L11 Membrane trafficking

What is membrane trafficking

Membrane trafficking is the process by which proteins, pathogens, and other macromolecules are distributed throughout the cell, and released to or internalised from the extracellular space.

Membrane trafficking uses membrane-bound vesicles as transport intermediaries.

This transport can take place within different organelles in the same cell, or across the cell membrane to and from the extracellular environment.

Membrane trafficking mediates the biosynthetic-secretory and endocytic pathways：

This vesicular traffic flows along highly organized, directional routes, which allow the cell to secrete, eat, and remodel its plasma membrane and organelles.

Transport directions: exocytosis (secretion) vs endocytosis

一、Vesicular transport

Vesicular transport consists of budding, targeting and fusion

Vesicles are membrane bound transport intermediates.

Principle: The orientation of the membrane and of membrane proteins does not change during vesicular transport processes.

Transport vesicles

• How are vesicles formed?

• How are vesicles budded from the original membrane?

• How does vesicle fusion occur?

• How is the whole process regulated?

1. Genetic analysis using yeast temperature-sensitive mutants

Multicopy suppression

• First, a known key gene for vesicular transport is mutated; to bypass this defect, an interacting protein that is produced to higher amounts will bind to the mutant protein vividly, to cure this defect.
• This is through library screening after transfecting of randomized plasmids into mutant yeast cells.
• Through this screening, important binding partners for a known vesicular transport protein can be identified.

2. Using biochemistry to study “vesicles”

The player in vesicle formation

Transport vesicles are characterized by a specific set of “coat” proteins, which drive formation of the vesicles.

What do vesicles need coat proteins?

Coat proteins perform two distinct functions:

• Inner layer: cargo recognition and concentration
• Outer layer: membrane deformation to generate a free vesicle

There are many players along vesicle-mediated transport: coat proteins, proteins functioning in vesicle fission, tether factor, motor molecules, proteins functioning in membrane fusion…

1. Various types of coated vesicles

The coat performs two main functions that are reflected in a common two-layered structure

1. First, an inner coat layer concentrates specific membrane proteins in a specialized patch, which then gives rise to the vesicle membrane. In this way, the inner layer selects the appropriate membrane molecules for transport.
2. Second, an outer coat layer assembles into a curved, basketlike lattice that deforms the membrane patch and thereby shapes the vesicle

The three major transport vesicles mediate transport

There are three well-characterized types of coated vesicles, distinguished by their major coat proteins: clathrin-coated, COPI-coated, and COPII-coated

Clathrin: transport proteins from the plasma membrane (endocytosis) and anterograde from trans-Golgi network (TGN) to early and late endosomes.

Both cis and trans faces are closely associated with special compartments, each composed of a network of interconnected tubular and cisternal structures: the cis Golgi network (CGN) and the trans Golgi network (TGN), respectively

Clathrin-coated vesicles, for example, mediate transport from the Golgi apparatus and from the plasma membrane, whereas COPI and COPII-coated vesicles most commonly mediate transport from the ER and from the Golgi cisternae

COP (coat-protein) I: Transport proteins in the retrograde direction between Golgi cisternae and retrograde from the cis-Golgi back to the ER (retrieval).

COP (coat-protein) II: Transport proteins from the ER to the cis-Golgi

COPI-coated vesicles and COPII-coated vesicles transport material early in the secretory pathway: COPI-coated vesicles bud from Golgi compartments, and COPII-coated vesicles bud from the ER

(1) Clathrin-coated pits and budding of CCVs at the PM

(2) Clathrin-coated vesicle (CCV)

The major protein component of clathrin-coated vesicles is clathrin (网格蛋白) itself, which forms the outer layer of the coat. Each clathrin subunit consists of three large and three small polypeptide chains that together form a three-legged structure called a triskelion

Clathrin triskelions assemble into a basketlike framework of hexagons and pentagons to form coated pits (buds) on the cytosolic surface of membranes (Figure 13–7). Under appropriate conditions, isolated triskelions spontaneously self-assemble into typical polyhedral cages in a test tube, even in the absence of the membrane vesicles that these baskets normally enclose (Figure 13–6C,D). Thus, the clathrin triskelions determine the geometry of the clathrin cage (Figure 13–6E).

“Clathrin” consists of 3 large and 3 small polypeptide chains, which assemble into a “three-legged” triskelion

• How would you call a protein complex that consists of 3 large and 3 small subunits?

(3) Clathrin-coated vesicle assembly and disassembly

Clathrin-coated vesicles, the first coated vesicles to be identified, transport material from the plasma membrane and between endosomal and Golgi compartments.

• Coat proteins sort the cargo proteins into the budding vesicle:
  • They link cargo selection to vesicle formation
    • Is it possible to form vesicles without cargo?

Questions related to vesicle-mediated transport:

• How to control when and where to form vesicles?
• How is membrane curvature induced?
• How are vesicles pinched-off (vesicle fission)?
• How are vesicles fused with target membrane (vesicle fusion)?

2. How are the cargo proteins recognized and packaged? – adaptor protein (AP) complexes

Adaptor proteins, another major coat component in clathrin-coated vesicles, form a discrete inner layer of the coat, positioned between the clathrin cage and the membrane

They bind the clathrin coat to the membrane and trap various transmembrane proteins, including transmembrane receptors that capture soluble cargo molecules inside the vesicle—so-called cargo receptors.

In this way, the adaptor proteins select a specific set of transmembrane proteins, together with the soluble proteins that interact with them,

Each type of adaptor protein is specific for a different set of cargo receptors. Clathrin-coated vesicles budding from different membranes use different adaptor proteins and thus package different receptors and cargo molecules

CCV cargo is recognized by tetrameric adaptor protein (AP) complexes

AP complexes:

• hetero-tetramer (4 different subunits, termed adaptins)

  • 2 large subunits (α-/β-adaptin)
  • 1 medium subunit (μ-adaptin)
  • 1 small subunit (σ-adaptin)

• 5 different APs exist

  • each of the 5 APs has its unique composition

• Bind to membranes, to cargo and to clathrin

APs are the central link between membrane, cargo & clathrin

phospholipids

Membranes contain a mix of phospholipids and each leaflet has a different phospholipid composition (Lecture 5)

• Membrane contains many different types of phospholipids
• Different types of phospholipids located unevenly between two layers

Different head groups of phospholipid allow specific interaction with proteins:

• Phosphatidylinositol (PI) at the inner/cytosolic leaflet: phosphorylation of PI by protein kinases.

AP complexes bind to phospholipids and to cargo protein

The adaptor protein AP2 serves as a well-understood example

When it binds to a specific phosphorylated phosphatidylinositol lipid (a phosphoinositide), it alters its conformation, exposing binding sites for cargo receptors in the membrane. The simultaneous binding to the cargo receptors and lipid head groups greatly enhances the binding of AP2 to the membrane

• Because several requirements must be met simultaneously to stably bind AP2 proteins to a membrane, the proteins act as coincidence detectors that only assemble at the right time and place

Upon binding, they induce membrane curvature, which makes the binding of additional AP2 proteins in its proximity more likely. The cooperative assembly of the AP2 coat layer then is further amplified by clathrin binding, which leads to the formation and budding of a transport vesicle

• Adaptor proteins found in other coats also bind to phosphoinositides which not only have a major role in directing when and where coats assemble in the cell, but also are used much more widely as molecular markers of compartment identity. This helps to control vesicular traffic,

• Lipid-induced conformation switch of AP2 to “open” the binding pocket

Reaction sequence:

1. Membrane binding to open the binding pocket for the cargo interaction

2. Cargo binding

3. Clathrin recruitment

Subcellular localization of phosphoinositides (PIPs)

PI can undergo rapid cycles of phosphorylation and dephosphorylation at the 3ʹ, 4ʹ, and 5ʹ positions of their inositol sugar head groups to produce various types of phosphoinositides (phosphatidylinositol phosphates, or PIPs)

The interconversion of phosphatidylinositol (PI) and PIPs is highly compartmentalized: different organelles in the endocytic and secretory pathways have distinct sets of PI and PIP kinases and PIP phosphatases (Figure 13–10).

• The distribution, regulation, and local balance of these enzymes determine the steady-state distribution of each PIP species.

