BIOMG 1350 Lecture 11 - Membrane Trafficking (Secretory and Endocytic Pathways)

Endomembrane System

• The collection of the ER, Golgi, endosomes, lysosomes, and plasma membrane is called the endomembrane system.

• Vesicle transport is the way in which proteins or lipids are transported to the various places in the endomembrane system. Vesicle transport is also known as "membrane trafficking."

Overview of Membrane Trafficking Pathways

• Vesicle transport pathways are from the ER, to the Golgi, and eventually to the plasma membrane or endosomes/lysosomes.

• Vesicles are the unit of transport of proteins to various organelles or to the extracellular space or plasma membrane.

• Selective transport of proteins (cargo) through the endomembrane system occurs by vesicles.

Mechanisms of Vesicle Budding, Movement, Tethering, and Fusion in the Life Cycle of a Vesicle

The life cycle of a transport vesicle: budding, movement, tethering, and fusion

• Budding is the process of the vesicle being pinched off from the donor compartment.

• Movement is the process of the vesicle moving along the cytoskeleton to reach its destination.

• Tethering is the process of the vesicle being targeted to the target compartment's membrane.

• Fusion is the process of the vesicle combining with the target compartment and releasing its contents inside.

Vesicles

• Selective transport of proteins (cargo) through the endomembrane system occurs by vesicles.

• Vesicles are formed by protein “coats” that select specific proteins for transport. This involves capture of cargo by receptor proteins, binding of adaptors to link the receptors to the coat, the formation of the coat to deform the membrane into a vesicle, the pinching off of the vesicle, subsequent shedding of the membrane coat, and fusion with the appropriate target membrane.

• Vesicles transport proteins from one organelle to another. The donor compartment, or a specific organelle can have a portion of their plasma membrane bud off to create a vesicle.

• The vesicles that bud off from a donor compartment can travel through the cytosol and undergo fusion to release their contents into the target compartment and combine with the plasma membrane.

• The asymmetry of the lipid bilayer is preserved as is the orientation of the membrane proteins despite the fusion and budding process for both the donor and target compartment.

Maintenance of Membrane Topology

• Vesicle transport maintains membrane topology. Specifically, the lumen of the ER, lumen of the transport vesicles, and lumen of the Golgi correspond topologically to the "outside" of the cell or the cellular membrane. Essentially they are made of the same plasma membrane with phospholipids and proteins.

• The regions of proteins with N linked glycosylation are on the luminal side and therefore topologically "outside" of the cell when they bind with the outer plasma membrane.

Vesicle Budding

Vesicle budding is the process by which a transport vesicle forms from a donor membrane through cargo selection and membrane deformation. Cargo adaptors recognize sorting signals on cargo proteins or receptors, while coat proteins help bend the membrane and facilitate vesicle formation and release.

• Vesicle budding includes cargo selection and membrane shaping. Package must be selected from the warehouse and then delivered in the proper manner.

• Brief Overview (expanded upon in a different flaschard): During vesicle budding, cargo receptors bind specific cargo proteins, while adaptor proteins recognize sorting signals on cargo proteins or receptors. Adaptors recruit outer coat proteins, which assemble to bend and shape the donor membrane into a bud. The bud separates from the donor membrane to form a transport vesicle, which then loses its coat through uncoating.

Process or Steps of Vesicle Budding

• Cargo receptors bind to the luminal protein (cargo) that must be transported. In ER export, a cargo receptor is a transmembrane protein. Its luminal domain binds the cargo whereas the cytosilic domain interacts with the vesicle forming machinery (coat).

• Cargo adaptors bind to the sorting signals in the cytosolic tails of the cargo receptors (or other transmembrane proteins that are transported as cargo).

• Coat proteins have cargo adaptors which are bound to the cargo receptors and these coat proteins shape the membrane into the vesicle shape allowing for the pinching off. This round vesicle is then able to be moved.

• Once the vesicle is uncoated, meaning the adaptors in the vesicle coat that attached to the adaptors dissociates from the vesicle membrane, the uncoated vesicle is then ready to fuse with a target membrane.

