Vesicle transport Flashcards
Vesicle
A membrane bilayer enclosed compartment with a hydrophilic interior. It is used to transport soluble proteins into, out of, and within the cell. Vesicles facilitate the cross of membranes via fusion. They also transport hydrophobic proteins to membranes via fusion. These hydrophobic proteins are stuck in the membrane of the vesicle
Cargo
The contents of vesicles
Vesicles vs micelles
Micelles are relevant to detergents and have a completely hydrophobic interior- they are not bilayer enclosed. On the other hand, vesicles are bilayer enclosed. They have a hydrophilic center and a hydrophobic region
Vesicle transport- budding and fusion
If cargo is being transported from the ER to another organelle, the vesicle would bud off of the ER membrane. The interior of the vesicle would contain hydrophilic cargo while hydrophobic cargo would be embedded in the vesicle membrane. The vesicle would travel through the cytoplasm and fuse with a target membrane, like the Golgi apparatus membrane. Once the vesicle fuses, it releases hydrophilic and hydrophobic proteins into the organelle
Exocytosis
Important for secreting protein cargo out of the cell. The vesicle membrane fuses with the cell membrane, and then the cargo can be released out of the cell. Also important for delivering proteins and lipids to the membrane to promote cellular expansion. Each time endocytosis occurs, lipids are lost from the membrane. Therefore, exocytosis must also occur to balance endocytosis and maintain the cell membrane
Biosynthetic-secretory pathway
Vesicles that bud from the ER and travel to a membrane. The vesicle buds off and fuses with the Golgi membrane. The contents go through the Golgi and another vesicle buds off the Golgi and travels to its final destination. Their final destination is usually endosomes or lysosomes, as well as the cell membrane for exocytosis. Vesicles generally deliver digestive enzymes to the endosome. If the vesicle fuses with the cell membrane, it’s contents will be secreted out of the cell
Endocytic pathway
Brings cargo from outside the cell at the membrane surface, usually bringing them over to the endosomes and lysosomes, fusing with these organelles. The vesicle takes in contents from outside the cell and pinches off the cell membrane. It fuses with the endosome, delivering the endocytosed material
Retrieval pathways
Bring membrane lipids and proteins back to their compartment of origin. Contents are returned to the Golgi apparatus or to the ER. This helps to “recycle” some components that were lost during the biosynthetic-secretory pathway
Endosomes and lysosomes function
They have enzymes that can break down endocytosed material
How do vesicles form?
They form from specialized, coated regions of the cell membrane, and they bud off as coated vesicles
Coated vesicles
The “coat” is a distinctive cage of structural proteins that surrounds the vesicle itself. The coat is extremely important to the formation of vesicles. Once the vesicle has formed and budded off a membrane, the coat is shed- the coat must be shed for the vesicle to fuse with other membranes
Functions of vesicle coats (2)
- The coat physically molds the forming and vesicle and it allows the vesicle to form
- Recruits or concentrates membrane proteins in a specialized patch. Some of the proteins are cargo receptors, which help to recruit soluble cargo proteins to the vesicle
3 types of coated vesicles
- Clathrin coated
- COP1 coated
- COP2 coated
How are the types of coated vesicles distinguished?
They are distinguished by the proteins that make up the coat. There are several types of each coated vesicle, and each one is specialized for different transport steps in different places of the cell. Some are specialized in forming vesicles at the ER, others are specialized in forming vesicles at the cell membrane. They incorporate different coat protein subunits that modify their properties
Clathrin coated vesicles
Major protein is clathrin. They mediate transport between the Golgi, lysosomes, endosomes, and cell membrane. The vesicle generally originates at the cell membrane and travels to one of the other organelles. This is important for the endocytic pathway
COP1 coated vesicles
Mediate transport from the Golgi cisternae (Golgi subunits) to the ER. This is important in the retrieval pathway. It helps to mediate transport between the different subunits of the Golgi. It can also mediate transport between the Golgi and the cell membrane
COP2 coated vesicles
Mediate transport from the ER to the Golgi cisternae. This is important for the biosynthetic-secretory pathway
Where do clathrin coated vesicles form?
They usually form at the cell membrane and usually fuse with the endosome or the Golgi compartments
Where do COP1 coated vesicles form? (2)
- Form in the Golgi and deliver things to the ER in the retrieval pathway
- Form in the Golgi and deliver things to the cell membrane in the second phase of the biosynthetic-secretory pathway
Where do COP2 coated vesicles form?
- Form in the ER and fuse with the Golgi in the first phase of the biosynthetic-secretory pathway
Clathrin structure
Contains subunits that are comprised of 3 large and 3 small polypeptide chains (heavy and light chains). It forms a triskelion (a 3 legged structure). The N terminals of the triskelions extend into the cage, forming contacts with adaptor proteins. The clathrin triskelions are the major structural proteins of the coat.
