C.Bio - Exocytosis

What conditions must be met before a protein is sent to the smooth ER?

The proteins have been synthesized in the ER and had the quality of their folding checked

Which coat protein facilitates the formation of transport vesicles for proteins exiting the ER?

COPII -- COPII is used to form COPII vesicles

Which coat protein facilitates the formation of transport vesicles for cargo entering the cell from the extracellular space?

Clathrin

Which coat protein facilitates the formation of transport vesicles for cargo within the Golgi?

COPI

What is a retromer

complex within cells that sorts and transports proteins (cargo) from the endosome to the trans-Golgi Network

What are all the important components of budding machinery?

1. Signal molecules -- defines where to form the vesicle

2. Adaptor proteins -- recognizes the signal and recruits the coat and cargo

3. Coat proteins

4. Membrane deforming proteins-- proteins that bend the membrane for bud formation (most energy demanding process)

5. Scission proteins -- cuts vesicle off of parental organelle/ donor membrane

6. Recycling molecules-- recycles coat for future vesicle formation

What is the specific budding machinery used in clathrin coated vesicles?

Signal: PI(4,5)P2 (phospholipid)
Adapter: AP-2
Coat: Clathrin
Invagination: BAR proteins, clathrin
Scission: Dynamin
Recycling: HSC70

What is the specific budding machinery used in COPII coated vesicles?

Signal: Sar1 (small GTPase)
Adaptor: Sec23/24
Coat: Sec13/Sec31
Invagination: Sar 1, Sec 23/24, Sec13/31
Scission: Sar1, Sec23/24, Sec13/31
Recycling: Sec13/31, Sec23, kinases
Note: THE SAME PROTEINS ARE USED TO BEND THE MEMBRANE AND FOR SCISSION

How did Randy Schekman discover the role of different proteins in secretion?

He used budding yeast! By making edits to the genomes of yeast using various forms of mutagenesis he was able to discover which gene mutations inhibited secretion -- recognized by accumulation of vesicles in the yeast

What is another name for the ER translocator?

Sec 61

Why is the Rab family of small GTPases so large?

Each membrane organelle has its own specific Rab GTPase which defines the identity of the specific membrane compartment

What molecule mediates the conversion of small GTPases from active to inactive?

GAP (GTPase Activating Protein) -- facilitates conversion of protein from GTP bound (active) to GDP bound (inactive) state by dephosphorylation

What molecule mediates the conversion of small GTPases from inactive to active?

GEF (Guanine nucleotide exchange factor) -- remove GDP from the inactive GTPase, which allows GTP ( exists in higher concentration within the cell) to diffuse into the small GTPase = activation!

What is a unique feature of the Rab family of small GTPases?

Many small GTPases in the Rab family have a prenyl anchor. This anchor is exposed when the small GTPase is active (embedded with the plasma membrane-- associates GTPase to plasma membrane) and hidden when the GTPase is inactive (protected by GDI-- Guanine nucleotide dissociation inhibitor)

What features of Sar1-GTP are analogus to the Rab family of small GTPases?

• Has two forms (active and inactive)
• Associates to the plasma membrane in the active form
• Transition to active and inactive form is driven by GEF and GAP

What features of Sar1-GTP are different from the Rab family of small GTPases?

Association to the plasma membrane is not mediated prenylation (prenyl anchor)-- it is mediated by the a amphiphilic helix! The helix has a hydrophobic side which is inserted into the plasma membrane and hydrophilic side which is exposed of the cytoplasm

Also there is no GDI (which makes sense because there is not prenyl anchor that need to be hidden)

What is the change in confirmation that occurs between active and inactive Sar 1-GTP?

In inactive form (GDP bound) the amphipathic helix is hidden in the body of the protein

In the active form (GTP bound) -- created by Sar-1's GEF ( Sec 12) -- the amphipathic helix is released and inserted into the membrane

What is the GEF for Sar1-GTP? What are its structural features?

Sec 12! It exists in the ER as a transmembrane resident ER protein

Resident ER protein

A protein that is present in the ER ( or its membrane if its a resident ER membrane/ transmembrane protein) and functions there

How does Sec12 function?

As a GEF for Sar 1, Sec 12 opens the nucleotide binding pocket of inactive Sar-1 so that GDP diffuses out and GTP from the cytoplasm is able to diffuse in and cause a confirmational change to Sar-1

What function does Sar-1 perform in its active form?

