Cell biology - The Membrane

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plasma membrane
separates cell contents from the outside environment, defines its boundaries, and maintains essential differences between the cytosol and the extracellular environment. Very stiff due to high levels of cholesterol
lipid bilayer
provides the basic structure for all cell membranes
most abundant membrane lipids
polar head group and 2 hydrophobic hydrocarbon tails. 1 tail usually is saturated the other unsaturated
main phospholipid, have a 3-carbon glycerol backbone
formation of bilayers
amphipathic and cylindrical in shape, phospholipids spontaneously form bilayers
2 leaflets=1 bilayer
contains a rigid ring structure and a single hydroxyl group and a short hydrocarbon chain. Orient themselves in the bilayer with their OH group close to the polar head groups of adjacent phospholipid molecules
lipid movements
phospholipids in synthetic bilayers very rarely "flip-flop" from the monolayer on one side to another. Unfavorable because charged head groups must pass through hyrdophobic layer. Required for lipid biosynthesis an maintaining proper lipid distribution. Occurs less than once/month, though cholesterol is the exception.
In contrast, lipids laterally diffuse very rapidly. Lipids are synthesized at the ER and added to cytosolic leaflet of the ER. Also lipids rotate and have flexible tails
bilayer fluidity
depends on content.
Increases fluidity:
short lipid tails
unsaturated fatty acids
high temperature
cholestorol effects vary

basically, more disorder=more fluid
cholesterol effects on membrane fluidity
at >30%, it acts as a spacer between phospholipids and increases lateral diffusion and fluidity
>50%, it makes the membrane more rigid
lipid asymmetry
functionally important; content of 2 leaflets are drastically different.
many proteins bind specifically to cytosolic lipid head groups, such as protein Kinase C (PKC), which is activated in response to various extracellular signals
e.g. phosphotidylserine is restricted to cytosolic leaflet; its presence on the extracellular leaflet is a signal for death by apoptosis
transmembrane proteins
amphiphilic; hydrophobic regions pass through the membrane, interact with hydrophobic region of lipids; hydrophilic regions exposed to water; hydrophobicity increased by covalent attachment of fatty acid chain
Contain sig seq for ER insertion (in additon to the possible leader sequence), which also consist of hydrophobic AA, can be recognized by SRP, bind to inside of translocon. However, not found at N-terminus, not cleaved by signal peptidase
glycosylphosphatifylinositol (GPI) anchor
added after the transmembrane segment of the protein is cleaved off in the ER, leaving the protein bound to the noncytolic surface of the membrane. Proteins bound to the membrane by it can be readily distinguished by the use of a specific enzyme which cuts these proteins free from their anchors. Built in the ER lumen. Can be removed by an enzyme.
peripheral membrane proteins
proteins which are bound to either face of the membrane by noncovalent interactions with other membrane proteins, or the head groups of membrane phospholipids. Can usually be removed from gentle extraction procedures.
Do not come in direct contact with the hydrophobic core of the lipid bilayer.
integral membrane proteins
transmembrane proteins and many proteins held in the bilayer that insert into the hydrophobic core of the lipid bilayer that cannot be released by gentle extraction procedures. They can span the bilayer completely or bend a portion of their strucutre into the bilayer.
Usually have transmembrane domains (TMDs), composed of alpha-helices of hydrophobic amino acids
Can be identified by analyzing the protein sequence with a computer or using a hydropathy plot to predict transmembrane alpha helices
identification of integral membrane proteins
1) analyze sequence
2) hydropathy plots predict hydrophobic transmembrane alpha-helices
lipid-linked proteins
can embed into either the cytosolic leaflet or the bilayer, or the extracellular/lumenal leaflet of the bilayer. 3 different types of lipids can be used to link proteins to the cytosolic leaflet of a membrane. Proteins attach via GPI anchor, the only type of lipid used to link proteins to the extracellular leaflet.
Behave like integral membrane proteins during purifications.
Purification of membrane proteins
need to be isolated from cells to study their functions
can be purified with detergents which replace the membrane bilayer
Src family
family of cytoplasmic protein tyrosine kinases in which mytistic acid, a saturated fatty acid is added to the N-terminal. A second lipid is then anchored to attach the proteins more firmly to the membrane; occurs in response to an extracellular signal.
Fluid-mosaic model
revised model of how phospholipis and proteins interact in a cell membrane
Membranes are patchy, patches are called lipid rafts, lipid layer is not homogenous, and more proteins that initially thought
Proteins also move within the membrane. Demonstrated using plasma membrane proteins in 2 different cells labeled with 2 different fluorescent molecules. When the cells were fused the 2 populations mixed together.
rough ER
has many ribosomes bound to its cytosolic surface; synthesize soluable and integral membrane proteins. Proteins transported into ER as they are being synthesized. ER membrane is unique in having ribosomes tethered to it.