Many proteins involved at different steps in vesicle transport contain domains that bind with high specificity to the head groups of particular PIPs

Local control of the PI and PIP kinases and PIP phosphatases can therefore be used to rapidly control the binding of proteins to a membrane or membrane domain. The production of a particular type of PIP recruits proteins containing matching PIP-binding domains

Differentially located phosphoinositides (PIPs) regulate vesicular transport

Specific phosphoinositides mark compartments and membrane domains

Specificity of vesicle formation by PIPs:

• Secretory vesicles (SVs) contain PI(4)P, after fusion with PM, a PM-localizing PI5-kinase converts PI(4)P to PI(4,5)P2
• PI(4,5)P2 recruits the AP complex for the formation of endocytic CCVs
• After CCV budding, a cytosolic PI(5)P phosphatase hydrolyses PI(4,5)P2, which promotes uncoating of the vesicle

As a clathrin-coated bud grows, soluble cytoplasmic proteins, including dynamin, assemble at the neck of each bud

Dynamin contains a PI(4,5) P2-binding domain, which tethers the protein to the membrane, and a GTPase domain, which regulates the rate at which vesicles pinch off from the membrane.

The pinching-off process brings the two noncytosolic leaflets of the membrane into close proximity and fuses them, sealing off the forming vesicle

• To perform this task, dynamin recruits other proteins to the neck of the bud. Together with dynamin, they help bend the patch of membrane—by directly distorting the bilayer structure, or by changing its lipid composition through the recruitment of lipid-modifying enzymes, or by both mechanisms.

Once released from the membrane, the vesicle rapidly loses its clathrin coat. A PIP phosphatase that is co-packaged into clathrin-coated vesicles depletes PI(4,5) P2 from the membrane, which weakens the binding of the adaptor proteins.

• In addition, an hsp70 chaperone protein (see Figure 6–80) functions as an uncoating ATPase, using the energy of ATP hydrolysis to peel off（剥离） the clathrin coat.
• Auxilin, another vesicle protein, is thought to activate the ATPase

Trigger Vesicle Formation

Vesicle formation triggered by small GTP-binding proteins (small G-proteins or GTPases)

(1) Small G-proteins

(2) Assembly and disassembly of coat by GTPases

While local production of PIPs plays a major part in regulating the assembly of clathrin coats on the plasma membrane and Golgi apparatus, cells superimpose additional ways of regulating coat formation

Coat-recruitment GTPases, for example, control the assembly of clathrin coats on endosomes and the COPI and COPII coats on Golgi and ER membranes.

GTP-binding proteins regulate most processes in eukaryotic cells. They act as molecular switches, which flip between an active state with GTP bound and an inactive state with GDP bound

Two classes of proteins regulate the flipping: guanine nucleotide exchange factors (GEFs) activate the proteins by catalyzing the exchange of GDP for GTP, and GTPase-activating proteins (GAPs) inactivate the proteins by triggering the hydrolysis of the bound GTP to GDP

Both monomeric GTP-binding proteins (monomeric GTPases) and trimeric GTP-binding proteins (G proteins) have important roles in vesicle transport

Coat-recruitment GTPases are members of a family of monomeric GTPases. They include the ARF proteins, which are responsible for the assembly of both COPI and clathrin coats assembly at Golgi membranes, and the Sar1 protein, which is responsible for the assembly of COPII coats at the ER membrane.

• When a COPII-coated vesicle is to bud from the ER membrane, for example, a specific Sar1-GEF embedded in the ER membrane binds to cytosolic Sar1, causing the Sar1 to release its GDP and bind GTP in its place

• GTP is present in much higher concentration in the cytosol than GDP and therefore will spontaneously bind after GDP is released.) In its GTP-bound state, the Sar1 protein exposes an amphiphilic helix, which inserts into the cytoplasmic leaflet of the lipid bilayer of the ER membrane. The tightly bound Sar1 now recruits adaptor coat protein subunits to the ER membrane to initiate budding

The coat-recruitment GTPases also have a role in coat disassembly. The hydrolysis of bound GTP to GDP causes the GTPase to change its conformation so that its hydrophobic tail pops out of the membrane, causing the vesicle’s coat to disassemble. Although it is not known what triggers the GTP hydrolysis, it has been proposed that the GTPases work like timers

• COPII coats accelerate GTP hydrolysis by Sar1, and a fully formed vesicle will be produced only when bud formation occurs faster than the timed disassembly process;

Once a vesicle pinches off, GTP hydrolysis releases Sar1, but the sealed coat is sufficiently stabilized through many cooperative interactions, including binding to the cargo receptors in the membrane, that it may stay on the vesicle until the vesicle docks at a target membrane. There, a kinase phosphorylates the coat proteins, which completes coat disassembly and readies the vesicle for fusion

Clathrin- and COPI-coated vesicles, by contrast, shed their coat soon after they pinch off. For COPI vesicles, the curvature of the vesicle membrane serves as a trigger to begin uncoating. An ARF-GAP is recruited to the COPI coat as it assembles. It interacts with the membrane, and senses the lipid packing density. It becomes activated when the curvature of the membrane approaches that of a transport vesicle. It then inactivates ARF, causing the coat to disassemble

Coat-recruitment GTPases:

• ARF (ADP-ribosylation factor) GTPases

  • COPI
  • clathrin

• Sar (secretion-associated Ras-superfamily related protein) 1 GTPase

  • COPII

• GTPase works like a timer and cause disassembly shortly after the budding is completed

IMPORTANT POINTS

• Assembly of coat proteins induces curvature in membrane and eventual budding

3. Tubular vesicles

Vesicles are not all spherical, and can be large irregular vesicle or tubule.

Not All Transport Vesicles Are Spherical

vesicle-budding from many intracellular membranes occurs preferentially at regions where the membranes are already curved

Transport vesicles also occur in various sizes and shapes. Diverse COPII vesicles are required for the transport of large cargo molecules.

• Collagen, for example, is assembled in the ER as 300-nm-long, stiff procollagen rods that then are secreted from the cell where they are cleaved by proteases to collagen, which is embedded into the extracellular matrix
• Procollagen rods do not fit into the 60–80 nm COPII vesicles normally observed. To circumvent this problem, the procollagen cargo molecules bind to transmembrane packaging proteins in the ER, which control the assembly of the COPII coat components

Tubules have a higher surface-to-volume ratio than the larger organelles from which they form

• They are therefore relatively enriched in membrane proteins compared with soluble cargo proteins. As we discuss later, this property of tubules is an important feature for sorting proteins in endosomes

4. Multiple factors control when and where to assemble coat proteins

Multiple factors confer location-specificity:

• Presence of specific phosphoinositides (PIPs) within a respective membrane of a cellular compartment can trigger coat recruitment
• Activation and recruitment of coat-recruiting GTPases:
  • ARF (ADP-ribosylation factor) GTPases:
    • responsible for clathrin coat assembly and for COP I coat assembly
  • Sar (secretion-associated Ras-superfamily related protein) 1 GTPases
    • Responsible for COP II coat assembly

5. Membrane-bending

How to apply force to a lipid bilayer:

• Membrane-bending proteins help deform the membrane during vesicle formation
• Cytoplasmic proteins can regulate the pinching-off and uncoating of coated vesicles

Membrane-Bending Proteins Help Deform the Membrane During Vesicle Formation

The forces generated by clathrin coat assembly alone are not sufficient to shape and pinch off a vesicle from the membrane. Other membrane-bending and force-generating proteins participate at every stage of the process.

Membrane-bending proteins that contain crescent-shaped domains, called BAR domains, bind to and impose their shape on the underlying membrane via electrostatic interactions with the lipid head groups

Some of these proteins also contain amphiphilic helices that induce membrane curvature after being inserted as wedges into the cytoplasmic leaflet of the membrane.

Finally, the clathrin machinery nucleates the local assembly of actin filaments that introduce tension to help pinch off and propel the forming vesicle away from the membrane

Membrane-bending proteins help deform the membrane during vesicle formation: BAR (Bin/Amphiphysin/Rvs) domains:

• coiled-coils that dimerize
• BAR domain-containing proteins are curved
• dimers have positively charged inner surface
• dimers interact/associate with negatively charged lipid headgroups
• induce curvature of membranes

Fission of the vesicle

Fission of the vesicle: Dynamin assists!