Types of Coat Proteins (Outer coat proteins and inner/adaptor proteins) in the Endomembrane System

• COPII is a coat protein that recognizes cargo receptors in the ER and transports them to the cis Golgi.

• COPI is another type of coat that recognizes cargo receptors on the cis Golgi and transports them to the ER.

• AP-1/Clathrin is a type of coat that recognizes cargo receptors on the trans Golgi and transports them to endosomes, lysosomes, or plasma membrane.

• AP-2/Clathrin is another type of coat that recognizes cargo receptors on the plasma membrane and transports them to the endosome or lysosome. This is the main coat system for receptor mediated endocytosis.

• These coat proteins and their specific adaptors that recognize specific cargo receptor sorting sequences allow the cell to specify where the vesicles should form and where they should go.

What is the difference between COPII and AP-1/clathrin as vesicle coat systems?

Both are vesicle coat systems, but their components differ:COPII: Refers to the entire coat complex, consisting of Sec23/Sec24 (inner coat) and Sec13/Sec31 (outer coat).

AP-1/clathrin: AP-1 is the adaptor complex, while clathrin is the outer coat protein. Together, they form the complete coat system.

Coat Complex

A protein assembly consisting of cargo-selecting adaptor/inner coat proteins and outer coat/cage proteins that work together to select cargo, bend the donor membrane, and facilitate transport vesicle formation during budding.

Adaptor

A protein that recognizes specific sorting signals on cargo proteins or cargo receptors and helps recruit outer coat proteins, ensuring the correct cargo is packaged into transport vesicles during budding.

The adaptor must recognize the specific cargo receptor. It is initially seperate from the outer coat protein and not in a complex with it.

Outer Coat or Cage Protein

A protein that assembles into a cage-like structure around a budding vesicle, interacting with inner coat or adaptor proteins to help bend and shape the membrane during vesicle formation.

The outer coat must recognize the proper adaptor. It is initially seperate and not in a complex with the adaptor protein.

Receptor Protein (For Adaptors)

A transmembrane protein that recognizes and binds specific cargo proteins, typically within the organelle lumen, and contains sorting signals on its cytosolic tail that are recognized by cargo adaptors, allowing the cargo to be selected and packaged into transport vesicles during budding.

The receptor protein must recognize the specific cargo protein that it must bind to and then its cytosylic tail can bind adaptin so that a vesicle can eventually be formed.

Vesicle Movement

• Many vesicles are moved along microtubules by molecular motors which are cytoskeletal elements. Dynein and Kinesin being two types of motors that move the vesicles toward the outer or interior regions of the cell. They transport vesicles to their destination organelle.

• Many organelles and vesicles are connected to molecular motors by Rab GTPase proteins. These Rab GTPases specify where the motor proteins should go and how the vesicles find their target. They also are  important for tethering of the vesicle as well.

• Rab GTPases on the outer vesicle membrane can help the vesicle recruit the correct motor protein for movement and also interacts with the tethering protein like a dynamic package label.

Vesicle Tethering and Docking

• Vesicles can interact with the molecular motor and they have the cargo receptor bound to the cargo. Vesicles also have V snares or vesicle snares.

• Snare proteins and Rab GTPases serve as recognition markers for the vesicle to fuse with the correct target membrane.

• Rab GTP is a small GTP binding protein that helps to target the vesicle to the target organelle on top of helping the vesicle recruit the proper motor protein, specifying where the vesicle should go. Many different Rab GTPases.

• Rab GTPase helps to tether the vesicle and then eventually helps it to dock with the target membrane.

• V and T snares also help with docking of the vesicle to the target membrane and the eventual fusion process.

Process by which Rab GTPase helps with Vesicle Tethering and Docking

• Rab GTP, bound in GTP form and active, can bind to the tethering proteins that sit on the target membrane.

• Tethering proteins are long, sometimes unstructured proteins that are tentacles into the cytosol so they can fish for the vesicles that carry Rab GTP.

• By random motion the vesicles bound to the tethering proteins by the Rab GTP come close to the membrane.