Clathrin coated pits
Clathrin assembles into a basketlike, convex framework of hexagons and pentagons to form coated pits on the cytosolic surface of membranes. The framework allows for the physical molding of that vesicle. A clathrin coated pit is when the vesicle is being molded and the clathrin is starting to assemble
Adaptor proteins
These proteins form a separate, second layer of the coat between the clathrin cage and the vesicle membrane. They physically bind the clathrin coat to the membrane and trap cargo receptors. Adaptor proteins recruit cargo receptors and therefore cargo to the forming vesicle. The receptors and cargo compartmentalize into membrane regions that form the vesicle
Cargo receptors
Transmembrane receptors that capture soluble cargo molecules in the vesicle. Once cargo receptors are recruited, they recruit and bind to soluble cargo proteins
Formation of clathrin coated vesicles (6 steps)
- Clathrin/adaptor protein complexes bind to cargo receptors, clustering them (selective recruitment)
- Cargo receptors bind to cargo molecules, recruiting the necessary cargo for the vesicle
- Sequential assembly of adaptor complexes and clathrin coat generates forces/curvature that result in vesicle formation by physically molding the vesicle
- The vesicle buds
- The clathrin coat is shed shortly after release and the components are reused in vesicle formation
- The vesicle can travel and fuse with a target membrane
Formation of COP1 and COP2 coated vesicles
COP1 and COP2 coats form similarly to clathrin. They form a basket-like lattice that induces membrane curvature. However, not all vesicle coats are like this
Retromer coat
A vesicle coat that only assembles on vesicles budding from endosomes, which function in returning transmembrane cargo proteins. Usually, these cargo proteins are acid hydrolase receptors that are being returned to the Golgi. The coat is different looking than clathrin, as clathrin coats are composed of many different clathrin molecules. Retromer coats form a dimer rather than a lattice
3 criteria for the formation of a retromer coat on endosomes
- It can bind cytoplasmic tails of cargo receptors
- It can interact directly with the curved membrane (endosome)
- It can bind phosphoinositides, a specialized type of membrane lipid which acts as an endosomal marker
All 3 criteria must be met for this coated vesicle to form
Coincidence detector
The retromer coat only assembles at the right time and place
Important domains of the retromer coat
The retromer coat is a multiprotein coat, where the proteins form dimers. The VPS35 domain binds the cytoplasmic tail of the cargo receptor. The PX domain binds to phosphoinositide. The BAR domain mediates dimerization and attachment to already curved membranes (like the endosome membrane).
Phosphoinositides (PIP)
Phosphorylated form of phosphatidylinositol (PI), which is a specialized membrane lipid. The head group of the lipid is a sugar ring. They can be phosphorylated on 3’, 4’, & 5’ carbon positions on the inositol sugar in different combinations. Depending on how they are phosphorylated, PIPs will be able to bind to different proteins (like cargo or adaptor proteins, and the vesicle coat proteins)
What determines the set of PIPs in each organelle?
Different organelles in the endocytic and biosynthetic-secretory pathways have distinct sets of PI and PIP kinases and phosphatases. Distribution of PIPs varies from organelle to organelle & membrane region to membrane region and the phosphorylation pattern varies. Results in compartmentalization, act as markers
Naming of PIPs
(PI(phosphorylation positions)P2)
The phosphorylation positions can be carbons 3, 4, and/or 5 on the sugar. P2 means that there are 2 phosphates, but there can be different numbers of phosphates as well
PIP binding proteins
Depending on how they are phosphorylated, PIPs will be able to bind to different proteins (like cargo or adaptor proteins, and the vesicle coat proteins). Each PIP is recognized by a specific PIP-binding protein. PIP binding proteins recognize specific regions of the membrane (on the organelle or cell surface). PIPs help to regulate vesicle formation since they come into contact with cargo receptors and adaptor proteins
How do PIPs help with vesicle formation?