ER tubules extend from the nucleus throughout the entire cytosol. Localization relies on in-tact MT array; if MT array is depolymerized, the ER network collapses towards the cell center
smooth ER
produces most of the lipid for the rest of the cell and functions as a store for Ca2+ ions
More abundant in certain cells, eg cells specialized in lipid metabolism, and accomadates the enzymes that make cholesterol to form the hormones.
Golgi Apparatus
consists of disc like compartments called Golgi cisternae. Recieves lipids and proteins from the ER and dispatches them to various locations, usually covalently modifying them en route.
Located close to the nucleus. Localization depends on MT array, which if depolymerized, the fargments disperse through the cell.
Cis-face main site of protein entry
Trans-face main site of protein exit
mitochondria and chloroplasts
generate most of the ATP that the cells use to drive reactions requiring an input of free energy; chloroplasts are a specialized versions of plastids, which can also store food and pigment molecules
contain digestive enzymes that degrade defunct organelles, as well as macromolecules and particles taken from outside the cell by endocytosis.
on their way to the lysosomes, endocytosed materil must first paass through these
small vesicular compartments that contain enzymes used in various oxidation reactions
nuclear pore complex (NPC)
mechanism by which the nucleus and the cytosol communicate with one another
gated transport
transport in which proteins move between the cytosol and the nucleus (which are topologically =) through NPCs. The NPCs function as selective gates that actively transport specific macromolecules, and passively diffuse smaller molecules
transmembrane transport
transmembrane protein translocators directly transport specific proteins across a membrane from the cytosol into a space that is topologically distinct. The transported protein must usually unfold into to fit through the translocator. Initial transport of selected proteins from the cytosol to the ER lumen occurs this way
vesicular transport
membrane-enclosed transport intermediates (either small vesicles or larger organelle fragments) ferry proteins from one compartment to another. Become loaded with cargo as they bud and pinch off from the membrane; they discharge their cargo into a 2nd compartment by fusing with its membrane and enclosing that compartment. Transfer of soluable proteins from ER to Golgi occurs this way. Can move proteins only between topologically equivalent compartments.
signal sequences
most reside in a stretch of amino acid sequence 15-60 residues long; often found at the N-terminus; recognized by the SRP; signal peptidases remove the signal sequence from the finished protein once the sorting process is complete. Can also be found in internal stretches of amino acids. Sometimes composed of multiple internal amino acid sequences that form a 3D arrangement of atoms called a signal patch.
Each specifies a particular destination in the cell.
Recognized by site in the translocon pore to open the pore and start transfer
Proteins destined for the ER
usually have signal sequence at their N-terminus, includes stretch of 5-10 hydrophobic AA. Many will pass to Golgi, but those with sequence of 4 AA at C-terminus will pass back to ER
Endoplasmic reticulum
ordered into a branching tubules and flattened sacs that extend throughout the cytosol which interconnect and are continuous with the outer nuclear membrane.
Has a central role in both lipid and protein biosynthesis, serves as a Ca ion store used in many signalling responses. The site of production of all the transmembrane proteins and lipids for most of the cell's organelles.
ER Lumen
formed by the internal space enclosed by the ER and nuclear membranes. AKA ER cisternal space. Occupies >10% of the cell volume.
Almost all proteins that will be secreted to the cell's exterior, and those destined for the Golgi or lysosomes, will first be delivered through it.
mammalian cells begin to import most proteins to the ER before complete synthesis of the polypeptide chain. The ribosome that is synthesizing the protein is attached directly to the ER membrane, allowing one end of the protein to be translocated into the ER while the rest of the chain is being assembled.
the import of the proteins into mitochondria, chloroplasts, nuclei, and peroxisomes
transitional ER
areas of smooth ER from which transport vesicles carrying newly synthesized proteins and lipids bud off for transport to the Golgi.
refers to both the orientation of the proteins within or with relation to the membrane and to the membrane lipids themselves. Maintained when the material is moved between compartments within the secretory system
The cytosolic leaflet of a membrane bilayer is always cytosolic and the non-cytosolic leaflet (lumenal or extracellular) is always non-cytosolic. The lumen is equivalent to the outside of the cell.
catalyzes the flipping of phospholipids to balance the # of lipid molefcules in the 2 leaflets leading to symmetric growth.