Dynamin:

• PI(4,5)-P2-binding domain tethers the protein to the membrane.
• GTPase domain regulates the rate of budding

1. Dynamin mediated budding of vesicles

Conformational changes in the GTPase domains of membrane-assembled dynamin drives a conformational change that constricts the neck of the bud

Dynamin pinches off CCVs at the PM

Deeply invaginated clathrin-coated pits form in the nerve endings of fly’s nerve cells, with a belt of mutant dynamin assembled around the neck

2. Targeted to specific sites for fusion

Rabs and SNAREs mediate the specificity during vesicle targeting and membrane fusion events: → recognition of the target membrane

Rab proteins:

1. Monomeric GTPases
2. Exhibit specific subcellular localization
3. Direct the vesicle to the correct target sites

SNARE proteins: soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor)

1. Exhibit specific subcellular distribution
2. Mediate recognition of the target membrane by SNARE-SNARE interactions
3. Trigger the fusion of the lipid bilayers

Rab protein family: monomeric GTPases

Rab Proteins Guide Transport Vesicles to Their Target Membrane

To ensure an orderly flow of vesicle traffic, transport vesicles must be highly accurate in recognizing the correct target membrane with which to fuse.

Because of the diversity and crowding of membrane systems in the cytoplasm, a vesicle is likely to encounter many potential target membranes before it finds the correct one.

Specificity in targeting is ensured because all transport vesicles display surface markers that identify them according to their origin and type of cargo, and target membranes display complementary receptors that recognize the appropriate markers. This crucial process occurs in two steps.

First, Rab proteins and Rab effectors direct the vesicle to specific spots on the correct target membrane.

• Rab proteins are also monomeric GTPases

Second, SNARE proteins and SNARE regulators mediate the fusion of the lipid bilayers

Their highly selective distribution on these membrane systems makes Rab proteins be able for identifying each membrane type and guiding vesicle traffic between them. Rab proteins can function on transport vesicles, on target membranes, or both

Monomeric GTPases with specific subcellular localizations

60 family members, each associates with one or more compartments of the biosynthetic secretory or endocytic pathways.

>30 Rabs on cytosolic surface

C-terminal regions are variable: Bind to other proteins, including GEFs

Protein	Organelle
Rab1	ER and Golgi complex
Rab2	cis Golgi network
Rab3A	Synaptic vesicles, secretory vesicles
Rab4/Rab11	Recycling endosomes
Rab5	Early endosomes, plasma membrane, clathrin-coated vesicles
Rab6	Medial and trans Golgi
Rab7	Late endosomes
Rab8	Cilia
Rab9	Late endosomes, trans Golgi

Rab GTPases are key players in membrane trafficking processes

Like the coat-recruitment GTPases, Rab proteins cycle between a membrane \and the cytosol and regulate the reversible assembly of protein complexes on the membrane. In their GDP-bound state, they are inactive and bound to another protein (Rab-GDP dissociation inhibitor, or GDI) that keeps them soluble in the cytosol; in their GTP-bound state, they are active and tightly associated with the membrane of an organelle or transport vesicle.

Membrane-bound Rab-GEFs activate Rab proteins on both transport vesicle and target membranes; for some membrane fusion events, activated Rab molecules are required on both sides of the reaction. Once in the GTP-bound state and membrane-bound through a now-exposed lipid anchor, Rab proteins bind to other proteins, called Rab effectors, which are the downstream mediators of vesicle transport, membrane tethering, and membrane fusion (Figure 13–16).

The rate of GTP hydrolysis sets the concentration of active Rab and, consequently, the concentration of its effectors on the membrane

The same Rab proteins can often bind to many different effectors. Some Rab effectors are motor proteins that propel vesicles along actin filaments or microtubules to their target membrane. Others are tethering proteins, some of which have long, threadlike domains that serve as “fishing lines” that can extend to link two membranes more than 200 nm apart;

Another GTP-switch:

1. Rab-GDP is inactive and cytosolic, usually bound to Rab-GDP-dissociation inhibitor (GDI)
2. Activated by membrane-bound Rab-GEF (Guanine nucleotide exchange factor) and becomes Rab-GTP (active form)
3. Once in active form, Rab-GTP exposes hydrophobic anchor to be membrane-bound and recruits other proteins e.g. Rab effectors

Recognizing the target membrane: tethering → docking → fusion:

• Activated Rab and Rab effectors tether
• SNARES trigger specific fusion
• GDI: (GDP dissociation inhibitor)

The tethering complex that docks COPII-coated vesicles, for example, contains a protein kinase that phosphorylates the coat proteins to complete the uncoating process

Coupling uncoating to vesicle delivery helps to ensure directionality of the transport process and fusion with the proper membrane.

The formation of a Rab5 domain on the endosomal membrane

Rab5, for example, assembles on endosomes and mediates the capture of endocytic vesicles arriving from the plasma membrane.

A Rab5 domain concentrates tethering proteins that catch incoming vesicles. Its assembly on endosomal membranes begins when a Rab5-GDP/GDI complex encounters a Rab-GEF. GDI is released and Rab5-GDP is converted to Rab5-GTP.

• Active Rab5-GTP becomes anchored to the membrane and recruits more Rab5-GEF to the endosome, thereby stimulating the recruitment of more Rab5 to the same site. In addition, active Rab5 activates a PI 3-kinase, which locally converts PI to PI(3)P, which in turn binds some of the Rab effectors including tethering proteins and stabilizes their local membrane attachment

Thus, while the Rab5 membrane domain receives incoming endocytic vesicles from the plasma membrane, distinct Rab11 and Rab4 domains in the same membrane organize the budding of recycling vesicles that return proteins from the endosome to the plasma membrane.

Formation of a Rab5 domain:

• activation and recruitment of Rab5-GTP
• recruitment of PI3-kinase to generate PI(3)P
• recruitment of protein that bind to PI(3)P (more Rab-GEF, tethering factors)

How can a compartment mature and change the identity?

Early endosomes possess Rab5 but late endosomes possess Rab7.

How can an early endosome mature into a late endosome?

Replacement of a RabA domain by a RabB domain

• maturation of early endosomes to late endosomes is Rab5 to Rab7 conversion

Rab Cascades Can Change the Identity of an Organelle

A Rab domain can be disassembled and replaced by a different Rab domain, changing the identity of an organelle

• Such ordered recruitment of sequentially acting Rab proteins is called a Rab cascade
  • Over time, for example, Rab5 domains are replaced by Rab7 domains on endosomal membranes. This converts an early endosome, marked by Rab5, into a late endosome, marked by Rab7.
    • This process is also referred to as endosome maturation. The self-amplifying nature of the Rab domains renders the process of endosome maturation unidirectional and irreversible
  • It alters the membrane dynamics, including the incoming and outgoing traffic, and repositions the organelle away from the plasma membrane toward the cell interior

3. SNAREs mediate specificity of membrane fusion

Recognition of the fusion target: Interaction between specific v- and t-SNAREs

• v-SNARE: single chain on vesicles
• t-SNARE: 2-3 chains on target membrane

The SNARE proteins (also called SNAREs, for short) catalyze the membrane fusion reactions in vesicle transport.

1. v-SNAREs usually found on vesicle membranes
   • A v-SNARE is a single polypeptide chain, whereas a t-SNARE is usually composed of three proteins
2. t-SNAREs usually found on target membranes

The resulting trans-SNARE complex locks the two membranes together. Biochemical membrane fusion assays with all different SNARE combinations show that t-SNARE pairing is highly specific

The trans-SNARE complexes catalyze membrane fusion by using the energy that is freed when the interacting helices wrap around each other to pull the membrane faces together, simultaneously squeezing out water molecules from the interface

In the cell, other proteins recruited to the fusion site, presumably Rab effectors, cooperate with SNAREs to accelerate fusion.