It recruits/ binds an effector -- Sec 23/24. This a dimer of two proteins, which acts as an adaptor protein of COPII vesicles

effector

a protein which can recognize specifically the ACTIVE form of a particular small GTPase

What specific part of Sec 23/24 recognizes active Sar-1 GTP?

Sec 23 recognizes/ interacts with the active Sar-1

What occurs after Sec 23/24 is recruited?

1. It docks on the membrane of the ER (through interaction with Sar-1) -- causing invagination of membrane simultaneously
2. It recruits cargo ( Sec 24 region of the adaptor has binding sites for specific signals on ER transmembrane proteins and can recruit them at the adaptor binding site)
3. It recruits coat proteins (Sec 13/31)

How to Sec 23/24 aid in membrane invagination?

Structurally Sec 23/24 has an intrinsic curvature-- visualized using electron micrographs. This results in the membrane curvature gradually increasing as multiple Sec 23/24 adapters are bound

How do Sec 13/31 coat COPII vesicles?

They create a coat by forming a tetramer. These tetramer are arranged in palindromic manner ( 31, 13,13,31) , together forming a rod-shaped structure with polymerizes as a cage formation

How to Sec 13/31 aid in membrane invagination?

The intrinsic curvature of the coat helps to further invaginate the plasma membrane, causing progressively more membrane curvature as more of the coat is recruited.

How is the COPII vesicle detached from the plasma membrane?

The current hypothesis is that this process is driven by GTP hydrolysis in Sar 1 -- however the molecular mechanism is still unclear… :(

What is the process of uncoating the COPII vesicle?

1. GAP for Sar-1 (Sec23) stimulates intrinsic GTPase activity of Sar-1
2. A phosphate is released and Sar-1 activity is inhibited -- it dissociated from the membrane and the coat detaches HOWEVER the coat itself is still polymerized

How can Sec 23 act as an adapter for Sar-1, but ALSO as its GAP?

When Sec23 binds to Sar-1 in the Sec 23/24 dimer complex it acts as an adaptor, recruiting Sec 13/31 as vesicle coating proteins. HOWEVER, when the Sec13/31 complex binds to the adapter it stimulates the GAP activity of Sec23, causing the hydrolysis of GTP in Sar-1 and eventually Sec23's own detachment

THIS IS AN INTERNAL NEGATIVE FEEDBACK LOOP

Why is formation of the COPII vesicle a time sensitive process?

In COPII budding machinery the Sec 23 is both the effector and the GAP for Sar-1. This creates an internal negative feedback loop. If the coat assembles quickly it can complete formation before Sec 23 causes complete uncoating of the vesicle. However, if the process occurs too slowly the partially formed coat results in activation of GAP activity in the adapter, inhibition of the Sar-1 signal, and dissociation of the coat before complete vesicle formation

What has to occur for Sec 13/31 subunits to dissociate from each other?

They need to be phosphorylated by recruited kinases which recognize the polymerized form of the protein.

Why kinds of proteins are recruited to COPII vesicles?

Sec 24 subunit of the adaptor complex can recruit

1. transmembrane cargo like nutrient receptors or signaling receptors
2. receptors for luminal cargo like soluble proteins that need to be secreted-- function in extracellular space ( ex. digestive enzymes)

Why do luminal cargo need receptors to mediate their integration into COPII vesicles?

Luminal cargo is polar, and therefore unable to associate through the plasma membrane, directly to the Sec 24 adapter. By using luminal cargo receptors (transmembrane proteins which on one side of the membrane bind the cargo and the other side bind the adapter) as an intermediary luminal cargo can associate with Sec 24

What allows transmembrane receptors or cargo to interact with Sec 24?

They have a signal sequence (small amino acid sequence) that allows them to be recruited by Sec24, the cargo binding subunit of the adapter complex

Why do resident ER proteins have -KDEL and -KKXX sequences?

Resident ER proteins (both luminal and transmembrane) can sometimes be captured into vesicles and removed from the ER during vesicle formation. -KDEL and -KKXX serve as 'return to ER signals' that mediate recapturing of these escaped proteins. This system is used to help maintain efficiency and not waste these proteins

What are -KDEL and -KKXX, and which types of ER proteins do they respectively belong to?