catalyzes the fliping of specific phospholipids to create an unequal distribution in the 2 leaflets (e.g. flippase at the plasma membrane ensures that all phosophotidylserine is moved to the cytosolic leaflet)
Require ATP hydrolysis. Many act at plasma membrane, contribute towards asymmetric distribution
hydropathy plot
generated by calculating the avg. hydrophobicity score for 20 AA, which is then moved by 1 AA and the score is calculated again. Process is repeated and plotting of the avg. scores created the plot. A peak represents the most hydrophobic 20 AA segment and indicated a possible transmembrane domain. Only predict transmembrane regions- every hydrophobic segment does not get a high score and some segments with high scores are not transmembrane domains
Outbound traffic/Secretory pathway
rough ER (protein synthesis) -> Golgi (protein modification) -> plasma membrane (or lysosome)
Proteins are moved between compartments in secretory vesicles. This pathway is used for membrane proteins that function in the ER, Golgi, plasma membrane, or lysosome as well as soluble proteins that are secreted or function in the lumen of these organelles
Inbound traffic/Endocytic pathway
Plasma membrane -> endosomes -> lysosomes
Extracellular membrane and soluble proteins will enter the endocytic pathway from the plasma membrane. The proteins are transported by endosomes (small vesicles) and eventually sent to the lysosome for degredation
Entry into the secretory pathway
1. Transport of proteins into the ER is co-translational
2. Proteins are transported into the ER in the unfolded state
3. Transport into the ER is unidirectional. Proteins do not leave the ER unless they fail to fold.
ER translocation of a lumenal/soluble protein
step 1) signal sequence is exposed during translocation and is required to guide the protein/ribosome complex to the ER
2) the signal recognition particle (SRP) binds to the signal sequence of the protein as its being translated and contacts the ribosome, which stops translation to prevent the protein from folding in the cytosol. The SRP guides the protein/ribosome complex to the ER membrane
3) SRP binds to the SRP receptor on the ER membrane, bringing the ribosome complex in contact with the translocon. SRP and the SRP receptor then dissociate from the ribosome-translocon complex
4) translation of the protein resumes upon contact of the ribosome with the translocon. The sig seq binds to the pore of the translocon to stabilize the interaction and open the pore to allow the protein to pass through. Protein folds in ER lumen.
5) when translation has finished, the signal peptide is cleaved off by a signal peptidsase and the protein is released into the lumen. The pore opens up to allow the sig seq to enter the membrane and it is degraded
signal recognition particle
complex of 6 proteins and a small RNA. Binds to the signal sequence of a newly synthesized protein as its being translated and contacts the ribosome. Causes a translational pause. Binds to the SRP receptor on the ER membrane. Links the translation machinery to the translocon
Found in all cells, indicating the mechanism evolved early and has been conserved
water-filled channel that the synthesized poplypeptide chain can pass through. translation of a protein resumes upon contact of the ribosome with it. Its pore can be opened when a signal sequence binds to it. Stays bound to the ribosome until translation is complete
free ribosomes in the cytosol and membrane bound ribosomes on the ER are equivalent and only differ in what kind of proteins they are making. Common pool of free ribosomes. Many times 1 mRNA contains multiple ribosomes that are translating at the same time (polyribosomes); those on the ER could be translating proteins into different translocons.
Types of transmembrane proteins
I: single transmembrane domain protein with the N-terminus in the lumen and the C-terminus in the cytosol
II: single transmembrane domain protein with the C-terminus in the lumen and the N-terminus in the cytosol
III/IV: Proteins with multiple transmembrane domains
signal sequence binding site
allows the signal sequence to bind to many different sequences; its a large hydrophobic binding pocket lined by methionines, which have unbranched, flexible sidechains. The pocket is sufficiently plastic to accomodate the hyrdrophobic signal sequences of different sizes and shapes
SRP structure
rodlike; wraps around the large ribosomal subunit with 1 end binding to the ER sig seq as it emerges as part of the newly made polypeptide chain. The other end blocks the elongation factor binding site at the interface between the large and small ribosomal subunits. The block halts protein synthesis.
The SRP RNA forms the backbone that links the domain of the SRP containing the signal sequence binding pocket to the domain responsible for blocking translation.
membrane-bound ribosomes
attached to the cytosolic side of the ER membrane; engaged in synthesis of proteins that are being concurrently translocated into the ER.
Free ribosomes
unattached to any membrane; synthesize all other proteins encoded by the nuclear genome not being concurrently translocated into the ER. Since many can bind to a single mRNA molecule, a polyribosome is usually formed, which becomes attached to the ER membrane, directed there by signal sequences on multiple growing polypeptide chains. The individual ribos. associated with the mRNA can return to the cytosol when the finish translation. The mRNA remains bound to the ER membrane by a changing population of ribosomes, each transiently held at the membrane by the translocator.
Method to study ER import
Step 1) Isolation of microsomes
step 2) Protein translation
step 3) protease protection assay
step 4) SDS-PAGE
Isolation of microsomes
1st step of method to study ER import.