In the process of regulated exocytosis, fusion is delayed until secretion is triggered by a specific extracellular signal

SNAREs proteins catalyze membrane fusion

SNARE-mediated bilayer fusion occurs in multiple steps

• Pairing between v- and t- SNAREs
• Lipid molecules in the two interacting (cytosolic) leaflets flow
• hemifusion, or half-fusion.
• Rupture and “sealing” of the new bilayer completes the fusion reaction.

Rab proteins, which can regulate the availability of SNARE proteins, exert an additional layer of control. t-SNAREs in target membranes are often associated with inhibitory proteins that must be released before the t-SNARE can function.

• Rab proteins and their effectors trigger the release of such SNARE inhibitory proteins.

4. Energy for Membrane Fusion

Interacting SNAREs Need to Be Pried Apart Before They Can Function Again

A crucial protein called NSF cycles between membranes and the cytosol and catalyzes the disassembly process.

• NSF is a hexameric ATPase of the family of AAA-ATPases

• The requirement for NSF-mediated reactivation of SNAREs by SNARE complex disassembly helps prevent membranes from fusing indiscriminately

Dissociation of SNARE pairs after membrane fusion requires ATP hydrolysis by the AAA-ATPase NSF

• NSF (N-ethylmaleimide-sensitive factor)
• SNARE (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors) protein

5. Special Membrane Fusion

Specificity of membrane fusion can be hijacked by viruses :

Entry of the enveloped HIV virus into cells occurs by specific fusion of the viral membrane with the PM of the cell

• Viral fusion proteins and SNAREs promote lipid bilayer fusion in similar ways.

• The entry of enveloped viruses into cells: Electron micrographs showing how HIV enters a cell by fusing its membrane with the plasma membrane of the cell.

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Small-GTPases, tethers and SNAREs determine specific vesicle formation and delivery to their destination

二、Transport in ER and Golgi

Transfer from one compartment to the next involves a delicate balance between forward and backward (retrieval) transport pathways.

Golgi Apparatus

1. Localization of the Golgi apparatus

In animal cells

• many stacks are linked together to a single complex close to the nucleus and to the centrosome, which is dependent on microtubule connection.

In plant cells

• Golgi stacks, termed dictyosomes（单核小体）, are individually, disperse in the cytoplasm.
• Cells contain hundreds of individual stacks

2. Structure of Golgi Apparatus

A “Golgi apparatus” is not a simple stack of pancakes!

The “Golgi apparatus” consists of an ordered series of functionally distinct compartments that contain distinct sets of enzymes.

Ordered series of Golgi apparatus

Golgins, Golgi matrix proteins, help organize the stack

A model of golgin function:

• Filamentous golgins anchored to Golgi membranes capture transport vesicles by binding to Rab proteins on the vesicle surface

3. Function of Golgi apparatus

1. Sorting and dispatching station for ER products → central sorting hub for proteins

2. Major site of carbohydrate synthesis and secretion of glycoproteins (lecture 10)

Post-translational attachment of lysosomal sorting signals on soluble proteins for lysosolmal targeting (only mammalian cells)

The Golgi is both a factory and a bus-station, it produces, modifies and distributes

It is a major site of carbohydrate synthesis, as well as a sorting and dispatching station for products of the ER.

• The cell makes many polysaccharides in the Golgi apparatus, including the pectin（果胶） and hemicellulose（半纤维素） of the cell wall in plants and most of the glycosaminoglycans（糖胺聚糖） of the extracellular matrix in animals

Transport from the ER through the Golgi

A subset of these oligosaccharide groups serve as tags to direct specific proteins into vesicles that then transport them to lysosomes

1. ER export

ER exit requires proper protein folding/assembly

Only properly folded and assembled proteins can leave the ER.

Molecular chaperons such as BiP (binding protein) and calnexin (not shown) facilitate protein folding and intermolecular assembly processes (lecture 10).

Misfolded soluble proteins can’t escape the calnexin cycle (they are bound to this membrane protein) that’s why they cannot exit the ER by bulk-flow!

Misfolded proteins rather will be degraded via the ERAD pathway in the cytosol

COPII coat

Proteins Leave the ER in COPII-Coated Transport Vesicles

• These vesicles bud from specialized regions of the ER called ER exit sites, whose membrane lacks bound ribosomes
• These cargo membrane proteins display exit (transport) signals on their cytosolic surface that adaptor proteins of the inner COPII coat recognize
  • Proteins without exit signals can also enter transport vesicles, including protein molecules that normally function in the ER (so-called ER resident proteins), some of which slowly leak out of the ER and are delivered to the Golgi apparatus.
• Some of these components act as cargo receptors and are recycled back to the ER after they have delivered their cargo to the Golgi apparatus.

ER exit sites:

Proteins leave the ER in COPII-coated transport vesicles:

Some transmembrane proteins that serve as cargo receptors for packaging some secretory proteins into COPII-coated vesicles are lectins that bind to oligosaccharides on the secreted proteins.

• One such lectin, for example, binds to mannose on two secreted blood-clotting factors (Factor V and Factor VIII), thereby packaging the proteins into transport vesicles in the ERs

Transport vesicles can fuse with each other:

• The formation of vesicular tubular clusters (VTCs) by homotypic（同型的） fusion of transport vesicles
  • Homotypic fusion: fusion occurs between vesicles from the same compartment
  • Heterotypic fusion: fusion occurs between vesicles from different compartments.
• Both are mediated by v-SNARE and t-SNARE interactions, also regulated by Rabs

Vesicular tubular clusters (VTCs)

Vesicular Tubular Clusters Mediate Transport from the ER to the Golgi Apparatus

• The fusion of membranes from the same compartment is called homotypic fusion, to distinguish it from heterotypic fusion, in which a membrane from one compartment fuses with the membrane of a different compartment.

The structures formed when ER-derived vesicles fuse with one another are called vesicular tubular clusters, because they have a convoluted appearance in the electron microscope

• The clusters move quickly along microtubules to the Golgi apparatus with which they fuse

Vesicular tubular clusters (VTCs) arise from homotypic fusion of COP II vesicles

• Vesicular tubular clusters (VTCs) are forming around an ER exit site (ERES).
• Many of the vesicle-like structures seen in the micrograph are cross sections of tubules that extend above and below the plane of this thin section and are interconnected.

Early endosomes (EE) originate from homotypic fusion of endocytic clathrin-coated vesicles

In mammals, VTCs mediate transport between the ER and the Golgi apparatus:

• VTCs are short lived and move quickly along the microtubule
• VTCs can bud off COP I vesicles on their own to send back the ER resident protein — a process called retrieval ( or retrograde) transport
  • Retrieval requires ER retrieval signals, which are recognized by COP I coats directly (on membrane proteins) or indirectly (on soluble proteins via sorting receptors (HDEL/KDEL receptor ERD2)

2. Retrograde transport from VTCs or Golgi back to the ER

COPI-coated vesicles are unique in that the components that make up the inner and outer coat layers are recruited as a preassembled complex, called coatomer

• They function as a retrieval pathway, carrying back ER resident proteins that have escaped, as well as proteins such as cargo receptors and SNAREs that participated in the ER budding and vesicle fusion reactions.
• The retrieval (or retrograde) transport continues as the vesicular tubular clusters move toward the Golgi apparatus.

