Lecture 7
Intracellular Membrane Traffic
Cells use an elaborate internal membrane system to add and remove cell-surve proteins, such as receptors, ion channels, and transporters.
Through exocytosis, the secretory pathway delivers newly synthesized proteins, carbohydrates, and lipids either to the plasma membrane or the extracellular space.
Through endocytosis, cells remove plasma membrane components and deliver them to internal compartments called endosomes, from where they can be recycled to the same or different regions of the plasma membrane or be delivered to lysosomes for degradation.
The secretory pathway leads outward from the ER toward the Golgi apparatus and cell surface, with a side route leading to lysosomes.
The endocytic pathway leads inward from the plasma membrane.
Retrieval pathways balance the flow of membrane between compartments in the opposite direction, bringing membrane and selected proteins back to the compartment or origin.
There are various types of coated vesicles
Most transport vesicles form from specialized, coated regions of membranes.
They bud off as coated vesicles, which have a distinctive cage of proteins covering their cytosolic surface.
Before the vesicles fuse with a target membrane, they discard their coat, as it is required for the two cytosolic membrane surfaces to interact directly and fuse.
The coat performs two main functions that are reflected in a common two-layered structure.
First, an inner coat concentrates specific membrane proteins in a specialized patch, which then gives rise to the vesicle membrane. The inner layer selects the appropriate membrane molecules for transport.
An outer coat layer assembles into a curved, basketlike lattice that deforms the membrane patch and thereby shapes the vesicle.
There are three types of coated vesicles, distinguished by their major coat proteins:
Clathrin-coated: mediate transport from the Golgi and from the plasma membrane
COPI-coated and COPII-coated: mediate transport from the ER and from Golgi.
Proteins leave the ER in COPII-coated transport vesicles
Entry into vesicles that leave the ER can be a selective process or can happen by default.
Many membrane proteins are actively recruited into such vesicles, where they become concentrated.
These cargo membrane proteins display exit signals on their cytosolic surface that adaptor proteins of the inner COPII coat recognize.
Some of these components act as cargo receptors and are recycled back into the ER after they have delivered their cargo to the Golgi.
Soluble cargo proteins in the ER lumen have exit signals that attach them to transmembrane cargo receptors.
Proteins without exit signals can also enter, including proteins that normally function in the ER (ER resident proteins), some of which slowly leak out and are delivered to the Golgi.
Only proteins that are properly folded and assembled can leave the ER
Proteins that are misfolded or incompletely assembled transiently remain in the ER, where they are bound to chaperone proteins.
SNAREs mediate membrane fusion
Once a transport vesicle has been tethered to its target membrane, it unloads its cargo by membrane fusion.
Membrane fusion requires bringing the lipid bilayers of two membranes within 1.5 nm of each other so they can merge.
For this approach, water must be displaced from the hydrophilic surface of the membrane, a process that is highly energetically unfavorable and required specialized fusion proteins.
The SNARE proteins catalyze the membrane fusion reactions in vesicle transport.
These transmembrane proteins exist as complementary sets, with v-SNAREs usually found on vesicle membranes, and t-SNAREs usually found on target membranes.
When a v-SNARE interacts with a t-SNARE, the helical domains of one wrap around the helical domains of the other to form a very stable four-helix bundle. This locks the two membrane together.
The trans-SNARE complex 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.
Rab proteins guide transport vesicles to their target membrane.
Specificity in targeting is ensured because all transport vesicles display surface markers that identify them according to their origin and type of cargo.
Target membranes display complimentary receptors that recognize the appropriate markers.
Rab proteins are GTPases.
Each Rab protein is associated with one or more organelles of the secretory pathway, and each of these organelles have at least 1 Rab protein on its cytosolic surface.
In their GTP bound state, they are tightly associated with the membrane of an organelle or transport vesicle.
Rab then binds to other proteins called Rab effectors, or tethering proteins, which initiate membrane fusion.
Rab proteins cooperate with SNAREs to accelerate fusion.
Rab proteins, which can regular the availability of SNARE proteins, exert an additional layer of control.
t-SNAREs in target membrane are often associated with inhibitory proteins that must be released before the t-SNARE can function.
Rab proteins and their effectors (tethering protein) trigger the release of such SNARE inhibitory proteins.
SNARE proteins are concentrated and activated in the correct location on the membrane, where tethering proteins capture incoming vesicles.
Rab proteins thus speed up the process by which appropriate SNARE proteins in two membrane find each other.
Interacting SNAREs need to be pried apart before they can function again
SNARE protein complexes need to be disassembled before they can mediate new rounds of transport.
A crucial protein called NSF cycles between membranes in the cytosol and catalyzes the disassembly process.
NSF is hexameric ATPase of the family of AAA-ATPases that use ATP hydrolysis to unravel the intimate interactions between the helical domains of paired SNARE proteins.
The requirement for NSF-mediated reactivation of SNAREs helps prevent membranes from fusing indiscriminately. If the t-SNAREs in a target membrane were always active, any membrane containing an appropriate v-SNARE might fuse whenever the two membranes made contact.