Open cells and homogenize. The ER will fragment into small closed vesicles called microsomes. Centrifugation can be used to separate the smooth microsomes that are light and found at the top of tube. Each microsome functions as a mini-ER
Protein Translation
step 2 of the method to study ER import
the factors needed for translation are added to the rough microsomes. Factors include mRNA, initiantion factions, elongation factors, tRNAs, amino acyl tRNA synthetases, nucleotides for energy, and AA that can be radioactive to allow for detection of newly synthesized proteins by autoradiography
Protease protection assay
step 3 of the method to study ER import
proteases added to degrade any accessible proteins. Membrane of microsome protects a protein from proteases. If a protein is imported successfully it will not be cleaved. A positive control to ensure that the protease is functional, the microsomes are treated w detergent to break up the membrane brefore treatment with the protease. Protein inside microsome will then be degraded since its not protected.
step 4 of the method to study ER import
The proteins from the experiment can be analyzed by SDS-PAGE to determine the size
Possible results from the method to study ER import
If microsomes are added after translation, there will be no co-translation translocation. The sig seq will not be cleaved. The protease will degrade the protein.
If microsomes are added before translation, co-translational translocation will occur, the sig seq will be cleaved, and the protein will not be degraded by protease (unless detergetn added)
Transmembrane protein type 1
the N-terminal domain within the ER lumen and a C-terminal domain in the cytosol (major type)
Transmembrane protein type 2
the C-terminus in the ER lumen and the N-terminus in the cytosol
Transmembrane protein types 3/4
Multipass membrane proteins
insertion of type 1 transmembrane protein containing a typical signal sequence and one internal sequence
N-terminal sig seq: acts like leader seq.
Second signal: stop transfer or signal anchor sequence.
The AA translated after the stop transfer sequence remain in cytosol. The sig seq is cleaved by signal peptidase, creating new N-terminus in the ER. Both hydrophobic sequences released into the membrane by the translocon. Signal peptide will be rapidly degraded and internal sequence will act as a membrane anchor
Insertion of a transmembrane protein with single internal signal sequence
internal sequence recognized by SRP and directed to ER. Sig seq binds to translocon and polypeptide threaded through translocon. Sig seq not cleaved, and can bind in 2 different orienations: C-terminus or N-terminus can be in the ER lumen. If SRP encounters more positively charged AA before the signal, C-term. will be in lumen. Mutating residues around the sig seq can reverse orientation
If there are more + AA following the hydrophobic core of the start-transfer sequence, the membrane protein will be inserted with its N-terminus in the ER lumen
Insertion of a protein with multiple internal sequences
multi-pass transmembrane domain proteins: orientation determined by orientation of N-term. Start as type 1 or 2. Only signal nearest N-term requires SRP binding and loading onto translocon; subsequent signals alternate, so if the 1st is a start transfer seq (eg signal peptide in a type I TMD protein), the next will be a stop transfer, etc. and the protein will be threaded from lumen to cytosol to the lumen to the cytosol
Insertion of a protein with two internal sequences
the internal sig seq will serve as start transfer seq. The next hydrophobic sequence will serve as a signal anchor sequence or stop transfer signal. Remainder of the protein will be in the cytosol. Neither sequence cleaved by signal peptidase. The protein will have 2 transmembrane regions
3 types of topogenic sequences create 3 different types of topologies
1. Signal peptide at N-term is a start sequence that is cleaved by signal peptidase
2. Internal sequence or start transfer sequence not at the N-term is not cleaved by signal peptidase
3. Internal signal anchor sequence or stop transfer sequence
Protein modifications in the ER
1. Cleavage of N-terminal signal sequence
2. N-linked glycosylation
3. GPI-anchor
4. Disulfide bond formation
N-linked glyosylation
effects 1/2 of euk. proteins; is the addition of a block of sugar groups to asp. residues. Sugar group is built on lipid anchor on cytosolic face of the lumen. More sugars are added and then the large sugar group is attached to an asp. resi of the protein by oligosacchyrl transferase (OST), physically associated w the translocon. Trimming of sugars associated with proper protein folding
Disulfide bond formation
disulfide bonds between cysteine residues can form spontaneously in the ER. If the wrong disulfide bonds from the protein will likely not fold properly. Protein disulfide isomerases in the ER remove the incorrect disulfide bonds and catalyze the formation of new disulfide bonds to help protein folding.
ER quality control
Chaperones in the ER recognized exposed hydrophobic patches of incompletely folded proteins and use ATP hydrolysis to ensure proteins are properly folded.
binding protein
Pulls post-translational proteins into the ER through the translocon. Recognizes incorrectly folded proteins, as well as subunits that have not yet assembled into their final form. It does so by Scanning for exposed hydrophobic patches that would normally be burried, and other sequences too. Hydrolyzes ATP to provide energy to help proteins translocate post-translationally into the ER, and helps these and other proteins fold
Calnexin and calreticulin
membrane bound and soluble respectively.