Retrograde transport: carry back the ER resident proteins that “leaked” out

Retrieval of membrane proteins and of soluble ER-resident proteins requires specific sorting signals

The retrieval pathway for returning escaped proteins back to the ER depends on ER retrieval signals

• Resident ER membrane proteins, for example, contain signals that bind directly to COPI coats and are thus packaged into COPI-coated transport vesicles for retrograde delivery to the ER

ER retrieval signals: di-lysine sequence in “KKXX” motif in ER membrane proteins, recognized by COP I coat

Membrane proteins in Golgi and ER have shorter Transmembrane (TM) domains (15 aa)

Soluble ER resident proteins, such as BiP, also contain a short ER retrieval signal at their C-terminal end, but it is different: it consists of a Lys-Asp-Glu-Leu or a similar sequence

• If this signal (called the KDEL sequence) is removed from BiP by genetic engineering, the protein is slowly secreted from the cell

H/KDEL sequence in soluble ER resident proteins, binds to sorting receptor ERD2 (H/KDEL) receptor

Unlike the retrieval signals on ER membrane proteins, which can interact directly with the COPI coat, soluble ER resident proteins must bind to specialized receptor proteins such as the KDEL receptor— a multipass transmembrane protein that binds to the KDEL sequence and packages any protein displaying it into COPI-coated retrograde transport vesicles

pH controls affinity of KDEL receptors

3. Intra Golgi transport

Intra Golgi transport: cisternal maturation vs vesicle transport

• Cisternal maturation model: New cisternae are continuously formed at the cis- and lost at the trans- face
• Vesicle transport model: Cisternae remain, cargo is transported by vesicles from cisternae to cisternae…

In vitro verification of vesicle-mediated intra-Golgi transport

Cell-free (in vitro) transport system to study vesicular transport intra Golgi transport, using Golgi stacks from two different cell populations

Experimental strategy:

• Both Golgi populations are incubated “at cellular conditions” (with cytosol and ATP and “detectable” radiolabeled sugars (3HGlcNAc)) to reconstitute transport

• Detection of 3HGlcNAc-reporters demonstrates transport

Analysis by tracing of green fluorescent (GF) protein fusions:

• Proteins were fused with the green fluorescent protein GFP and the red fluorescent protein RFP, respectively, and then visualized by time-lapse fluorescence microscopy

A temperature-sensitive mutant secretory membrane protein:

Transport is monitored over time after the shift of temperature

Tool:

• VSVG: Vesicular stomatitis virus G protein
• ts045 (VSVG–GFP): thermo reversible folding mutant
• at 40°C misfolds and is retained in the ER
• at 32°C moves as a synchronous population to the Golgi complex before being transported to the plasma membrane

Summary: ER → Golgi transport

COP (coat protein) II-coated transport vesicle (COP II vesicles) mediate protein transport from the ER to the Golgi

COP II vesicles bud from ER exit sites (ERES) at the ribosome-free transitional ER (tER)

Membrane proteins display sorting signals (exit signals) on their cytosolic domain for ER exit

COP II coat can recognize the exit signals on membrane proteins directly

Soluble secretory proteins can be exported by “bulk-flow” and do not need export signals to leave the ER in COPII vesicles (Need/existence of membrane proteins that act as “export receptors” for soluble proteins has not been unequivocally demonstrated yet)

Soluble ER-resident proteins require the tetrapeptide HDEL as signal for COP I-mediated retrieval for their ER localization

三、Transport from Golgi to lysosomes

Lysosomes are membrane-enclosed organelles filled with soluble hydrolytic enzymes that digest macromolecules

• All are acid hydrolases; that is, hydrolases that work best at acidic pH.
• A vacuolar H+ ATPase in the lysosome membrane uses the energy of ATP hydrolysis to pump H+ into the lysosome, thereby maintaining the lumen at its acidic pH
• The lysosome H+ pump belongs to the family of V-type ATPases and has a similar architecture to the mitochondrial and chloroplast ATP synthases (F-type ATPases), which convert the energy stored in H+ gradients into ATP

Lysosomes are heterogeneous:

• coated pits (CP, 有被小窝; 被膜小窝)
• early endosome (EE, 早期内体)
• late endosome (LE, 晚期内体)
• lysosome (LY, 溶酶体)

Structure of Lysosomes

Features:

• Protein rich membranes
  • Lamp/Limp family of glycoproteins

Most of the lysosome membrane proteins, for example, are highly glycosylated, which helps to protect them from the lysosome proteases in the lumen

• Unusual lipids

  • lyso-bisphosphatidic acid
  • thought to protect lysosomal membrane lipids from action of lumenal lipases

• Multivesicular bodies (MVB, late endosomes/lysosomes)

  • invagination of the limiting membrane
  • forms internal membranes
  • TM proteins can segregate into limiting or internal membranes
  • limiting membrane can be recycled, internal is not

Lysosomes contain a variety of digestive enzymes that would chew up cell components:

Lysosomes Are Heterogeneous

• Lysosomes are found in all eukaryotic cells
• The diversity reflects the wide variety of digestive functions that acid hydrolases mediate, including the breakdown of intra- and extracellular debris, the destruction of phagocytosed microorganisms, and the production of nutrients for the cell

Their morphological diversity, however, also reflects the way lysosomes form

• Late endosomes containing material received from both the plasma membrane by endocytosis and newly synthesized lysosomal hydrolases fuse with preexisting lysosomes to form structures that are sometimes referred to as endolysosomes, which then fuse with one another.
• When the majority of the endocytosed material within an endolysosome has been digested so that only resistant or slowly digestible residues remain, these organelles become “classical” lysosomes

Plant and Fungal Vacuoles Are Remarkably Versatile Lysosomes

Most plant and fungal cells (including yeasts) contain one or several very large, fluid-filled vesicles called vacuoles

• Vacuoles are related to animal cell lysosomes and contain a variety of hydrolytic enzymes, but their functions are remarkably diverse
• The plant vacuole can act as a storage organelle for both nutrients and waste products, as a degradative compartment, as an economical way of increasing cell size, and as a controller of turgor pressure (the osmotic pressure that pushes outward on the cell wall and keeps the plant from wilting (枯萎；衰弱))
• The same cell may have different vacuoles with distinct functions, such as digestion and storage
• The vacuole is important as a homeostatic device, enabling plant cells to withstand wide variations in their environment
• They do so by changing the osmotic pressure of the cytosol and vacuole—in part by the controlled breakdown and re-synthesis of polymers such as polyphosphate in the vacuole, and in part by altering the transport rates of sugars, amino acids, and other metabolites across the plasma membrane and the vacuolar membrane

Protein sorting to lysosome

1. Mannose-6-phosphate

A Mannose 6-Phosphate Receptor Sorts Lysosomal Hydrolases in the Trans Golgi Network

• In animal cells they carry a unique marker in the form of mannose 6-phosphate (M6P) groups, which are added exclusively to the N-linked oligosaccharides of these soluble lysosomal enzymes as they pass through the lumen of the cis Golgi network
• Transmembrane M6P receptor proteins, which are present in the TGN, recognize the M6P groups and bind to the lysosomal hydrolases on the lumenal side of the membrane and to adaptor proteins in assembling clathrin coats on the cytosolic side.

Mannose-6-phosphate (M6P): post-translational glycan modification:

• The synthesis of the lysosomal sorting signal on a soluble hydrolase

Mannose-6-phosphate targets proteins to lysosomes

M6P group cause the lysosomal hydrolases to dissociate from these receptors, making the transport of the hydrolases unidirectional.

• Lysosomal sorting signals on soluble lysosomal proteins emerge in the TGN

2. Sorting Process

• Lysosomal sorting signal: mannose 6-phosphate (M6P)

• Lysosomal sorting receptor: mannose 6-phosphate receptor (MPR)

• M6P receptor protein binds best at pH 6.5-6.7 in the TGN lumen, and release at pH 6.0 (early endosome), which is the pH in the lumen of endosomes

Thus, after the receptor is delivered, the lysosomal hydrolases dissociate from the M6P receptors, which are retrieved into transport vesicles that bud from endosomes

These vesicles are coated with retromer, a coat protein complex specialized for endosome-to-TGN transport, which returns the receptors to the TGN for reuse

• M6P is then hydrolyzed inside endosome

Transport in either direction requires signals in the cytoplasmic tail of the M6P receptor that direct this protein to the endosome or back to the TGN

These signals are recognized by the retromer complex that recruits M6P receptors into transport vesicles that bud from endosomes

• The recycling of the M6P receptor resembles the recycling of the KDEL receptor discussed earlier, although it differs in the type of coated vesicles that mediate the transport

Not all the hydrolase molecules that are tagged with M6P get to lysosomes. Some escape the normal packaging process in the trans Golgi network and are transported “by default” to the cell surface, where they are secreted into the extracellular fluid

As lysosomal hydrolases require an acidic milieu to work, the can do little harm in the extracellular fluid, which usually has a neutral pH of 7.4.

pH influenced sorting example:

• The low pH in the endosome induces transferrin to release its bound iron, but the iron-free transferrin itself (called apotransferrin) remains bound to its receptor.
• The receptor–apotransferrin complex enters the tubular extensions of the early endosome and from there is recycled back to the plasma membrane. When the apotransferrin returns to the neutral pH of the extracellular fluid, it dissociates from the receptor and is thereby freed to pick up more iron and begin the cycle again
• Thus, transferrin shuttles back and forth between the extracellular fluid and early endosomes, avoiding lysosomes and delivering iron to the cell interior, as needed for cells to grow and proliferate

Since all glycoproteins leave the ER with identical N-linked oligosaccharide chains, the signal for adding the M6P units to oligosaccharides must reside somewhere in the polypeptide chain of each hydrolase

Genetic engineering experiments have revealed that the recognition signal is a cluster of neighboring amino acids on each protein’s surface, known as a signal patch

Since most lysosomal hydrolases contain multiple oligosaccharides, they acquire many M6P groups, providing a high-affinity signal for the M6P receptor.