- Cargo receptors bind to soluble cargo
- An adaptor protein can bind to PIP, which would allow the protein to undergo a conformational change
- The conformational change allows the adaptor protein to bind to a cargo receptor, which in turn is bound to cargo
- Then, the other parts of the vesicle coat are recruited
- Ultimately, PIPs interacting with adaptor proteins helps with vesicle coat formation
Coat-recruitment GTPases
Help to control vesicle assembly in addition to PIPs. Some are monomeric and some are trimeric, but all need to hydrolyze GTP to function. They are found in high concentrations in the cytosol in GDP bound form
Monomeric GTPases
Monomeric GTP-binding proteins. These are the most prevalent GTPases. 2 examples are Arf proteins and Sar1 proteins
Trimeric GTPases
Trimeric GTP-binding proteins (G-proteins) contribute to vesicle assembly too but are less well understood
Arf proteins
GTPases, important for COP1 coat assembly and clathrin coat assembly at Golgi membranes
Sar1 proteins
GTPases, important for COP2 coat assembly at the ER membrane
Sar1 protein mechanism
There are specific Sar1 Guanine nucleotide exchange factors (GEFs) in the ER membrane, which bind cytosolic Sar 1 (GDP-GTP) and give it GTP. When given GTP, the Sar1-GTP molecule undergoes a conformational change where it exposes an amphipathic helix (which has hydrophobic qualities). The hydrophobic effect takes over, so Sar1 is stuck in the closest membrane, which is the ER membrane in this case. Sar1-GTP inserts into the cytoplasmic leaflet of the ER membrane. The tightly bound GTP-Sar1 recruits COP2 coat protein subunits to the ER membrane to initiate coat formation and vesicle budding. Other GEFs & coat-recruitment GTPases operate similarly in other membranes/ compartments. This, along w/ phosphoinositide binding and binding to cytoplasmic tails of receptors induces coat assembly
Guanine nucleotide exchange factors (GEFs)
Help to charge Sar1 proteins with GTP
Sar1-driven COP2 assembly (7)
- Sar1 GEF exchanges GDP for GTP on Sar1, causing a conformational change and exposing the hydrophobic portion
- Due to hydrophobic effect, the hydrophobic portion of GTP-Sar1 inserts into the ER membrane
- GTP bound Sar1 recruits Sec23 (which binds it) and Sec24, which binds the cytoplasmic tail of the cargo receptor. These proteins also recruit cargo receptors and soluble cargo
- GTP-Sar1 and adaptor proteins recruit the structural COP2 proteins Sec13/31
- Once the coat begins to assemble, it begins to physically mold the vesicle
- The vesicle buds
- The COP1 coat is lost
2 adaptor proteins for COP2 assembly
Sec23 and Sec24
2 COP2 proteins making up the COP2 coat
Sec13 and Sec31. The proteins form a basket-like lattice
Arf protein mechanism
Arf works similarly to Sar1. When GTP is bound, it has an exposed hydrophobic portion. In contrast to Sar1, Arf has 2 hydrophobic portions- it has a covalently attached fatty acid that adds to the hydrophobicity
What mediates uncoating of vesicles?
Coat-recruitment GTPases mediate uncoating as well. GTP hydrolysis causes a conformational change that buries the hydrophobic portion of the Arf or Sar1 protein into the protein. Once GTPase is popped out, the coat disassembles
Vesicle pinching
When the vesicle is budding off and beginning to be fully formed. Dynamin assists with this process. Vesicle pinching fuses the outer leaflets of the membrane, releasing the vesicle.
Dynamin and other recruited proteins help bend a patch of membrane and directly distort the bilayer by changing lipid composition via lipid-modifying enzymes
Dynamin
A specialized protein that helps vesicle pinching to occur- it assembles as a ring around the neck of the vesicle bud. This forces the fusion of the bilayer of the membrane and causes the vesicle to bud off. Once the vesicle buds off, GTPase is removed and the vesicle coat disassembles. Dynamin has a PI(4,5)P2 binding domain, which tethers the protein to the membrane. Dynamin also has a GTPase domain which allows it to hydrolyze GTP and regulates the rate of vesicle pinching
Dynamin mechanism (3)
- The GTPase of dynamin is able to hydrolyze GTP
- GTP hydrolysis creates a conformational change of dynamin, making it wrap itself around the neck of the vesicle and squeeze the membranes together
- Ultimately, this squeezing causes the vesicle to bud off
Vesicle uncoating
Uncoating occurs due to PIP phosphatase activity & coat recruitment GTPases. Hydrolysis of GTP causes the hydrophobic portions of Sar1 and Arf proteins to be buried. With PIP, the lipids lose their phosphates, which releases the hold of PI on the vesicle coat
Vesicle uncoating of clathrin coated vesicles
A PI(4,5)P2 phosphatase is co-packaged into clathrin-coated vesicles, as it is necessary to dephosphorylate PI- this allows for coat disassembly. It depletes PI(4,5)P2, weakening binding of adaptor proteins. Chaperone proteins like Hsp70 functions as an uncoating ATPase. They use ATP hydrolysis to physically peel off the clathrin coat. Auxillin (vesicle coat protein) activates ATPase
Vesicle targeting
Vesicles must be targeted to a specific membrane when they leave their site of origin. Targeting must be specific since a vesicle will encounter many membranes. The correct target membrane must be found for proper fusion. Rab proteins (monomeric GTPases) are important to targeting specificity
Rab proteins
Monomeric GTPases that are important to the specificity of targeting. There are over 60 members of the protein family. Each Rab protein is associated with 1 or more membrane-enclosed organelles of the biosynthetic-secretory & endocytic pathways. Different Rab proteins are found in different membrane locations, and they are associated with vesicles that bud from certain organelles. They act as markers for the target membranes the vesicles will be fusing to. Companion Rab proteins are also present on the vesicle membranes, so interactions with the Rab proteins on both sides ultimately creates the specificity of targeting