Calcuim-dependent ER chaperones that bind to the last glucose on the N-liked oligosaccharide and help the protein fold. Prevent incompletely folded proteins from becoming irreversibly aggregated. Glucosidase then trims off the end glucose residues. If the protein is still unfolded, glucosyl transferase will add more glucose residues to N-linked oligosaccharide of proteins to repeat the cycle of chaperone-assisted folding until the protein is correctly folded. Also promote the as sociation fo incompletely folded proteins with another ER chaperone which binds to cysteines before disulfide bonds.
last resort for improperly folded proteins. Proteins exported from the ER, deglycosylated, ubiquinated, and ultimately degraded by the proteasome.
unfolded protein response (UPR)
transcriptional response to many misfolded proteins in the ER. The signal induces the transcription of chaperones, ubquitination machinery, and degradation machinery.
ER lipid synthesis
ER is the site of most lipid synthesis in the cell. Lipid-synthesizing enzymes are found on the cytosolic side of the ER, components found in the cytosol. Fatty acids brought to the ER by proteins, activated w CoA. Series of reactions --> phospholipids.
Secretory or biosynthetic pathway
outbound traffic. Cargo molecules are secretory proteins (transmembrane & soluble proteins, newly synthesized material), carbs, and lipids. Move from ER to Golgi to plasma mmebrane or lysosome through secretory vesicles.
Type of transport in secretory pathway; if the target compartment is the plasma membrane. The transport vesicles are exocytic vesicles
Endocytic pathway
inbound traffic; transports molecules from plasma membrane to internal compartments via endocytic vesicles. The final destination can be lysosome or recycling back to plasma membrane. Cargo includes membrane proteins, lipids, and soluble proteins. Not the exact opposite of exocytosis because the molecules follow different routes (proteins end up in lysosomes instead of Golgi or ER)
moves material along the secretory and endocytic pathways. Requires budding and fusing; bud from donor membranes, fuse with acceprot or target membranes in a manner that maintains topology of cargo molecules
Steps in vesicular transport
1. Cargo selection
2. Budding
3. Scission
4. Uncoating
5. Transport
6. Targeting
7. Docking
8. Fusion
9. Disassembly
Vesicular transport Cargo Selection
1st step. Specific cargo must be selected. Membrane proteins have sorting signal in the cytoplasmic tail of the protein (not hydrophobic), which is recognized by cytosolic coat proteins, which cause clustering of the membrane proteins. Each type of vesicle has its own type of cargo and coat. 1) clathrin: golgi and plasma mem, 2) COPI- Golgi, 3) COPII- ER
Coat proteins give size and shape to vesicles
sorting signal
membrane proteins have one in their cytoplasmic tail. The signal sequence is not hydrophobic. Its recognized by cytosolic coat proteins.
Coat proteins
Recognizes the sorting signal. Causes clustering of the membrane proteins. 3 major types: clathrin, COPI, COPII
Help cause bending of the membrane to create a bulge. Do not bind directly to cargo molecules, but need adaptor proteins.
coating protein that coats vesicles from the Golgi and Plasma membrane
coating protein that coats vesicles from the Golgi
coating protein that coats vesicles from the ER
Budding in Vesicular transport
2nd step. In order for a vesicle to form, the donor membrane must deform the membrane to create curvature and form a bud. Binding of coat and adaptor proteins recruits additional proteins to form a complete vesicle.
Coat recruitment proteins
Recruit coat proteins to the correct membrane. Arf1 for COPI and sometimes clathrin for Golgi, Arf6 for clathrin to plasma membrane, Sar1 for COPII to ER.
Monomeric GTPases that are cytoplasmic in the GDP-bound state, associated with target membrane in GTP-bound state. Guanine nucleotide exchange factor on the target membrane activates the recruitment protein by causing GDP to be released and exchanged for GTP.
Sar1 Activation and Recruitment
Recruits COPII.
1) Sar1 GDP located in cytosol, has fatty acid tucked into structure
2) Sar1 GEF in the ER membrane activates it by removing GDP, replaced by GTP.
3) GTP binding causes conformation change resulting in fatty acid becoming accessible and inserting into ER membrane
4) Sar1 assembles COPII coat proteins, causing the bulge necessary for vesicle budding.
Scission in vesicle transport
3rd step.
At ER and Golgi, coat recruitment proteins provide enough energy to cause spontaneous scission. The plasma membrane needs dynamin, which forms a ring around the neck and tightens to pinch the vesicle.
Forms rings around the neck of a clathrin-coated vesicle on the plasma membrane, and tightens to pinch the vesicle causing scission. GTPase activity is required for function. Mutant dynamin that cant hydrolyze GTP causes paralysis in flies because the vesicle scission is required in the neurons for activation of the muscles
Uncoating in vesicle transport
4th step.