3. Diseases

Rare diseases associated with failure in TGN-lysosome transport: Lysosome storage diseases (LSD):

• I-cell disease (I-细胞疾病): defect in GlcNAc-phosphotransferase, resulting in failure of lysosomal hydrolases to be transported to lysosome, instead, they are secreted out of the cell.

• Hurler’s disease (赫尔勒氏症): defect in a certain enzyme important for breakdown of glycosaminoglycan chains

• Albinism (白化病): defect in exocytosis of pigment lysosomes such as melanocytes

Inclusion (I)-cell disease

GlcNAC-phosphotransferase phosphorylates mannose on lysosomal proteins

Trafficking to lysosome

1. Multiple Pathways Deliver Materials to Lysosomes

Three pathways lead to degradation in the lysosome:

1. The best studied of these degradation paths is the one followed by macromolecules taken up from extracellular fluid by endocytosis.
2. A similar pathway found in phagocytic cells, such as macrophages and neutrophils in vertebrates, is dedicated to the engulfment, or phagocytosis, of large particles and microorganisms to form phagosomes
3. A third pathway called macropinocytosis specializes in the nonspecific uptake of fluids, membrane, and particles attached to the plasma membrane.
4. A fourth pathway called autophagy originates in the cytoplasm of the cell itself and is used to digest cytosol and worn-out organelles

2. Autophagy

Introduction

All cell types dispose of obsolete parts by a lysosome-dependent process called autophagy, or “self-eating.”

• It helps restructure differentiating cells, but also in adaptive responses to stresses such as starvation and infection. Autophagy can remove large objects—macromolecules, large protein aggregates, and even whole organelles—that other disposal mechanisms such as proteasomal degradation cannot handle
• In the initial stages of autophagy, cytoplasmic cargo becomes surrounded by a double membrane that assembles by the fusion of small vesicles of unknown origin, forming an autophagosome

The whole process occurs in the following sequence of steps:

1. Induction by activation of signaling molecules: Protein kinases (including the mTOR complex 1, discussed in Chapter 15) that relay information about the metabolic status of the cell, become activated and signal to the autophagic machinery.
2. Nucleation and extension of a delimiting membrane into a crescent-shaped cup: Membrane vesicles, characterized by the presence of ATG9, the only transmembrane protein involved in the process, are recruited to an assembly site, where they nucleate autophagosome formation.
   • ATG9 is not incorporated into the autophagosome: a retrieval pathway must remove it from the assembling structure.
3. Closure of the membrane cup around the target to form a sealed double-membrane-enclosed autophagosome.
4. Fusion of the autophagosome with lysosomes, catalyzed by SNAREs.
5. Digestion of the inner membrane and the lumenal contents of the autophagosome.

Selective / Nonselective

Autophagy can be either nonselective or selective

In nonselective autophagy, a bulk portion of cytoplasm is sequestered in autophagosomes. It might occur, for example, in starvation conditions

In selective autophagy specific cargo is packaged into autophagosomes that tend to contain little cytosol, and their shape reflects the shape of the cargo. Selective autophagy mediates the degradation of worn out, or otherwise unwanted, mitochondria, peroxisomes, ribosomes, and ER; it can also be used to destroy invading microbes

• The selective autophagy of worn out or damaged mitochondria is called mitophagy
  • Damaged mitochondria cannot maintain the gradient, so protein import is blocked. As a consequence, a protein kinase called Pink1, which is normally imported into mitochondria, is instead retained on the mitochondrial surface where it recruits the ubiquitin ligase Parkin from the cytosol
    • Mutations in Pink1 or Parkin cause a form of early-onset Parkinson’s disease,

四、Endocytosis

Two major types of endocytosis:

1. Phagocytosis (large particles 250 nm in diameter)
2. Pinocytosis (∼ 100 nm in diameter), Pinocytosis ingests small amount of fluid and plasma membrane

• A macrophage is “eating” two red blood cells
• Macrophages can eat 1011 old red blood cells/day

Factors triggering endocytosis

“Eat-me” signal: Factors triggering endocytosis:

1. Antibody coated pathogens.
2. Complements in pathogens.
3. Oligosaccharides in microorganisms
4. Phosphotidylserine (PS) in apoptotic cells

Phagocytosis engulfs large particles and microorganisms

• Pseudopod extension and phagosome formation are driven by actin polymerization and reorganization, which respond to the accumulation of specific phosphoinositides in the membrane of the forming phagosome:
  • PI(4,5)P2 stimulates actin polymerization (promotes pseudopod formation)
  • PI(3,4,5)P3 stimulates depolymerizes actin filaments at the base.

In endocytosis, the material to be ingested is progressively enclosed by a small portion of the plasma membrane, which first invaginates and then pinches off to form an endocytic vesicle containing the ingested substance or particle

• Most eukaryotic cells constantly form endocytic vesicles, a process called pinocytosis (“cell drinking”); in addition
• some specialized cells contain dedicated pathways that take up large particles on demand, a process called phagocytosis (“cell eating”).

Specialized Phagocytic Cells Can Ingest Large Particles

Phagocytosis is a special form of endocytosis in which a cell uses large endocytic vesicles called phagosomes to ingest large particles such as microorganisms and dead cells. Phagocytosis is distinct, both in purpose and mechanism, from macropinocytosis

Phagocytosis is important in most animals for purposes other than nutrition, and it is carried out mainly by specialized cells—so-called professional phagocytes.

• In mammals, two important classes of white blood cells that act as professional phagocytes are macrophages and neutrophils
  • These cells develop from hemopoietic stem cells and they ingest invading microorganisms to defend us against infection.
• Macrophages also have an important role in scavenging senescent (变老的) cells and cells that have died by apoptosiss

1. Pinocytosis

Caveolae-mediated pinocytosis

This is Clathrin-independent endocytosis

Pinocytic Vesicles Form from Coated Pits in the Plasma Membrane

Pinocytosis varies between cell types, but it is usually surprisingly high

• Since a cell’s surface area and volume remain unchanged during this process, it is clear that the same amount of membrane being removed by endocytosis is being added to the cell surface by the converse process of exocytosis

The endocytic part of the cycle often begins at clathrin-coated pits. These specialized regions typically occupy about 2% of the total plasma membrane area

• The lifetime of a clathrin-coated pit is short: within a minute or so of being formed, it invaginates into the cell and pinches off to form a clathrin-coated vesicle

Not All Pinocytic Vesicles Are Clathrin-Coated

In addition to clathrin-coated pits and vesicles, cells can form other types of pinocytic vesicles, such as caveolae (from the Latin for “little cavities”), originally recognized by their ability to transport molecules across endothelial cells that form the inner lining of blood vessels

• Caveolae, sometimes seen in the electron microscope as deeply invaginated flasks, are present in the plasma membrane of most vertebrate cell types

• They are thought to form from lipid rafts in the plasma membrane, which are rich in in cholesterol, glycosphingolipids, and glycosylphosphatidylinositol (GPI)-anchored membrane proteins

  The major structural proteins in caveolae are caveolins, a family of unusual integral membrane proteins that each insert a hydrophobic loop into the membrane from the cytosolic side but do not extend across the membrane

  On their cytosolic side, caveolins are bound to large protein complexes of caving proteins, which are thought to stabilize the membrane curvature.

• In contrast to clathrin-coated and COPI or COPII-coated vesicles, caveolae are usually static structures and they can be induced to pinch off and serve as endocytic vesicles to transport cargo to early endosomes or to the plasma membrane on the opposite side of a polarized cell (Transcytosis).