Rab protein mechanism
- Since they are GTPases, Rab proteins undergo a conformational change once they are bound to GTP, which exposes a hydrophobic portion of the protein (the hydrophobic lipid anchor). Rab-GDP is soluble in cytosol
- Once the hydrophobic lipid anchor is exposed, the protein can insert into vesicles and target membranes
- Membrane-bound Rab-GTP interacts w/ Rab effectors, which facilitate vesicle transport, membrane tethering, & assist in fusion
Rab effectors
Rab effectors and Rab proteins interact with each other on both sides- the vesicle and the target membrane. Their structures vary greatly, and they can have one of 3 different functions based on their structures. Some are motor proteins, some are tethering proteins, and some interact with SNAREs
Motor Rab effectors
Physically propel vesicles along actin or microtubules tracts of the cytoskeleton
Tethering Rab effectors
Act as “fishing lines” that capture and reel in vesicles to target membrane for fusion. These are
large complexes that link vesicles already close to target membrane for fusion
SNAREs
Proteins directly responsible for membrane fusion. They overcome the energy barrier to catalyze fusion of the vesicle to the target membrane, squeezing out the water molecules. SNAREs also provide an added layer of targeting specificity since there are different SNAREs in different target membranes. There are at least 35 different SNAREs in animal cells, and each is associated with a particular organelle in the biosynthetic and secretory pathways
Motor Rab effector mechanism (3)
- Rab proteins are bound to vesicles. Motor Rab effectors will bind to the Rab protein on the vesicle
- The Rab effector will undergo repeated conformational changes to “walk” the Rab and vesicle complex down the microtubule (part of the cytoskeleton)
- This is the mechanism by which vesicles move throughout the cell
Tethering Rab effector mechanism (2)
- Rab proteins are bound to vesicles. As the vesicle and Rab protein get closer to the target membrane, the Rab effector interacts with the Rab protein
- The Rab effector “reels in” the vesicle closer to the membrane, so it can fuse with the membrane and deliver its cargo
Rab effectors interacting with SNARE proteins mechanism (2)
- The Rab effector interacts with the Rab protein on the vesicle and the SNARE proteins on the target membrane
- The SNARE proteins wrap around each other to cause vesicle fusion and the delivery of cargo
Why is vesicle fusion necessary?
It is needed for the vesicle cargo to be delivered and unloaded
Vesicle fusion mechanism (4)
- For fusion to occur, the vesicle and target membranes must be in very close proximity to each other (within 1.5 nm)
- This distance allows lipids to flow from 1 bilayer to another so the fusion event can occur
- Fusion is the coming together of 2 hydrophobic structures. Therefore, the presence of water between the 2 membranes in energetically unfavorable. The membranes must be so close together in order to squeeze all water molecules out from the interface between the 2 structures
- SNARE fusion proteins overcome the energy barrier to catalyze fusion of the structures, squeezing out the water molecules
2 complementary sets of SNARE proteins
- vSNAREs- found on vesicle membrane (1 polypeptide chain)
- tSNAREs- found on target membrane (3 polypeptide chains), quaternary structure
SNARE structure
Both vSNAREs & tSNAREs have helical domains that wrap around 1 another, forming a stable 4-helix bundle called the Trans-SNARE complex. This complex locks membranes together. vSNAREs are attached to the vesicle, while tSNAREs are attached to the target membrane. As they wrap more tightly around each other, the 2 membranes are brought in closer proximity. This movement also helps to squeeze out water from the interface
SNARE mediated fusion
- vSNAREs and tSNAREs wrap, pulling membranes together and squeezing out water from the interface
- Once the 2 membranes are close together, the lipids in the outer leaflets flow between the membranes, creating a stalk-like structure (like the neck in vesicle budding)
- Lipids in the inner leaflets interact, and the stalk widens (this event is called hemifusion)
- Rupture of the fused bilayer completes fusion, joining the membrane interiors
NSF
After vesicle fusion, the SNAREs are still wrapped around each other. At this point, they have fulfilled their function and are not necessary anymore. NSF is a protein that uses ATP hydrolysis to unravel the interacting SNAREs so they can be used in other fusion processes
Which molecules does the biosynthetic-secretory pathway deal with? (3)
- Handles proteins translocated into ER lumen & lipids synthesized in ER lumen
- Proteins and lipids destined for membrane
- Proteins destined for secretion out of the cell
Function of the Golgi apparatus
The next stop for proteins leaving the ER is the Golgi apparatus. The Golgi apparatus is a major site of carbohydrate synthesis. It is also a sorting/dispatching station for products of ER, products can leave here and go to the lysosomes or the cell membrane. It is responsible for glycosylation of proteins & lipids and other protein modifications
ER exit sites
Parts of the ER membrane that do not contain ribosomes and can be considered smooth ER. COP2 coated vesicles bud off from this area
Transport from ER to Golgi vesicles
Transport between these organelles concerns COP2 vesicles, which bud off from ER exit sites.