Signals that are encoded at the very C-terminus of luminal and transmembrane resident ER proteins.
-KDEL ( lysine, aspartic acid, glutamic acid, leucine) = luminal
-KKXX ( 2 lysines and any 2 amino acids) exists on cytoplasmic side = transmembrane

What machinery is used to recognize -KDEL and -KKXX sequences for successful return back to the ER?

-KDEL and -KKXX receptors are of the transmembrane proteins recruited in COPII vesicles! In later compartments they capture the escaped ER resident proteins and return them to the ER

What is ERGIC (or vesicular tubular cluster)?

ERGIC ( ER-Golgi intermediate compartment) is a compartment formed by many uncoated COPII vesicles fusing with each other. ERGIC travels to the Golgi through microtubules to deliver its cargo

When does the recycling process occur for ERGIC?

While ERGIC is traveling to the Golgi! The escaped proteins and receptors for luminal cargo are returned to the ER to either stay their or be used again as receptors for other load of cargo

What coat protein facilitates the recycling process for ERGIC?

COPI. COPI is formed on the Golgi only so it function to pinch off vesicles from all Golgi compartments ONLY

What is the process for COPI vesicle formation?

1: The signal to assemble the coat is a small GTPase ( Arf-1 GTP -- also from the Rab family)
2: Arf-1 is activated by Gea (its GEF) Gea is associated with the Golgi membrane
3: Arf-1 associates with the Golgi membrane
4: Active Arf-1 recruits adapter for the COPI vesicle coat (which unlike COPII -- where it is formed sequentially-- is PREASSEMBLED into the COPI complex and recruited simultaneously)
5: GAP for Arf-1 is recruited from the cytoplasm
6: Arf-1 is deactivated by phosphorylation and the coat is subsequently disassembled by phosphorylation

the rest of the features are the same as for COPII , the intrinsic curvature of the complex bends the membrane and it is pinched off the Golgi membrane, hypothetically by the energy derived from GTP hydrolysis of Arf-1

How does the recycling of ER resident proteins occur?

The -KKXX sequence of transmembrane ER proteins is recognized by the COPI coat

Transmembrane resident ER proteins and cargo receptors have the -KKXX sequence on them.

For luminal ER proteins, they have -KDEL at their C terminus--> those -KDEL sequences have -KDEL receptors --> those transmembrane -KDEL receptors have -KKXX sequences that COPI recognizes

What regulatory system/ technique is used to facilitate when receptors bind to their substrates and when they don't?

A pH gradient. ERGIC begins to receive and influx of proteins via a proton pump. This makes the system more acidic as it goes from ER (neutral) to ERGIC (slightly acidic) and through the compartments of the Golgi

• Provides DIRECTIONALITY for transport of cargo proteins forward and escaped proteins backward*

Describe the anatomy of the Golgi compartment?

The Golgi is asymmetric. One side of the Golgi contains the cis Golgi network and the central cisterna (cis, medial and trans cisterna) which appear more flat and pancake like. The peripheral side ( trans Golgi network) consists of membrane networks. These compartment CAN be connected to each other via tubules but can also be separate.

Proteins enter the cis face of the Golgi and exit at the trans face

How does the ER distribution in the cell compare to that of the Golgi complex?

While the ER is distributed all throughout the cell as well as enveloping the nucleus, the Golgi complex is concentrated near the nucleus -- this also means that when protein are synthesized in the ER they need to travel across the entire cell to accumulate in the Golgi!

What is the function of the Golgi?

To add post-translational modifications to newly synthesized proteins. The main post-translational modification that occurs is glycosylation -- this can vary of addition of complex oligosaccharides OR addition of simpler high-mannose oligosaccharides to the core n-linked oligosaccharide depending on the protein

Why is post-translational modification especially important for luminal proteins and others exposed to the extracellular space?

This process is especially important for the parts of the proteins which will be exposed to the extracellular environment and luminal proteins-- they need to be glycosylated to resist harsh environment outside the cell (or equivalent to the outside of the cell for luminal proteins)

What substrate does post-translational modification occur on?

The N-linked precursor oligosaccharide.