Process by Which V and T Snare Proteins Interact for Vesicle Docking and Fusion

• Once the vesicle is brought close enough to the target membrane by the tethering protein attached to the Rab GTP, the V snare can interact with the T snare protein and bind to one another.

• Once the V and T snare are bound together, the vesicle is bound and fully docked (no longer tethered) close to the target membrane.

• Docking of the vesicle by the V and T snares brings the Rab GTP close to the Rab GAP and this Rab GAP forces the Rab GTP to hydrolyze into Rab GDP releasing it from the vesicle. Then the Rab GDP can drift back into the cytosol where it can be activated again by a Rab GEF, ready to bind to the next vesicle.

• V and T snares come in many different types with ones being specific to their target membrane and the corresponding vesicle that must fuse.

Vesicle Fusion

• Once the vesicle is tethered to the target membrane, the V and T snare are really close together.

• These V and T snares can interact with one another, wrapping around one another in a zipper like fashion similar to coiled coil structures.

• Zipper formation between T and V snares squeezes the vesicle into the target membrane. The zippering provides the energy to force the vesicle membrane to squeeze and fuse with the target membrane.

• The cargo inside of the vesicle can be released inside of the target membrane once the vesicle becomes one with the target membrane, disappearing.

Mutated Membrane Trafficking Proteins and their Consequences

• Mutated membrane trafficking proteins are often present in many forms of disease. Specific genes can totally disrupt the vesicle transport by disrupting the membrane trafficking proteins that allow for proper transport.

• There is a constant exchange of proteins via vesicles and cells have to make sure that they can control the composition of these organelles making sure the right proteins are being transported to the right places.

• Cells also have mechanisms to recognize proteins that are incorrectly transported and transport them back usually via the correct vesicle coats to bring them back.

Golgi Apparatus Structure

• The Golgi apparatus is a stack of flattened cisterna or membrane closed sacks. It contains a cis face that is facing the ER and a trans face which is facing the cell membrane. The medial Golgi is simply the middle portion of the Golgi.

• Vesicles come into the Golgi on the cis face and then are modified and then exit to the plasma membrane or endosomes/lysosomes on the trans face.

• It is the main packaging and modification center of the endomembrane system.

Golgi Apparatus Function

There are 2 main functions of the Golgi.

• Modification of the N linked oligosaccharide chains (glycosylation) which change protein function.

• Sorting of proteins to multiple destinations in the trans Golgi Network. Principally 2 different locations: exocytosis on the cell membrane or endosomes and then eventually lysosomes.

• The overall trans Golgi network is composed of a variety of different functional elements.

  • On the rough ER, proteins are synthesized. In the ER lumen those proteins are folded and glycosylated if they have important signals. Golgi modification and proper sorting occurs in all 3 layers of the Golgi (cis, medial, and trans).

  • Then selective transport to the Golgi complex by COPII coated vesicles occur.

  • Finally there is transport, delivery, and fusion of the released Golgi vesicles at the appropriate destination.

2 Main Types of Golgi Exocytosis

For expelling things from the Golgi into the environment there are 2 pathways. One is the constitutive secretion and the other is regulated secretion (both forms of exocytosis). Basically some things are expelled continuously or by default and other things are expelled when a signal is detected.

• Newly synthesized soluble proteins are often destined for constitutive secretion.

• Concentrated secretory proteins that are more niche in the cell are only released in the process of regulated secretion after a signal transduction occurs.

2 Primary and Different Locations of Proteins exiting the Golgi Complex

The two primary destinations of proteins exiting the Golgi complex are:

1. Plasma membrane: Proteins are transported to the plasma membrane for membrane insertion or secretion outside the cell (exocytosis).

2. Endosomes/Lysosomes: Proteins are transported to endosomes, where some are subsequently delivered to lysosomes for functions such as intracellular digestion.

Endosome and their Function

An endosome is a membrane-bound compartment that acts as a sorting station for material brought into the cell by endocytosis. It sorts cargo so that some materials are recycled back to the plasma membrane, while others are transported onward, often toward lysosomes for degradation.