Exit signals
Protein or sugar signals present on the cytosolic side of cargo receptors and recognized by coat proteins. They are displayed on the cargo and recognized by cargo receptors
Selective recruitment
There is selective recruitment in COP2 vesicle transport from the ER to the Golgi, so adaptor proteins selectively recruit cargo receptors that then selectively recruit soluble cargo proteins.
Vesicle transport from ER to Golgi mechanism
- Cargo receptors bind to the exit signals on the soluble cargo proteins
- In turn, the exit signals on the cargo receptors are recruited to and bind to the adaptor proteins in the vesicle that’s forming
- This is selective recruitment. This is also how vesicle coat formation helps to recruit cargo for the vesicle that’s forming
What must happen to protein structure so they can leave the ER?
Proteins must be properly folded. Also, if the proteins have quaternary structure, the proteins must be assembled. If not, the chaperones will be bound to the proteins. This blocks the exit signal and promotes folding and assembly. The chaperone will only release the proteins once they are properly assembled
Vesicular tubular clusters
Multiple vesicles leave the ER at once to head to the Golgi. After leaving the ER and shedding their coat, vesicles fuse with one another (homotypic fusion). Each vesicle has their own vSNAREs and tSNAREs, which help them to fuse together. These clusters are short lived, they travel along to microtubules to the Golgi, fuse, and deliver their contents
Homotypic fusion
A vesicle fusing with another vesicle
Heterotypic fusion
A vesicle fusing with a target membrane
Vesicle retrieval pathway
Returns proteins and lipids to their original compartments, as well as resident proteins that have escaped non-specifically. This is pathway is between the ER and Golgi. COP1 coated vesicles function in retrieval. COP1 vesicles and their adaptor proteins recognize retrieval signals for the proteins
Retrieval signals for proteins (2)
- For ER membrane proteins, the sequence would be C-terminus KKXX sequence (lysines as well as variable amino acids). They bind to COP1 coat proteins
- For soluble ER proteins, the sequence would be the KDEL sequence (letters represent the amino acids). It binds to the KDEL receptor- this receptor binds to COP1 proteins
KDEL receptor-retrieval pathway (4)
- ER proteins with the KDEL sequence are traveling toward the Golgi, although they are not supposed to be traveling there
- KDEL receptors recognize the KDEL sequence
- The KDEL receptors get recognized by the adaptor proteins of the COP1 coat
- The vesicle buds off and returns the membrane and soluble proteins back to the ER
Which factors change KDEL affinity?
Different ionic conditions and pH may change KDEL affinity. Different conditions may promote binding of the KDEL receptors in the Golgi but promote the release of those interactions in the interior of the ER
After the ER, what is the next step on the vesicle pathway?
The Golgi apparatus is the next step
Cisternae
Stacks of flattened, membrane enclosed compartments. There are also tubular connections that exist between the stacks, linking many of them and creating one linked organelle. The stacks are precisely arranged and are navigated using microtubules and motor proteins like the rest of the cell
Cis face of the Golgi apparatus
Where vesicles enter/fuse, the side closest to the ER
Cis Golgi network (CGN)
A collection of fused, vesicular tubular clusters
Trans face of the Golgi apparatus
The exit, closest to the cell membrane. This is where vesicles would bud off
Trans Golgi network (TGN)
Proteins and lipids exit from here bound for the cell membrane or another compartment (like lysosomes)
Trimming and glycosylation
Part of this process occurs in the ER and part occurs in the Golgi. During trimming, the oligosaccharide gets trimmed back to the “core” oligosaccharide. In some cases, other sugars like trisaccharides are added. All different sugars can be part of the glycosylation.
