Cell bio Midterm 2

Structure of ancestor prokaryotic cell

• eukaryotic cell evolved from a prokaryotic cell

• ancient basic prokaryotic cell has a membrane but there is not a lot of compartmentalization

• DNA was tethered to the inside of the membrane

• Protein synthesis was associated with the membrane

• During evolution the membrane started to fold into the inside of the cell and then folded more to start organizing the cell

  • This gave rise to the completely enclosed structure of the nucleus containing the DNA, and other organelles

Origin of compartments

• Nucleus and cytosol— derived from early cytosol in the ancient prokaryotic cell

  • Nucleus inner membrane was continuous with the ER membrane

  • Nuclear pores

• ER, golgi, vesicles, lysosomes— lumens— derived from the outside of the original cell + the early plasma membrane that enclosed these vesicles

• Mitochondria— early aerobic prokaryote

  • early prokaryotes had electron transport machinery— makes ATP

How do proteins know where to go?

• Protein synthesis ALWAYS starts in the cytoplasm

• ER makes little buds that can transport proteins; it can fuse with the membrane of the Golgi and diffuse the proteins that are in the vesicle into the Golgi

• when the vesicles buds, the orientation of all of the stuff stays the same— the outer leaflet of the membrane is still on the outside and the inner leaflet is still on the inside, transmembrane proteins stay in the same orientation as well

• Any soluble proteins that are not in the membrane can be brought along in the inside of the vesicle

• When the vesicle fuses with the target compartment, everything stays in the same orientation

  • the outer leaflet of the vesicle fuses with the outer leaflet of the target compartment (the Golgi)

Protein sorting and vesicle transport pathways

• All proteins begin in cytoplasm

• can go to the nucleus, mitochondria, or ER

Sorting methods

Gated transport, transmembrane, vesicle transport

Gated Transport

• Transporting from the cytosol to the nucleus (it is possible to go from nucleus to cytosol as well)

• The proteins that are being transported are always folded into 3D structures

Transmembrane sorting

• Cytosol to mitochondria

  • proteins are UNfolded, post translational— even though its been fully translated, it’s not going to fold up into its 3D structure

• Cytosol to ER

  • Co-translational— not folded because it is in the process of being translated as it is moved

  • ribosome in the cytoplasm translates and ejects it into the ER at the same time

Vesicle transport

• Proteins are moved around in vesicles between different compartments (like ER to golgi budding process)

Sorting mechanism

• Sorting sequence (SS)— 15-60 amino acids (consecutive/all in a row)— amino acids in the primary amino acid sequence of the protein, the information of where to send the protein is in the amino acid sequence of the protein itself

  • can work for nucleus, er or mitochondria

• Sorting patch (SP)— non-consecutive amino acids— spread out a little bit in the protein sequence, the primary sequence folds up into the 3D structure and these proteins come together to form a small patch in the 3D structure.

  • sorting patch can only work for cytosol to nucleus transportation because it has to be folded

• Signal sequence receptors and sorting patch receptors

  • proteins that read the sequences and understand where the proteins go

What is the feature of amino acid sequence that means the protein will be sorted into the nucleus?

It will have a stretch of basic or positively charged amino acids

LYSINE AND ARGININE

What is the feature of amino acid sequences that means the protein will be sorted into the mitochondria?

Has a bunch of basic positively charged amino acids (lysine and arginine) but is different from import to nucleus because they are spaced out and have hydrophobic residues between them (not lysine and arginine)

What is the feature of amino acid sequence that means the protein will be sorted into the ER?

ALL hydrophobic

NO CHARGED

Cytosol to nucleus transportation

• Gated transport

• protein folded into 3D shape

• could have simple sorting sequence or could have patch

• Nuclear pore complex is how proteins get in and out

Nuclear pore complex features

• proteins in the nuclear pore are nucleoporins

  • have little patches on them called FG repeats

• Gateway for the gated mechanism

• The center of the nuclear pore complex forms an aqueous channel

  • very narrow— almost no proteins can move through just by diffusion

• The center is full of proteins

Protein import to the nuclear pore complex

• NLS— nuclear localization signal (the sorting signal/patch)

  • Most often positively charged (lysine and arginine)

• NIR— nuclear import receptor

  • binds to the sequence on the protein

  • has 3 binding sites:

    • NLS (of the cargo protein—protein that’s gonna be imported)

    • FG repeats of nucleoporins

    • Ran GTPase (binds GTP)

Protein export from the nuclear pore complex

• NES— nuclear export signal

  • different from NLS

  • basically same process as import

• NER— nuclear export receptor

GTPases

• protein family

• all binds and hydrolyze GTP

• see drawing 2/19

Ran GTPase

• GAP is in the cytosol

• GEF is in the nucleus

• Ran is pretty much always bound to GDP because the Ran gap is in the cytoplasm so there is a high concentration of Ran bound to GDP

• Then it runs into the GEF which causes it to drop its GDP and then immediately bind to GTP so now there is a high concentration of ran GTP in the nucleus