• The glucoses are removed prior to transport into the Golgi
• 6 of the 9 mannoses are removed at some point during transport to the Golgi

Glycosylation occurs on what is left of the N-linked oligosaccharide (named the core n-linked oligosaccharide)

How is the order of monosaccharides added to the core n-linked oligosaccharide determined/defined?

The order of monosaccharides in the polysaccharide is determined by the arrangement of enzymes in the asymmetric Golgi stack. Monosaccharides that need to be added earlier are present in the cis-Golgi compartment. Those that need to be added later (and other modifications that need to happen later) are positioned in the trans-Golgi compartment.

THIS GOLGI SYSTEM EVOLVED IN THIS WAY TO ARRANGE SUGARS IN THE PROPER ORDER ( this is essentially what mRNA is for a polypeptide)

How does the Golgi stack prevent proteins from being modified identically?

The enzymes that perform modifications in the Golgi stack have to recognize the protein as their substrate. If a certain protein is not recognized by enzymes in certain compartments it can pass through unmodified.

What are the two mechanisms of transport through the golgi apparatus?

A) Vesicle Transport Mechanism
B) Cisternal Maturation Mechanism

How does post-translational modification occur through the vesicle transport mechanism?

When ERGIC arrives at the Golgi, cargo-containing vesicles are pinched off from ERGIC and delivered to the cis-Golgi network. After processing in the cis-Golgi network, they are pinched off again and sent to the cis-Golgi cisterna, and so on. The cargo-containing vesicles are essentially pinched off and re-inserted into each Golgi compartment to accumulate modifications.

MEANWHILE, cargo receptors are sorted to be returned to their original compartment. This is mediated by pH as the pH gradient adds directionality to transport.

In this mechanism, enzymes that perform modifications remain in their original compartment (they don't move anywhere) -- only cargo moves forward, and cargo receptors move backward.

How does post-translational modification occur through the cisternal maturation mechanism?

When ERGIC arrives to the cis face of the Golgi it becomes the cis-Golgi network. In order to fully become the cis-Golgi network, it receives enzymes from the previous cis-Golgi network. Cargo proteins remain in ERGIC, and the cargo receptors are removed as the compartment gets acidified.

Following, the former cis-Golgi network, following the dispatch of its enzymes to ERGIC, receives cis-cisterna enzymes, which are brought by vesicles, and becomes the new cis-cisterna.

EACH COMPARTMENT CHANGES ITS IDENTITY -- SIMILAR TO ENDOCYTOSIS.

What factors distinguish whether cargo proteins will undergo post-translational modification through the vesicle transport mechanism or the cisternal maturation mechanism?

Size!

Small cargo -- which can easily be put into vesicles-- is typically seen going through the vesicle transport mechanism (faster)

Large cargo -- like collagen and large proteins-- which can't fit in vesicles, are transported through the cisternal maturation mechanism ( moves slowly with the changing identity of the compartments until it reaches the trans-Golgi network, where the whole compartment is moved to its intended destination)

NOTICE IN CISTERNAL MATURATION, THE # OF STACKS OF THE GOLGI REMAINS THE SAME BECAUSE AS ONE COMPARTMENT IS ADDED, ANOTHER IS REMOVED

What is the name for enzymes delivered to endosomes from the trans-Golgi network (give specific examples) ?

Acid Hydrolases: a series of enzymes that can disrupt polymers into monomers and remove post-translational modifications like phosphate and sugars
Ex: Nucleases, Proteases, Glycosidases, Lipases, Phosphatases, Sulfatases, Phospholipases

Proton Pump: important for maintaining appropriate pH for membrane organelles/ compartments

What signal is used for proper transport of acid hydrolases to the endosome?

A phosphated mannose on the N-linked oligosaccharide. The signal is called M6P (mannose 6-phosphate)

How is M6P attached to acidic hydrolases?

There is an enzyme in the Golgi (GIcNAc phosphotransferase) that can recognize acidic hydrolases-- these enzyme recognize the signal patch that is forms when lysosomal hydrolases completely fold (patch only forms when the protein in PROPERLY FOLDED).