2 styles of trimming
- Minimal trimming- high mannose sugar is created when some mannoses are trimmed a bit, no new sugars are added in the Golgi
- An oligosaccharide is trimmed back to the core and a trisaccharide is added. The copies of trisaccharide can vary, the core can be heavily glycosylated or lightly glycosylated
What happens when proteins arrive at the Golgi
- Proteins arrive at the Golgi from the ER. Different things happen in the cisternae
- Trimming takes place at the cis face of the Golgi. Mannoses are removed. Trimming can be minimal or more extensive (in this case more sugars will be added)
- Addition of extra sugars occur at the trans face
What determines the extent of trimming?
It depends on the accessibility of the sugars. If the oligosaccharide is more closely associated with the surface of the protein in question, it will be less accessible to enzymes that can remove those sugars. This would become a high mannose oligosaccharide because there would be less trimming and less sugars added. If the oligosaccharide is less closely associated and is more accessible, there will be more trimming and addition of more sugars
O-linked glycosylation
Occurs when sugars are being added to proteins as they move through the Golgi. It is called O linked because sugars are added to the hydroxyl group of the amino acids serine or threonine. The sugars here are added one at a time, like an extension of N-linked oligosaccharides. When a protein undergoes O-linked glycosylation, that usually means that it is heavily glycosylated. This type of glycosylation is heaviest on mucins and proteoglycan core proteins.
Mucins
Glycoproteins found in our mucus secretions
Proteoglycans
Chief components of the extracellular matrix. It becomes a proteoglycan when a glycosaminoglycan (GAG) sugar chain is added to it.
Glycosaminoglycan (GAG)
Long unbranched sugar polymer of repeating disaccharides. GAG is heavily sulfated in the trans Golgi network, which adds a negative charge to proteins
Why are proteins glycosylated? (4)
- Serves as signal of folding state of proteins in the ER
- Makes certain proteins more resistant to proteases
- Facilitates cell-cell recognition. Components of the membrane or ECM are recognized by lectins
- Modifies antigenic properties of a particular protein. Added sugars change the shape of a protein which can impact receptor recognition
2 models for how proteins travel through the Golgi
- Vesicular transport model
- Cisternal maturation model
It is unclear which model is correct or whether there is a combination
Vesicular transport model
In this model, COP1 coated vesicles move proteins between the individual cisternae. These vesicles move forward, although they also function in the retrieval pathway (retrograde transport). There is stepwise fusion- a vesicle releases and fuses to the next stack, releases and fuses to the stack after, and so on. Different adaptor proteins in the coat may confer directionality
Cisternal maturation model
Vesicles from the ER fuse, forming vesicular tubular clusters, which morphs into the cis Golgi Network. The CGN then matures into the cisternae and TGN. In this model, COP1 coated vesicles function exclusively in retrograde transport
Lysosomes
Digestive organelles that contain digestive enzymes (acid hydrolases) that are present in the lumen. They are responsible for endocytosis, autophagy, and phagocytosis. All cells can carry out endocytosis and autophagy
What happens to proteins when they leave the lysosomes?
The TGM sorts fully matured proteins and ships them off. One possible destination is the lysosomes
Acid hydrolases
Digestive enzymes present in the lysosomes. There are specific hydrolases that are responsible for breaking down specific molecules. They are called acid hydrolases because they function at a low pH.
V-type ATPase
Uses ATP hydrolysis to actively pumps protons into the lumen of the lysosomes. The more protons, the more acidic the environment becomes. Therefore, the V-type ATPase maintains a low pH
Acidification of lysosomes
The V-type ATPase is a piston-shaped pump. It conducts multiple rounds of ATP hydrolysis which causes rotation around the “rotor” in the membrane. Protons progressively bind to the rotor until all spots are filled, and then the protons are pumped across the membrane
Endocytosis
Large molecules are taken up into early endosomes, where some lysosomal hydrolases are delivered. It then matures into a late endosome, where the pH gets lowered due to the V-type ATPase. Then, the endosome fuses with the lysosome and the pH is at the perfect range for the hydrolases. Once fusion with the lysosome occurs, the hydrolases attack and destroy the macromolecule that was endocytosed
Autophagy
Like endocytosis but from within the cell. It helps to remove senescent or damaged proteins or organelles from the cell. The proteins are found in the cytoplasm, and then a vesicle forms around them called the autophagosome. The autophagosome then fuses with the lysosome and lysosomal hydrolases degrade the damaged organelle
Phagocytosis
Not all cells are capable of phagocytosis, only professional phagocytes. This is a specialized process for the removal of pathogens or pathogenic material. They are taken up in a phagosome which then fuses with the lysosome. The hydrolases then degrade the pathogen