After a vesicle is released from donor membrane, coat is no longer needed and must be removed before fusion with acceptor membrane. For COPI and COPII, the GTP hydrolysis of coat recruitment proteins starts the coat disassembly. Clathrin coats use chaperones that use the energy from ATP hydrolysis to remove coat.
Transport step in vesicle transport
5th step.
To get to target membrane, vesicles can bind to a motor protein that attaches to a cytoskeletal filament that acts as a road. Most commonly, vesicles bind to kinesin (outward pathway) or dynein (inbound pathway) and move on MTs. At the periphery of cell, actin can be used.
Targeting in vesicle transport
6th step.
initial recognition and connection of vesi to target membrane is mediated by Rab proteins. GDP dissociation inhibitors (GDIs) prevent Rab activation to control timing of vesicle fusion. On target membrane, active Rav activates Rab effector proteins. Many different Rabs, each specific for different organelle.
Rab proteins
GTPases that are cytosolic when inactive and bound to a specific target membrane when active. Mediates the initia recognition and connection of a vesicle to a target membrane. GDP dissociation inhibitors (GDIs) prevent Rab activation, controlling the timing of vesicle fusion. On target membrane, active Rab activates Rab effector proteins. Rab 1 associates with ER and Golgi, Rab3A on secretory vesicles, Rab 4 located on recycling vesicles, Rab 5 localizes to early endosomes.
Docking in vesicle transport
7th step.
Strengthens the recognition and connection of the vesicle to target membrane. Mediated by SNARE proteins, which act as a tight zipper.
SNARE proteins
transmembrane proteins with cytosolic alpha helices that can wrap around each other, forming a 4-helix bundle athat acts like a zipper, needed for vesicle docking. They form complimentary pairs with 1 v-SNARE (on vesicle membrane) or t-SNARE (found on target membrane). The toxins that cause botulism and tetanus cleave SNARE proteins, interfering with vesicle trafficking. Also help with membrane fussion. Separated by interacting proteins. Recycled and used again.
Fusion in vesicle transport
8th step.
SNARE proteins help by bringing the 2 membranes so close together water is excluded and lipids of the outer leaflets mix together (hemifusion). Inner leaflets rupture to cause complete membrane fusion
Disassembly in vesicle transport
9th and last step.
Tightly bound SNARE proteins separated by interacting proteins such as NSF. the SNARES are recycled to be used again.
Vesicle fusion and viruses
Viruses often us mechanisms close to vesicle budding and fusion to enter and infect cells. eg HIV, which fuses with the plasma membrane using CD4 and chemokine receptor to dock. The virus reveals a hydrophobic region that inserts into the plasma membrane, brings the viral membrane in contact w/ plasma membrane. Leads to fusion and release of the viral contents into the cytosol.
start-transfer signal
The 2nd time the signal sequence is recognized, by a binding site on the pore on the translocon (the 1st is by an SRP in the cytosol). It opens the pore of the translocon. Dual recognition may help to ensure only aappropriate proteins enter the lumen of the ER.
stop-transfer signal
anchors the protein in the membrane after the ER signal sequence (the start-transfer sig) has been cleaved off and released from the translocon. Transferred into the bilayer by the lateral gating mechanism
difference between start and stop transfer signals
whether a sig seq functions as a start or stop depends on location in the polypeptide chain. SRP begins scanning chain for hydrophobic segments at N-terminus, towards C-terminus (direction protein is synthesized). The first hydrophobic sequence that is recognized is the "reading frame". The next recognized hydropohibic sequence is a stop, causing the region to be threaded across the membrane.
Protein orientation in the lipid bilayer
membrane proteins are always inserted from the cytosolic side of the ER, so all copies of the same polypeptide chain will have the same orientation in the lipid bilayer. Generates asymmetrical ER membrane; protein domains exposed on 1 side different from those on other. Asymmetry maintained during transport from ER to other membranes. Asymmetry not inherent property of the protein, but the process by which proteins are inserted into the ER membrane from cytosol.
1/2 of all Euk proteins are glycosylated- most ofthe soluble and membrane-bound proteins made in ER (including those destined for Golgi, plasma mem, or extracellular space)
Very few cytosolic proteins are glycosylated, and those that are carry a much simpler modification
lipid that holds the precurso oligosaccharide in the ER lumen during protein translocation. The precursor oligosac. is held to it by a high-energy pyrophosphate bond, which provides the activation energy that drives the glycosylation reaction.
slow trimming of mannose on the core-oligosaccharide tree by this enzyme in the ER is thought to create a new oligosaccharide structure the retrotranslocation apparatus recognizes. Proteins that fodl and exit from the ER faster than the enzyme's action would escape degredation.
adaptor proteins
major coat component in clathrin-coated vesicles; form a discrete second layer of the coatm positioned between clathrin cage and the membrane. They bind the clathrin coat to the membrane and trap various transmembrane proteins and cargo receptors.