GIcNAc phosphotransferase binds the lysosomal hydrolase in one binding site and recruits a substrate (UDP-GIcNAc-- a monosaccharide with two phosphates, pyrophosphate, and a uridine nucleotide). Disruption of the pyrophosphate bond releases energy, which is used to link the GlcNAc-P to one of the manoses on the N-link oligosaccharide. In this form, the phosphotransferase releases the acid hydrolase, and all mannoses, except for the one with GlcNAc-P bound, are removed. When GlcNAc sugar is removed in a later compartment of the Golgi, the M6P region is exposed and can be recognized by an M6P receptor

Describe the process of lysosomal/ acid hydrolase maturation

1) Lysosomal hydrolases enter the Golgi with no modification to the mannoses
2) Early in the cis-Golgi a phosphate is added to the N-linked oligosaccharide together with the sugar GlcNAc
3) The partially modified lysosomal hydrolase travels through the rest of the Golgi until it reaches the trans-Golgi compartment, where the sugar (GIcNAc) is removed -- mannose is now phosphorylated without being blocked by a monosaccharide
4) In trans-Golgi network M6P is recruited by the M6P receptor
5) The M6P receptor with bound M6P modified lysosomal hydrolases is recruited and sorted into clathrin-coated vesicles (used AP1 as an adapter-- specifically recruits M63 receptors)
6) The vesicles are pinched off and get uncoated, and deliver their cargo to the endosome
7) Due to the difference in pH between compartments, the M6P receptors release their cargo and are recycled back to the Golgi by a retromer coat (the retromer coat operates in the endosome)
8) The phosphate on the lysosomal hydrolase+ M6P is removed, and at a sufficiently acidic pH the hydrolase will gain enzymatic activity, digesting polymers

What are the two types of secretory pathways that ER synthesize proteins can be extocytosed through?

A) Constitutive Secretory Pathway
B) Regulated Secretory Pathway

What is the purpose of the constitutive secretory pathway vs. the regulated secretory pathway?

The constitutive secretory pathway functions for proteins that need to be constantly delivered to the plasma membrane ( ex: structural membrane proteins, receptors in the plasma membrane that are constantly being used) and for proteins that the cell doesn't know what to do with

The secretory pathway is for proteins that need to be secreted only on demand (ex: digestive enzymes -- they are constantly synthesized by exocrine cells, but are stored in the cytoplasm until food is consumed. Only then are these proteins secreted). These proteins are stored in high concentration with secretory vesicles, which are formed in the cytoplasm.

Why might a protein be retained in the ER even if it is not a resident ER protein?

Due to protein misfolding! Remember, proteins are only allowed to go to ER exit sites if they are folded properly and released by the ER chaperone protein. If the protein is misfolded, it remains in the cycle until proper folding occurs. Ex: VSVG (a fluorescent temperature-sensitive protein) is misfolded and 40 C, and therefore is retained in the ER. It is only when the temperature is switched to 32 C that the protein is able to be secreted and trafficked normally to the plasma membrane through the constitutive secretory pathway

What is the process for packing of secretory proteins into mature secretory vesicles?

1) The cargo is recruited from the trans-Golgi network into clathrin-coated vesicles
2) The vesicles are uncoated and fuse to form an immature secretory vesicle-- where the cargo proteins are loosely packed and contain a lot of luminal contents, which are unnecessary and take up space (called condensing granules in the George Palade experiment)
3) Unnecessary components are pinched off and sorted into clatrin-coated retrieval vesicles, after which they are returned to the trans-Golgi network
4) Process 3 continues until the proteins are highly concentrated and tightly packed into a mature secretory vesicle-- this is so the vesicle takes up less space in the cell and can be stored for a sufficiently long time (called zymogen granules in the George Palade experiment)

Give one real-world example of regulated secretory pathway

Release of Insulin
Insulin is a hormone that regulates sugar levels in the blood. Insulin is produced in beta cells of the pancreas and stored there in secretory vesicles. It is released only when blood sugar levels in the extracellular space reach a certain threshold. High glucose in the extracellular environment induces a signal that results in the secretion of insulin. This stimulates the transport of glucose from the blood across the cell membrane.

How is glucose taken up?

Glucose is transported across the cell membrane through a channel, which functions at the plasma membrane.

These channels are normally present at the plasma membrane of tissue cells in small amounts

however, when high concentrations. of insulin appears in the extracellular space, it induces a signaling cascade through insulin receptors, which triggers the secretion of glucose transporters to the plasma membrane.

Describe the structure of glucose transporters

Multipass transmembrane proteins with many alpha helices, which form a barrel/channel. They are stored in the membrane of secretory vesicles.