Mannose 6-phosphate tag
Lysosomal hydrolases are an example of proteins that are packaged in the Golgi and sent to the lysosomes. All proteins destined for the lysosome are given a mannose on one of their N-linked oligosaccharides right as they are leaving the TGN (phosphorylation and addition of the mannose occurs here). Once the tag is added, proteins can now bind to cargo receptors. The cargo receptors (M6P receptors) bind to adaptor proteins which in turn bind to clathrin coat proteins. The vesicle then leaves the Golgi, loses its coat, and fuses with the early endosome. The early endosome will later mature into a lysosome. The lower pH (pH 6) encourages the proteins to dissociate from the receptor, and they are then in their correct location. When the cargo is released, the hydrolases are de-phosphorylated, and the M6P receptor is returned to the Golgi in a COP1 coated vesicle
Mannose 6-phosphate tagging mechanism
- Only proteins with a mannose 6-phosphate tag will be recognized by cargo receptors in the Golgi membrane
- Cargo receptors bind to adaptor proteins and the clathrin coat forms
- This is how proteins destined for lysosomes are recruited from the Golgi
- Fusion with the lysosome means that lysosomal hydrolases are delivered. The pH is lowered
- Therefore, the proteins are released from the M6P receptors
- The hydrolases will now be active as long as the pH remains low
Types of endocytosis (3)
- Phagocytosis- occurs only in specialized phagocytic cells- used for large molecules or whole cells
- Pinocytosis- fluid and solutes. This is always happening
- Receptor mediated endocytosis- specific macromolecules
Process of phagocytosis (6)
- A pathogenic organism binds to 1 of 4 phagocytosis receptors
- Binding triggers exocytosis so the membrane surface area will expand for necessary pseudopod formation to occur
- Binding then triggers actin polymerization. Rho GTPases turn on phosphoinositides (PI) kinases, and actin polymerizes in response to PI(4,5)P2
- Reorganization of the actin portion of the cytoskeleton allows for pseudopod formation
- The phagosome is sealed when PI(4,5)P2 is converted to PI(3,4,5)P3
- Actin is depolymerized and the pathogenic cell is taken in
Phagocytosis receptors (4)
- Pattern recognition receptors (PRPs)- recognize common antigens
- Fc receptors- recognize antibody opsonized pathogens
- Complement receptors recognize complement opsonized pathogens
- Phosphatidylserine (PS) receptors- remove apoptotic cells
Pseudopod formation
Phosphoinositides are important here for the binding of the actin machinery. Once the actin machinery binds, the cytoskeleton can rearrange and form pseudopods around the pathogen. This is an important part of phagocytosis. This process is also concurrent with exocytosis so the membrane will expand enough for the actin rearrangement to occur
Pseudopod formation mechanism (4)
- Phosphoinositides (PI(4,5)P2) form, which allows the cell machinery (N-WASP) to bind. N-WASP is what activates actin polymerization
- The Arp2/3 complex binds to N-WASP, as N-WASP is an activator of Arp2/3
- Once Arp2/3 is activated, actin rearrangement can occur
- The actin rearrangement forms pseudopods
Fate of phagocytosed material (5)
- The pathogen is taken into a phagosome and undergoes acidification
- Lysosomes fuse with the phagosome, forming a phagolysosome
- The lysosomes have already delivered their acid hydrolases, which become active
- NADPH oxidases from the lysosomes help to form reactive oxygen species to attack the pathogen in the lysosome
- Myeloperoxidase- another lysosomal enzyme which forms bleach
Respiratory burst
NADPH oxidases from the lysosomes help to form reactive oxygen species to attack the pathogen in the lysosome
Myeloperoxidase
A lysosomal enzyme that produces hypochlorous acid (HOCl aka bleach) from hydrogen peroxide and chloride. It partially dissociates into hypochlorite (OCl-) in aqueous solution
Pinocytosis
A constitutive (does not need to be triggered), non-specific process that occurs for small nutrients, solutes, and fluids (like fat droplets, vitamins, antigens). It is important for nutrient and fat absorption, and Ag uptake. The molecules are taken up into clathrin-coated pits, forming small pinocytic vesicles (100-200 nm in diameter). Animal cells ingest 1-3% (fibroblast- macrophage) of their surface every minute. Membranes are kept in balance by an almost equal rate of exocytosis. This replenishes lipids and proteins in membrane
Steps of pinocytosis (6)
- Attraction of positively charged molecules to the negatively charge membrane
- The cell membrane changes shape
- The membrane forms a clathrin coated pit, which helps to mold the vesicle
- Dynamin pinches off the clathrin coated pinocytic vesicle
- The clathrin is shed and the pinocytic vesicle fuses with the lysosome
- The contents of the vesicle are degraded by hydrolases
Pinocytosis vesicle content degradation
This process occurs in the lysosomes and requires ATP. Hydrolases degrade the content
Pinocytosis vs phagocytosis
Pinocytosis is like a pit that forms, like an invagination. In phagocytosis, reaching pseudopods form