• Some animal viruses such as SV40 and papillomavirus (which causes warts) enter cells in vesicles derived from caveolae

• Cholera toxin (discussed in Chapters 15 and 19) also enters the cell through caveolae and is transported to the ER before entering the cytosol

Clathrin-mediated pinocytosis

Receptor-mediated endocytosis (RME)

• 25 different receptors mediate this type of endocytosis
• Many different receptors cluster in the same pit
• The similar receptors can cluster in different pits

2. Macropinocytosis

Clathrin-independent endocytosis

Macropinocytosis is another clathrin-independent endocytic mechanism that can be activated in practically all animal cells

In most cell types, it does not operate continually but rather is induced for a limited time in response to cell-surface receptor activation by specific cargoes, including growth factors, integrin ligands, apoptotic cell remnants, and some viruses

• These ligands activate a complex signaling pathway, resulting in a change in actin dynamics and the formation of cell-surface protrusions, called ruffles（皱纹、褶皱）
• When ruffles collapse back onto the cell, large fluid-filled endocytic vesicles form, called macropinosomes

Macropinocytosis is a dedicated degradative pathway: macropinosomes acidify and then fuse with late endosomes or endolysosomes, without recycling their cargo back to the plasma membrane

3. Receptor-Mediated Endocytosis

A low-density lipoprotein (LDL) particle

Cholesterol uptake by low-density lipoprotein (LDL) receptors on the cell surface

Structure of the LDL

• Atherosclerosis: leads to stroke and heart attack. It occurs by Failure to take up LDL into cells, which causes accumulation of LDL in the blood, which then forms Atherosclerotic plaques.

Receptor-mediated endocytosis: cholesterol uptake

Cholesterol uptake by low-density lipoprotein (LDL) receptors on the cell surface

Most cholesterol is transported in the blood as cholesteryl esters in the form of lipid–protein particles known as low-density lipoproteins (LDLs)

Once in the plasma membrane, the LDL receptors diffuse until they associate with clathrin-coated pits that are in the process of forming

• There, an endocytosis signal in the cytoplasmic tail of the LDL receptors binds the membrane-bound adaptor protein AP2 after its conformation has been locally unlocked by binding to **PI(4,5)P2 **on the plasma membrane

Specific Proteins Are Retrieved from Early Endosomes and Returned to the Plasma Membrane

Early endosomes are the main sorting station in the endocytic pathway, just as the cis and trans Golgi networks serve this function in the secretory pathway.

• In the mildly acidic environment of the early endosome, many internalized receptor proteins change their conformation and release their ligand, as already discussed for the M6P receptors.

Endocytosis of LDL by normal and mutant LDL receptors

If too much free cholesterol accumulates in a cell, the cell shuts off both its own cholesterol synthesis and the synthesis of LDL receptors, so that it ceases both to make or to take up cholesterol.

pH dependent interactions

affects protein-protein interactions in endolytic compartments

Lysosomes and endosomes

1. Once generated at the plasma membrane, most endocytic vesicles fuse with a common receiving compartment, the early endosome, where internalized cargo is sorted: some
   1. cargo molecules are returned to the plasma membrane
   2. directly or via a recycling endosome
   3. others are designated for degradation by inclusion in a late endosome
2. Late endosomes form from a bulbous, vacuolar portion of early endosomes by a process called endosome maturation.
   • This conversion process changes the protein composition of the endosome membrane, patches of which invaginate and become incorporated within the organelles as intralumenal vesicles, while the endosome itself moves from the cell periphery to a location close to the nucleus

1. Lysosomes maturation

Late endosome-lysosome pathway mediated by multivesicular bodies (MVBs)/late endosomes (LEs): endosome maturation

Multivesicular body (MVB): Intralumenal vesicles formed via membrane invagination become incorporated within the lysosome.

The sequestration of endocytosed proteins into intralumenal vesicle of MVB：

Each of the stages of endosome maturation—from the early endosome to the endolysosome—is connected through bidirectional vesicle transport pathways to the TGN

Early Endosomes Mature into Late Endosomes

1. The distribution of the molecule after its uptake reveals the sequence of events. Within a minute or so after adding the tracer, it starts to appear in early endosomes, just beneath the plasma membrane
2. By 5–15 minutes later, it has moved to late endosomes, close to the Golgi apparatus and near the nucleus.

Early endosomes have tubular and vacuolar domains. Most of the membrane surface is in the tubules and most of the volume is in the vacuolar domain

• During endosome maturation, the two domains have different fates:
  1. the vacuolar portions of the early endosome are retained and transformed into late endosomes;
  2. the tubular portions shrink

Maturing endosomes, also called multivesicular bodies, migrate along microtubules toward the cell interior, shedding membrane tubules and vesicles that recycle material to the plasma membrane and TGN, and receiving newly synthesized lysosomal proteins

As they concentrate in a perinuclear region of the cell, the multivesicular bodies fuse with each other, and eventually with endolysosomes and lysosomes

Many changes occur during the maturation process

1. The endosome changes shape and location, as the tubular domains are lost and the vacuolar domains are thoroughly modified
2. Rab proteins, phosphoinositide lipids, fusion machinery (SNAREs and tethers), and microtubule motor proteins all participate in a molecular makeover（打扮、装饰） of the cytosolic face of the endosome membrane, changing the functional characteristics of the organelle
3. A V-type ATPase in the endosome membrane pumps H+ from the cytosol into the endosome lumen and acidifies the organelle.
4. Intralumenal vesicles sequester（扣押） endocytosed signaling receptors inside the endosome, thus halting the receptor signaling activity.
5. Lysosome proteins are delivered from the TGN to the maturing endosome.

Most of these events occur gradually but eventually lead to a complete transformation of the endosome into an early endolysosome.

In addition to committing selected cargo for degradation, the maturation process is important for lysosome maintenances

2. The fate of endocytosed receptor proteins

1. Recycling directly back to the same PM domain via an recycling endosome
2. Transcytosis “recycling” to a different PM domain
3. Degradation via ESCRT pathway

Recycling of the LDL-receptors

Plasma Membrane Signaling Receptors are Down-Regulated by Degradation in Lysosomes

A second pathway that endocytosed receptors can follow from endosomes is taken by many signaling receptors, including opioid receptors and the receptor that binds epidermal growth factor (EGF)

• Unlike LDL receptors, EGF receptors accumulate in clathrin-coated pits only after binding their ligand, and most do not recycle but are degraded in lysosomes, along with the ingested EGF
• This is a a process called receptor downregulation, that reduces the cell’s subsequent sensitivity to EGF

Receptor downregulation is highly regulated. The activated receptors are first covalently modified on the cytosolic face with the small protein ubiquitin

• Unlike polyubiquitylation, which adds a chain of ubiquitins that typically targets a protein for degradation in proteasomes, ubiquitin tagging for sorting into the clathrin-dependent endocytic pathway adds just one or a few single ubiquitin molecules to the protein—a process called monoubiquitylation or multiubiquitylation, respectively.
• Ubiquitin-binding proteins recognize the attached ubiquitin and help direct the modified receptors into clathrin-coated pits
• After delivery to the early endosome, other ubiquitin-binding proteins that are part of ESCRT complexes (ESCRT = Endosome Sorting Complex Required for Transport) recognize and sort the ubiquitylated receptors into intralumenal vesicles, which are retained in the maturing late endosome

In this way, addition of ubiquitin blocks receptor recycling to the plasma membrane and directs the receptors into the degradation pathways

• Other receptors don’t recycle but are degraded in lysosomes instead (e.g. EGFR opioid receptors).
  • This process is called receptor down-regulation.