Vesicular tubular clusters mediate transport from the ER to the Golgi apparatus
After transport vesicles have budded from the ER exit sites and have shed their coat, they begin to fuse with one another.
The fusion of membranes from the same compartment is called homotypic fusion. This requires a set of matching SNAREs.
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.
They are generated continually and function as transport containers that bring material from the ER to the Golgi.
As soon as vesicular tubular clusters form, they begin to bud off transport vesicles of their own. These vesicles are COPI-coated.
COPI-vesicles are unique in that the components that make up the inner and outer coat layers are recruited as a pre assembled 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 COPI coat assembly begins only seconds after the COPII coat has been shed.
The retrieval pathway to the ER uses sorting signals
Resident ER proteins contain signals that bind directly to COPI, and are thus packaged into COPI coated vesicles for retrograde transport back to the ER.
Soluble ER resident proteins contain a KDEL sequence.
When the KDEL sequence is deleted, these proteins are secreted from the cell.
If the KDEL sequence is transferred to a sequence that is normally secreted, it is returned back to the ER.
Soluble ER resident proteins must bind to KDEL signal receptors
These are multipass transmembrane proteins that bind to the KDEL sequence and package any protein that contains the signal into COPI-coated vesicles.
To do this, KDEL receptors have to travel back and forth from the ER to the Golgi, so its affinity for the KDEL sequence must differ between the two organelles.
It needs to be able to pick up proteins in the Golgi, and release them in the ER.
It is hypothesized that differences in the pH level in the organelles is what alters the affinity.
The Golgi is regulated with H+ pumps that keep it acidic. The KDEL receptors can only bind to the KDEL sequence at a low pH.
The pH will increase as the vesicle approaches the ER, which is neutral, and the proteins are released.
The Golgi apparatus consists of an ordered series of compartments
During their passage through the Golgi, transported molecules undergo an ordered series of covalent modifications.
Each Golgi stack has two distinct faces:
A cis face (entry face)
A trans face (exit face)
Proteins entering the cis face can either move on further in the Golgi, or be returned back to the ER.
Proteins exiting from the trans face move onwards and are sorted according to their next destination.
N-linked oligosaccharides are attached to many proteins in the ER, and then trimmed.
The oligosaccharide intermediates created by trimming help proteins fold, and help misfolded proteins get to the cytosol to be degraded by proteasomes.
Once the protein is moved to the Golgi, the oligosaccharide plays other roles.
heterogenous oligosaccharide structures seen in mature proteins are generated.
Proteins arrive in the cis face, the first modification compartment.
They move through the Golgi, ending in the trans face where glycosylation is completed and the proteins are packaged for transport.
Each layer of the Golgi contains different enzymes, depending on what modifications are being done there. AKA, sugars are being added/remove depending on what enzymes are present.
N-linked glycosylation promotes protein folding in 2 ways:
It makes folding intermediates more soluble, preventing them from aggregating.
Also, the sequential modifications establish a “glyco-code” that marks the progression of protein folding and mediates the binding of chaperone proteins
Most proteins synthesized in the rough ER are glycosylated by the addition of a common N-linked oligosaccharide.
About half of the soluble and membrane bound proteins that are processed in the ER are glycoproteins that are modified in this way.
In the ER, a precursor oligosaccharide is added to the protein.
The transfer is catalyzed by a membrane bound enzyme complex called an oligosaccharyltransferase.
Oligosaccharides are used as tags to mark the state of protein folding
Some proteins require N-linked glycosylation in the ER for proper folding.
Monomeric GTPases control coat assembly
GTP binding proteins 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
GEFs activate the proteins by catalyzing the exchange of GDP for GTP.
GAPS inactivate the protein by triggering the hydrolysis of the bound GTP to GDP.
The Sar1 GTPase is responsible for the assembly of COPII coats at the ER membrane.
When a COPII coated vesicle is to bud from the ER membrane, a Sar1 GEF embedded in the ER membrane binds to cytosolic Sar1, causing it to release its GDP and bind to GTP.
GTP is present in much higher concentration in the cytosol and will therefore spontaneously bind once GTP is released.
Once bound to GTP, Sar1 will expose an amphiphilic helix tail that inserts into the cytosolic leaflet of the ER membrane.
The tightly bound Sar1 now recruits adapter coat protein subunits to the ER membrane to initiate budding.
Sar1 binds to the COPII coat proteins Sec23/24 to form the inner coat. Sec23 is bound to Sar1, which recruits Sec24. Sec24 binds to the cargo receptor.
Then, Sec 13/31 are bound to Sec23/24, forming the outer coat and pinching off occurs.
Sec13/31 can assemble into a symmetrical cage even alone.
The coat recruitment GTPases also have a role in coat disassembly.
The hydrolysis of bound GTP to GDP causes the Sar1 GTPase to change its conformation so that the tail pops out of the membrane.
This causes the vesicle’s coat to disassemble.