Receptor mediated endocytosis
Since it is receptor mediated, it is more specific than pinocytosis. Ligands are selectively captured by specific receptors, increasing the efficiency of internalization of correct and specific ligands. More than 25 receptors participate in RME. It results in the formation of a clathrin coated pit
RME of low density lipoprotein (LDL)
This is 1 way in which the cell obtains cholesterol for membranes. LDL is taken up into an endosome, which fuses with a lysosome. The cholesteryl esters from LDL are hydrolyzed to free cholesterol that can be used for the cell
Types of receptor mediated endocytosis (2)
- Stimulated endocytosis- this is most common
- Constitutive endocytosis- vesicles form continuously
Stimulated endocytosis
The most common form of RME. The vesicle only forms when receptors are bound
Steps of receptor mediated endocytosis (7)
- AP2 adaptin binds PIP2
- Clathrin-coated pit forms
- Dynamin wraps around and pinches off vesicle
- Some contents are returned to membrane
- Some contents remain in the endosomes, and some endosomes contain multivesicular bodies
- Glycocalyx forms around the inner leaflet of the endosome. This protects the endosome membrane when it fuses with the lysosome
- Endosome fuses with lysosome and the contents of the endosome are degraded
Multivesicular bodies
Endosomes with luminal vesicles- basically an endosome with smaller vesicles inside. Sometimes this occurs during receptor mediated endocytosis
Autophagy
A pathway specialized for the removal of damaged proteins or damaged organelles. An autophagosome forms around the damaged organelle, lipids are delivered and basically form a vesicle around the damaged organelle. The autophagosome will fuse with a lysosome, where the hydrolases will degrade the damaged organelle.
Process of autophagy
- An autophagosome begins to form around the damaged organelle. Vesicles containing the ATG9 protein begin to deliver lipids that will fully form the autophagosome
- The class 3 PI3K complex forms a PI3 phosphate around the autophagosome. This promotes the next step (fusion)
- Fusion- SNARE proteins are also important here, they wrap around each other in a trimeric fashion and bring the vesicles closer together so they can fuse
- The hydrolases can destroy the contents of the autophagosome
Class 3 PI3K complex
Important during autophagy, includes Beclin-1 and AMBRA1 proteins. The complex produces a PI3 phosphate which is a phosphoinositide. It changes some of the lipids in the autophagosome
SNARE proteins important in autophagy
- STX17 on the autophagosome
- VAMP8 on the lysosome
- SNAP29
All of these proteins wrap around each other in a trimeric fashion, with SNAP29 forming a “bridge” between the other 2
Exocytosis
For molecules leaving from the TGN that are going to the cell surface or outside of the cell. There are 2 kinds of pathways- the constitutive secretory pathway and the regulated secretory pathway. Exocytosis replenishes the plasma membrane, balancing out endocytosis
Constitutive secretory pathway
All cells are capable of this. The pathway delivers lipids and proteins to the membrane, such as ECM proteins. Like pinocytosis but in reverse (and pinocytosis is balanced out by an equal rate of exocytosis). Some of the protein release is just constitutive secretion of proteins
Regulated secretory pathway
Only found in specialized secretory cells, where proteins are stored in specialized secretory vesicles. This vesicles still bud off of the TGN. This includes hormones, neurotransmitters, digestive enzymes, and cytokines. These vesicles must wait for a signal, and they will only fuse with the cell membrane and cause exocytosis when they receive the signal
Secretory vesicles
Wait near the cell membrane for the stimulus. Usually, the signal is an influx of calcium that is localized to that part of the cytoplasm. Calcium binding to specific sensors triggers fusion. This occurs with mast cells and cytotoxic T cells
Neurotransmitters and chemical synapses
Neurotransmitter vesicles wait near the membrane of the presynaptic neuron’s axon terminal. They are triggered to fuse with the membrane, partially due to an action potential that comes down the membrane of the neuron. Once the vesicles fuse, they release neurotransmitters into the synapse. The neurotransmitters open channels on the postsynaptic cell
Chemical synapses
When a change in membrane potential in the axon (due to an action potential) triggers exocytosis of neurotransmitters. The action potential opens a voltage gated calcium ion channel, and the calcium disrupts the storage interactions of secretory vesicles with the cytoskeleton. The secretory vesicles are no longer locked in place, and they fuse with the axon membrane via SNAREs
What happens to neurotransmitters after the postsynaptic neuron is stimulated?
They can be rapidly degraded in the synapse or rapidly taken up by presynaptic neuron
Formation of synaptic vesicles
Synaptic vesicles re-form in the presynaptic neuron via a modified endocytic pathway. They do not turn into lysosomes. The vesicles contain membrane proteins that are specialized for uptake of neurotransmitters