• Because the ran GEF is in the nucleus, all of the ran will have gtp bound

• Then the ran gtp wants to go outside to follow its concentration gradient

• goes into the nucleus if bound to GDP, goes out of the nucleus if bound to GTP— constant cycle that drives nuclear import and export

Mechanism of nuclear import

• NLS of Cargo protein binds to the receptor to form a dimer

• then the import receptor binds to FG repeats on the nuclearporins

• then it worms its way down through the nuclear pore by consecutivelt binding to FG repeats

• emerges into the nucleus as a dimer between receptor and cargo

• when the dimer gets into the nucleus, RanGTP (high concentration in the nucleus) binds to the import receptor and the cargo protein falls off because the import receptor is not capable of binding to both the ran GTP and the NLS of the cargo at the same time (mutually exclusive binding)

• the purpose of this is that the receptor has now delivered the cargo sp ot can now go do its job

• now the Ran GTP gradient comes in

• we have a dimer between ran GTP and receptor

• the concentration gradient of ran GTP drived the import receptor back through the nuclear pore and into the cytoplasm (uses FG repeats again but is driven by concentration gradient)

• The RanGTP is now going to run into the Ran GAP which is in the cytoplasm which then hydrolyzes the GTP into GDP

• Ran associated with GDP cannot bind to the receptor anymore— can ONLY bind when GTP is attached

• As soon as it runs into the GAP, the Ran comes off of the receptor so it is now released and can go bind another protein

Mechanism of nuclear export

• starts inside the nucleus

• the nuclear export receptor is different from the nuclear import receptor

  • WANTS to bind to both RanGTP and the cargo at the same time to form a trimer—cooperative binding

• wants to go out of the nucleus because of the concentration gradient (high concentration of RanGTP inside the nucleus)

• trimer runs into GAP and GTP is hydrolyzed

• now that Ran is not binding to the receptor anymore the cargo comes off too because it wants to be a trimer

• RanGTP is constantly moving the exporters out so the exporters are more concentrated on the outside so they want to xome back in

• the export receptor interacts with the FG repeats in the same way that the import receptor does

Proteins that go from the cytosol to the mitochondria

• very small amount of proteins in the mitochondria are synthesized there since the mitochondria has its own small genome

• the vast majority of proteins in the mitochondria come from the cytosol

• proteins to get in must have a signal sequence (SS) that is an amphipathic alpha helix

  • remember that the amino acid structure will be partially charged and partially hydrophobic— arginine and lysine with other residues between them

• the SS binds to a receptor

• the protein is unfolded when it goes to the mitochondria— not in its 3D shape post-translationally

• the polypeptide stays unfolded because of proteins called cytosolic hsp70

Machinery for import from the cytosol to the mitochondria

• signal sequence at the end terminus of the protein— amphipathic alpha helix, cleaved off after it gets imported into the mitochondrial matrix by signal peptidase

• Translocators: a protein that moves a whole polypeptide chain across a membrane

  • TOM—translocator outer membrane

  • TIM— translocator inner membrane

Mechanism for import from the cytosol to the mitochondria

• cytosolic hsp70 bind along the length of the polypeptide chain that is going to be imported and stops it from being folded

• SS binds to TOM on the outer membrane

• SS gets transferred from the TOM to the transportation channel

  • as the chain gets through the channel, the cytosolic hsp70s are removed otherwise the chain would not be able to fit through (ATP driven)

• Hooks up with TIM

• coordinated threading of the chain through both TOM and TIM

• gets put into the mitochondria, can fold up, signal sequence is cut off

• Process uses a lot of energy

Where is energy used in the mechanism for import from cytosol to mitochondria?

• ATP is used to strip off cytosolic hsp70

• Membrane potential of the inner membrane— pulls the protein through TIM

  • inner membrane is a lot more positively charged on the outside and negative inside— this attracts the SS through because the SS is positive and the inside is negative

• ATP— Import ATPase pulls the protein into the matrix

  • grabs hold of the polypeptide chain and pulls it through

Transport from cytosol to the endoplasmic reticulum

• ribosomes cotranslationally import proteins into the ER

Key players of cytosol to the ER transport

• cotranslational (once a protein gets into the ER it cannot come out to the cytosol again—now it is in vesicle transport)

• Signal sequence—start transfer sequence (SRP signal sequence)

  • composed of at least 8 hydrophobic residues (amino acids)

• signal recognition particle (SRP)— acts as the receptor for SRP signal sequence

  • has three binding sites

    • SRP signal sequence binding site

    • ribosome binding site

    • SRP receptor— in the ER membrane

• Translocation channel—translocator

SRP binding sites

1. SRP signal sequence binding site

2. ribosome binding site

3. SRP receptor in the ER membrane

How does SRP work?

• SRP binds signal sequence then wraps around the ribosome

• SRP binds to ribosome and stops the translation of the protein that the ribosome is making

• Ribsome SRP complex gets located to the ER

• The thirs binding site is for the receptor which has a binding site for SRP

• after it binsd to the receptor then the whole ribosome gets moved over onto a translocator channel

• receptor and SRP get released and the SRP can find another ribosome

What happens after the ribosome reaches the translocator channel?