Several types, each specific for a different set of cargo receptors. Lateral interactions between adaptor complexes and clathrin molecules aid in forming the vesicle
cargo receptors
transmembrane receptors that capture soluble cargo molecules inside the vesicle
Golgi stacks
each stack has a cis face where proteins enter and a trans face where they exit. Proteins move progressively through the Golgi, modified progressively by glycosylation, which started in the ER and is completed in the Golgi. Golgi is a major sorting station and proteins exiting the Golgi are destined for the lysosome, plasma mem, secretory vesicles, or may be returned to ER
Overview of ER to Golgi transport
1. Cargo selection- based on AA sequences in the cytoplasmic tails of membrane proteins
2. Vesicle budding- COPII interacts w adaptors and coat recruitment proteins
3. Scission of the vesicle
4. Uncoating- Sar1 hydrolyzes GTP to GDP and the COPII disassembles
5. Transport along MTs
6. Tethering of the vesi to the Cis-Golgi using Rabs
7. Docking- binding of t-SNARE on cis-Golgi and v-SNARE on vesi
8. Fusion with help of t-SNAREs
9. Disassembly of SNARes by NSF
ER exit sites
specialized regions of the ER where vesicles form; face the cis-Golgi and do not have ribosomes
Selection of cargo- Membrane proteins
they have a sorting signal in the cytosolic tail recognized by adaptor and coat proteins that allow ER exit
Selection of cargo- Soluble proteins
they may have an ER exit signal that allows them to bind to the lumenal part of a membrane protein containing a sorting signal on the cytoplasmic take. Can also be transported by bulk transport if the concentration is high enough that the proteins will be randomly trapped in vesicles as they form from the ER
Quality Control mechanisms
cells have stringent mechanisms to ensure that defective proteins are not allowed to leave the ER. Chaperones make sure the proteins are folded properly and protein complexes are complete (eg antibodies).
Cystic fibrosis transmembrane regulator (CFTR)
The QC of the ER are so stringent that one that is slightly misfolded but still functional is not allowed to leave and reach the plasma membrane. Is a chloride channel required in epithelial cells to keep extraceullular mucus less stick. Patients w cystic fibrosis have mucus build up b/c their CFTR does not reach the plasma mem.
Vesicular tubular clusters
formed between ER and Golgi by fusion of COPII vesicles; an intermediate compartment that can hold more material than individual vesicles. Fusion of COPII vesis from the same compartment (ER) is ex. of homotypic fusion.
Clusters then fuses with the cis-Golgi network. Both compartments are part of the checkpoint to look for proteins that have escaped the ER
ER retrieval pathway
returns ER resident proteins that have escaped to the Golgi. eg BiP escapes by bulk transport and SNAREs travel to Golgi during vesi transport, both proteins need to be returned to ER. Proteins are returned by COPI coated vesicles from the vesicular tubular cluster or the cis-Golgi
ER retention or retrieval signal
must be contained by recycled proteins. For mem prots, signal is KKXX, and for soluble proteins, the signal is KDEL.
KDEL receptor
transmembrane protein found in the ER, the vesicular tubular cluster, and the Golgi. Has a signal to travel to the Golgi in the cytosolic tail and a signal to return to the ER (KKXX). The KDEL receptor binds tightly to the soluble KDEL cargo in the Golgi, then binds to adaptor and coat proteins to form a vesicle. After transport to the ER, the KDEL receptor releases the KDEL cargo in the ER lumen. Receptor has lower affinity for the cargo in the ER due to high levels of calcium and higher pH.
Transit through the Golgi and Glycosylation
1) progressive modification (glycosylation) which is sequential and ordered
2) each cistern contains a specific set of modifying enzymes
3) Golgi enzymes are membrane bound
4) The order of glycosylation is mirrored by the order of enzymes in the Golgi stacks
N-linked glycosylation
modifies most glycoproteins. High mannose oligosaccharides may undergo trimming in the Golgi, but do not have additional sugars added. Complex oligosaccharides have more sugar residues added in the Golgi.
O-linked glycosylation
Sugar residues can be added to Ser/Thr residues exclusively in the Golgi
Important functions of Glycosylation
1) proper folding and ER exit (N-linked)
2) cell-cell recognition (especially in immune system)
3) protection from proteases
4) structural support tissues
5) ensure that extracellular matrix is a hydrated gel
6) serve as co-receptors for signaling molecules
Golgi organization controversy
Vesicular transport model vs. cisternal maturation model.
Evidence exists to support both. They are not mutually exclusive and both probably happen. Small cargo = maybe vesicular transport, large cargo = maybe cisternal maturation
Vesicular transport model
Theoretical model in which the Golgi has fixed, static cisternae and vesicles move between the stable cisternae. Each compartment has unique enzymes.
