the membrane, and when they move into a semi-formed pit, an LDL particle will bind to it and a clathrin-coated pit begins to form, eventually forming a clathrin-coated vesicle; the clathrin coat then comes off via an ATP-dependent event; this uncoated vesicle is now called an early endosome, which will fuse with a late endosome; in the late endosome, the LDL releases from the receptor and the receptor is recycled back to the plasma membrane; the late endosome then matures into a lysosome and the LDL particles are degraded into free cholesterol; free cholesterol is exported from the lysosome and then inhibits the activity of HMG-CoA reductase, stopping cholesterol synthesis in the cell
How precise is the regulation of cholesterol in cells?
It is very precise, with cells regulated the amount of cholesterol in their membranes with a variance of only about 0.1%; the process of cholesterol synthesis is constantly being regulated
What cells are mainly responsible for free cholesterol storage and how do they store it?
Adipocytes; the free cholesterol is stored in an organelle called a lipid droplet; almost the entire volume of an adipocyte is a lipid droplet (multiple microns in diameter)
Do lipid droplets only store lipids?
No, there are also many proteins involved with lipid metabolism associated with lipid droplets and other metabolic processes are organized at lipid droplets
How do viruses that enter cells via endocytosis do it?
They can enter via clathrin-dependent endocytosis or via clathrin-independent endocytosis (form caveolae)
What is Transcytosis?
It is a combination of endocytosis and exocytosis; it is how molecules are moved across cell layers (ex. in the cells lining the small intestine); endocytosis occurs at the apical domain, bringing in nutrients, and an endosome forms; rather than maturing into a lysosome, the endosome traffics to the opposing side of the cell to the basolateral domain and exocytosis occurs, releasing the contents into the interstitial fluid
How do nutrients release from the receptors they are bound to in transcytosis?
The pH of the intestinal lumen is lower (6) than the pH of the blood/interstitial fluid (7), so the ionization state of the side chains of the proteins change, causing it to release