Retromer-mediates receptor recycling back to Golgi :

• Working model of Retromer

Transferrin receptor (转铁蛋白受体) and opioid receptor are sorted differently：

The transferrin receptor follows a similar recycling pathway as the LDL receptor, but unlike the LDL receptor it also recycles its ligand

(1) Transcytosis in polarized epithelial cells

Receptors on the surface of polarized epithelial cells can transfer specific macromolecules from one extracellular space to another by transcytosis

The transcytotic pathway from the early endosome back to the plasma membrane is not direct

• The receptors first move from the early endosome to the recycling endosome
• many receptors also possess sorting signals that guide them into the appropriate transport pathway

recycling endosomes play an important part in adjusting the concentration of specific plasma membrane proteins

• Different pH in two sides of cells decides association and dissociation between receptor and its ligand

• Different sorting signals in receptors may play a role in transcytosis

(2) Storage of plasma membrane proteins in recycling endosomes

Recycling endosomes can serve as an intracellular storage site for specialized plasma membrane proteins that can be mobilized when needed

• Example: regulated transport of glucose transporters to the PM:

(3) Selective down-regulation of PM proteins through endocytosis

Cells can actively regulate receptor mediated endocytosis for downregulation/degradation of receptors (signal termination):

1. Mono- and multi-ubiquitination of receptors
2. Ubiquitin binding（泛素结合） proteins recognizes these receptors
3. Direct clathrin coat assembly

(4) Ubiquitination tags

Ubiquitination tags serves as recognition marks for formation of multivesicular body

ESCRT proteins

ESCRT Protein Complexes Mediate the Formation of Intralumenal Vesicles in Multivesicular Bodies

• As endosomes mature, patches of their membrane invaginate into the endosome lumen and pinch off to form intralumenal vesicles
• Because of their appearance in the electron microscope such maturing endosomes are also called multivesicular bodies

ESCRT proteins recognize both ubiquitination and PIPs signals during multivesicular body formation

• Endosomal sorting complex required for transport (ESCRT)
• Mutations in ESCRT proteins are associated with prolonged signaling and cancer

Clathrin-mediated endocytosis takes up specific proteins from plasma membrane

1. Clathrin-coated vesicles fuse with endosomes where low pH dissociates receptors from cargos.
2. Receptors returned to plasma membrane and Cargos delivered to lysosome.
3. Multivesicular bodies process receptors for degradation in lysosomes.

五、Secretion and exocytosis

(From TGN to plasma membrane)

• Formation of secretory particle
• Signaling to release secretory content
• Membrane lipid and protein after exocytosis

The formation of the secretory vesicles

Many Proteins and Lipids Are Carried Automatically from the Trans Golgi Network (TGN) to the Cell Surface

• A cell capable of regulated secretion must separate at least three classes of proteins before they leave the TGN:

  1. those destined for lysosomes (via endosomes)

     Proteins destined for lysosomes are tagged with M6P for packaging into specific departing vesicles, and analogous signals are thought to direct secretory proteins into secretory vesicles

  2. those destined for secretory vesicles

  3. those destined for immediate delivery to the cell surface

• The nonselective constitutive secretory pathway transports most other proteins directly to the cell surface. Because entry into this pathway does not require a particular signal, it is also called the default pathway

Secretory Vesicles Bud from the Trans Golgi Networks

Concentration of cargo is achieved by:

• Aggregation of cargo in the TGN
• Removal of membranes by retrograde transporting CCVs

Electron micrograph of pancreas β-cells Showing secreting vesicle formation. Antibody to clathrin is conjugated to gold

Exocytosis of secretory vesicles

Cells that are specialized for secreting some of their products rapidly on demand concentrate and store these products in secretory vesicles (often called densecore secretory granules because they have dense cores when viewed in the electron microscope)

• Secretory vesicles form from the TGN, and they release their contents to the cell exterior by exocytosis in response to specific signals.

The secreted product can be either a small molecule (such as histamine or a neuropeptide) or a protein (such as a hormone or digestive enzyme).

At least three signal in TGN should be clearly recognized:

1. Signal-mediated diversion to lysosomes (via endosomes)
2. signal-mediated diversion to secretory vesicles (for regulated secretion)
3. constitutive secretory pathway

It is unclear how the aggregates of secretory proteins are segregated into secretory vesicles

Initially, most of the membrane of the secretory vesicles that leave the TGN is only loosely wrapped around the clusters of aggregated secretory proteins.

Morphologically, these immature secretory vesicles resemble dilated trans Golgi cisternae that have pinched off from the Golgi stack. As the vesicles mature, they fuse with one another and their contents become concentrated

Precursors of secretory proteins

Proteolysis is necessary to liberate the active molecules from these precursor proteins. The cleavages occur in the secretory vesicles and sometimes in the extracellular fluid after secretions

Additionally, many of the precursor proteins have an N-terminal pro-peptide that is cleaved off to yield the mature protein. These proteins are synthesized as pre-pro-proteins, the pre-peptide consisting of the ER signal peptide that is cleaved off earlier in the rough ER

The same polyprotein may be processed in various ways to produce different peptides in different cell types

Pre-pro-peptide

Why is proteolytic processing so common in the secretory pathway?

1. Some protein, such as enkephalins (five-amino-acid neuropeptides with morphine-like activity), are undoubtedly too short in their mature forms to be co-translationally transported into the ER lumen or to include the necessary signal for packaging into secretory vesicles
2. In addition, for secreted hydrolytic enzymes—or any other protein whose activity could be harmful inside the cell that makes it, delaying activation of the protein until it reaches a secretory vesicle, or until after it has been secreted

• the delay prevents the protein from acting prematurely inside the cell in which it is synthesized.

Constitutive secretion vs regulated secretion

1. Constitutive secretion

Exists in all eukaryotic cells

Cargo molecules of this transport are:

• membrane proteins
• lipid molecules
• proteins from the extra cellular matrix (ECM)

2. Regulated secretion

Exists mainly in specialized secretory cells

Cargo molecules of this transport are:

• hormones, histamine, etc.
• Neurotransmitters
• digestive enzymes

Scale models of a brain presynaptic terminal sand a synaptic vesicle:

How to trigger secretory vesicle to release its content?

Example 1: in nerve cells, voltage-gated Ca2+ channel influx Ca2+, which triggers nerve cells to release neurotransmitters. (lecture 8)

Example 2: ligand binding stimulate mast cells to release histamine

(1) Ca2+ influx trigger neurotransmitter release

Formation of synaptic vesicles

Nerve cells (and some endocrine cells) contain two types of secretory vesicles.

1. As for all secretory cells, these cells package proteins and neuropeptides in dense-cored secretory vesicles in the standard way for release by the regulated secretory pathway
2. In addition, however, they use another specialized class of tiny (≈50 nm diameter) secretory vesicles called synaptic vesicles.

• These vesicles store small neurotransmitter molecules, such as acetylcholine, glutamate, glycine, and γ-aminobutyric acid (GABA), which mediate rapid signaling from nerve cell to its target cell at chemical synapses

• When an action potential arrives at a nerve terminal, it causes an influx of Ca2+ through voltage-gated Ca2+ channels, which triggers the synaptic vesicles to fuse with the plasma membrane and release their contents to the extracellular space

• The speed of transmitter release (taking only milliseconds) indicates that the proteins mediating the fusion reaction do not undergo complex, multistep rearrangements

• After vesicles have been docked at the presynaptic plasma membrane, they undergo a priming step, which prepares them for rapid fusion. In the primed state, the SNAREs are partly paired, their helices are not fully wound into the final four-helix bundle required for fusion

  Proteins called complexins freeze the SNARE complexes in this metastable state

  The brake imposed by the complexins is released by another synaptic vesicle protein, synaptotagmin, which contains Ca2+-binding domains.

  A rise in cytosolic Ca2+ triggers binding of synaptotagmin to phospholipids and to the SNAREs, displacing the complexins

Synaptic Vesicles Can Form Directly from Endocytic Vesicles

Secretion can be locally restricted or can occur all over in the cell

membrane lipids and proteins

What happens to the membrane lipids and proteins after exocytosis?

1. Transiently fused with plasma membrane: forward flow
2. then recycled or to lysosomes for degradation— retrograde flow

Examples to expand membrane:

1. sorting plasma membrane proteins

Two ways of sorting plasma membrane proteins in a polarized epithelial cell

Proteins from the ER destined for different domains travel together until they reach the TGN, where they are separated and dispatched in secretory or transport vesicles to the appropriate plasma membrane domain

The apical plasma membrane of most epithelial cells is greatly enriched in glycosphingolipids, which help protect this exposed surface from damage