• The secretory pathway and endocytic pathways intersect in endosomes.

• This just means that the secretory pathway which involves the secreted proteins from the golgi and endocytic pathway which involves extracellular material being ingested by the cell both deliver proteins and other materials to endosomes, making endosomes a common meeting point for the two pathways.

Lysosome and their Function

• The lumen of the lysosome is acidic and full of hydrolytic enzymes.

• The lumen of the lysosome is acidic as the membrane of the lysosome contains many protons pumps which pump hydrogen ions.

• Lysosomes contain hydrolytic enzymes, that only function at acidic temperatures, that can break down any biological molecule.

• Lysosomal enzymes function optimally at pH of 5.0 and are collectively called acid hydrolases.

• Lysosomes contain membrane transporters so the products of the lysosome degradation can be reused by the cell.

Cargo Sorting and Vesicle Coat Specificity

• Different vesicle coats with different adaptor proteins mediate the transport of vesicles to and from different organelles. Coats and their associated cargo adaptors define the protein composition of transport vesicles.

• Many ER cargos have more than one sorting signal to reach their final destination. They may have one signal to go from the ER to Golgi and additional signals to go from the Golgi to endosome and further.

• Each cargo adaptor and vesicle coat is associated with specific sorting signals. Depending on where the cargo protein is being transported, it may have a particular sorting signal that is recognized by cargo receptors and/or adaptors associated with the appropriate coat protein. Some are listed below.

Endocytosis

• Process by which material is taken in (uptake) via the invagination of the plasma membrane (membrane bending inwards forming a pocket).

• Materials taken up by the plasma membrane via endocytosis can be transported to the endosome to be processed and sent elsewhere or to the lysosome for degradation and recycling.

• Endocytosis is also a receptor mediated process.

Process of LDL Uptake into Cells via Receptor Mediated Endocytosis

One example of receptor mediated endocytosis is the intake of LDL and how LDL receptors are recycled.

• This receptor mediated endocytosis pathway involves:

  • (a) binding of LDL to the surface LDL receptor

  • (b) internalization of the receptor-LDL complex from a coated pit into a coated vesicle

  • (c) delivery of the receptor-LDL complex to the early endosome

  • (d) under the mildly acidic conditions of the early endosome, the LDL and LDL receptor dissociate

  • (e) return of the LDL receptor to the cell surface

  • (f) movement of the free LDL from the early endosome to the late endosome, and then to the lysosome

  • (g) in the lysosome, the protein parts of LDL are degraded and the cholesterol (one example) released for use in membrane biosynthesis.

• Internalization of the LDL receptor in cell surface coated pits involves an interaction of the tail of the receptor with adaptin-2, which interacts with the outer coat protein, clathrin. Clathrin shapes the membrane into a vesicle, and the membrane then undergoes fission to generate a free vesicle. The vesicle then then fuses with an early endosome for LDL processing.

Low Density Lipoprotein (LDL)

• LDL (low density lipoprotein) is a "particle" made of proteins and lipids, and is used by the body to transport fat, lipids, and cholesterol through the bloodstream. The LDL basically is like a mini vesicle filled with stuff your body needs.

Receptor Mediated Endocytosis (RME)

• One example of receptor mediated endocytosis involves the uptake of LDL from the blood or extracellular environment and into the cell as it contains materials essential to the cell such as cholesterol.

• Many other proteins have specific receptors that can be taken up by receptor mediated endocytosis.

• Receptor mediated endocytosis is also used to remove membrane proteins from the surface of the cell, when no longer needed, one example is the Epidermal Growth Factor Receptor.

How do Viruses Often Enter the Cell?

Endocytosis is often exploited by many viruses to gain entry into the cell. The virus can bind to the cell surface receptor and exploit the acidic pH of the endosome to escape into the cytosol.

Then the virus often goes to the nucleus in order to hijack the DNA replication mechanisms in order to make more copies of itself. These copies depending on the viral pathway (lytic or lysogenic) escape from the cell to poliferate further iterations of themselves.