Cisternal maturation model
Theoretical model in which the Golgi is dynamic and the cisternae move progressively through the Golgi maturing and migrating to become the next cisternae. Budding of vesicles collects glycosylation enzymes and delivers them to the earlier cisternae to maintain the progressive system. Vesicular tubular clusters become the CGN and the TGN becomes multiple vesicles that are transported to the plasma membrane or lysosome.
Components of cell-free biochemical assay to study Golgi Transport
1) Vesicular stomatitus virus (VSV) infected cells expressing the glycoprotein VSV-G
2) mutant cells that do not express GlcNac transferase which glycosylated proteins in the Golgi
3) WT cells
Experimental details of the cell-free biochemical assay to study Golgi transport
Mix Golgi purified from uninfected cells (which have no VSV-G and normal GlcNac transferase) w Golgi purified from infected cells that lack GlnNac transferase (but express VSV-G). Add ATP (energy source), cytosol, and radioactive GlcNac (to detect glycosylation). After the assay, immunoprecipitate VSV-G and detect incorporation of radioactive GlcNac.
Results and Interpretation of the cell-free biochemical assay to study Golgi transport
Results: Radioactive GlcNac is attached to VSV-G and requires GTP and cytosol.
Interpretation: Transport vesicles carry VSV-G from the mutant/infected Golgi to the normal Golgi with the GlcNAc transferaase (consistent with vesicular transport model) OR transport vesicles carry GlcNAc transferase from the normal Golgi to the mutant/infected Golgi (consistent w cisternal maturation model)
Lysosomal/Acid hydrolases
The main cargo carried from TGN to lysosomes (via endosomes) are lysosomal or acid hydrolases, which are powerful degredative enzymes including nucleases, proteases, glycosidases, lipidases, phosphatases, sulfatases, and phospholipases. The lysosomal hydrolases are active only in an acidic environment of pH4.5-5, which is maintained by vacuolar ATPases that push protons into the lysome. Therefore, lysosomal hydrolases are active in the lysosome, but not in any other comparments in the cell, protecting the cell from damage. Lysosomal proteins are protected by hydrolase activity by lots of glycosylation.
Protein Transport from Golgi to Lysosomes
1) Lysosomal hydrolases are synthesized and N-glycosylated in the ER
2) a signal patch on lysomal hydrolases is recognized by GlcNac phosphotransferases
3) In the TGN the terminal GlcNac is cleaved off exposing the mannose-6-phosphate (M6P) that is a specialy "tag" for transport to the lysosome
4) M6P tags are recognized by the M6P receptor,a membrane proteins that resides in the TGN
5) M6P binds to an adaptor protein that binds to Clathrin
6) Clathrin coated vesicles with M6P receptors and the M6P cargo bud off from the TGN
7) the clathrin coated vesicles travel to and fuse with endosomes
8) The M6P cargo separates from the M6P receptor in the late endosome because the environment is more acidic than the Golgi
9) M6P receptor recycles back to Golgi using the retromer coat protein
10) a phosphatase removes the phosphate and the late endosome with lysosomal hydrolases fuse with the lysosome
11) the lysosomal hydrolases are active in the acidic environment of the lysosome.
Effect of pH on transport from Golgi to Lysosomes
Relatively neutral pH of ER and Golgi prevents lysosomal hydrolases from degrading proteins. 2nd, the acidic environment of the endosomes helps separate M6P cargo and M6P receptors. (opposite of KDEL receptor which release the cargo in the less acidic environment)
Lysosomal storage diseases
caused by defects in transport to the lysosome that lead to accumulation of undigested material in the lysosome. eg Inclusion cell (I-Cell) disease
Inclusion cell disease
I-Cell disease, caused by a mutation in the GlcNac transferase that adds the M6P tag to the lysosomal hydrolases. If the M6P tag is not present in the cargo, it wont be recognized by M6P receptors and will be trafficked to the plas mem. Inclusion bodies characterize I-cell disease. lysosomal membrane proteins which dont depend on M6P tag are still transported to lysosome. All cells affected by mutations in GlcNac transferase, but neurons especially. Symptoms include developmental delay and mental retardation.
Transport from Golgi to Plasma Membrane
delivered by the default (constitutive) pathway or signal-mediated (regulated) pathway. For constitutive, no signals are needed and there is a steady stream of proteins transported to plasma mem.
The regulated requires a signal.
signal-mediated pathway from Golgi to Plasma membrane (regulated)
requires a signal for fusion of vesicles with the plasma membrane, common in cells specialized for secretion, eg neurons and release of synaptic vesicles. Release of synaptic vesicles can be regulated by Ca levels. In neurons, synaptic vesicles are not made in Golgi and transported to the end of the acon, but the raw materials re transported by bulk transport to the end of the axon and the vesicles are assembled locally, allowing for rapid response to signals.