• The signal sequence gets stuck inside the translocator

• Its N-terminus is facing towards the cytoplasm

• Translation resumes and the protein gets pushed through the translocator channel

• signal peptidase inside the ER lumen can cut off the signal sequence that is stuck in the channel so that the protein can be delivered into the ER lumen and can fold up into its 3D shape

Soluble ER lumen protein

N terminus…start transfer sequence…X(cleavage site)…C terminus

• SS is 8 hydrophobic amino acids

Single pass membrane proteins

• alpha helix that spans the membrane

• hydrophobic and at least 20 amino acids

• integral membrane protein

• N terminus………………STS………………CT

  • No cleavage site

  • The STS has to be at least 20 hydrophobic residues

  • SRP binds to it but it’s also the alpha helix that spans the membrane

  • N terminus in the cytoplasm

• N terminus………STS…X…….….StopTS…….CT

  • STS only has to be 8 amino acids

  • StopTS= stop transfer signal— greater than or equal to 20 hydrophobic amino aicds so this is the part that spans the membrane

  • X= cleavage site

  • N terminus in the ER lumen

Two pass membrane protein

NT………STS……….StopTS……CT

• two alpha helices span the membrane

• STS is at least 20 hydrophobic residues because it is one of the alpha helices

• StopTS at least 20 hydrophobic because it is also going to be an alpha helix spanning the membrane

• No cleavage site

• N terminus and the C terminus are in the membrane

Single pass ER membrane protein (no StopTS) insertion

• The SS gets stuck in the translocator and the most N-terminal part of it is facing the cytoplasm at the top

• The ribosome pushes the protein through as it gets translated and you end up with the C terminus of the protein in the lumen of the ER

• the alpha helix spans the membrane

• ALWAYS ORIENTS WITH THE N TERMINUS IN THE CYTOPLASM AND THE C TERMINUS IN THE LUMEN

Single pass ER membrane protein (with StopTS) insertion

• The STS and the StopTS are stuck in the translocator

• The ribosome is stull there translating the protein and the ribosome detaches and is floating around and finishes making the protein

• Cleaved at the cleavage site and the protein is released into the lumen

• N terminus in the lumen and C terminus in the cytosol

• opposite to other single pass orientation

Multi-pass ER membrane protein insertion (2 pass)

• STS and StopTS get stuck in translocator

• N terminus and the C terminus in the cytoplasm and a domain between the transfer signals is in the ER lumen

Protein glycosylation

• sugars are covalently attached to a protein in the ER lumen

• oligosaccharide attached covalently to a protein makes it a glycoprotein

• a lipid in the ER membrane—dolichol—ends up with oligosaccharide on it

• In the ER lumen there is a protein called oligosaccharyl transferase that adds the sugars to the proteins

  • adds the sugar onto asparagine (aka Asn aka N)— N-linked glycosylation

• Branched sugar chain ends up in the ER lumen and the protein that attaches sugar is also in the lumen

• Protein is getting synthesized by a ribosome and it has an Asn on it that gets recognized by the enzyme to put the sugar on

• Takes the branched sugar molecule off the lipid and puts it onto the residue

• Branched sugars can give a protein a new signal to be sorted to somewhere else

GPI anchor covalent attachment to ER proteins

• a way a protein can be a peripheral membrane protein

• GPI anchors also get attached to proteins in the ER lumen

Where does membrane lipid synthesis occur?

In the ER membrane

endocytosis

• things coming into the cell

• these vesicles are going to end up at a lysosome which is where complex molecules can be broken down into simpler molecules that the cell can use

exocytosis

• things coming out of the cell

• ER makes a vesicle, goes to the Golgi, then the vesicle buds off again and from there two things can happen

  • vesicles can go to the plasma membrane to be secreted

  • the vesicle could go to the lysosome

Retrieval pathways

• membrane balance: vesicles can come from the golgi and go back to the ER— take some membrane and some protein back so that when the vesicles are made the proteins and membrane aren’t depleted

• Returning missorted proteins— some of these proteins should not have left the ER/ended up in the golgi by mistake

• recycling specific proteins— snares, receptors

Vesicle coats

• coats are a bunch of different proteins on the outside of a vesicle, form a shell around the membrane

• Particular types of coated vesicles are only made in certain locations

  • Vesicles with COPII are only make in the smooth ER so these vesicles are all going to the Golgi

  • Clathrin coats are involved in endocytosis so they form at the plasma membrane surface and bring nutrients into the cell

  • COPI coats are vesicles for retrieval pathway, formed at the surface of the Golgi and sent back to the ER

Primary function of vesicle coats

• Drive vesicle formation

• Target vesicle to target membrane

Clathrin

• made up of triskelion proteins

• triskelions come together and form a cage-like structure

• involved in endocytosis

• form at the PM surface and bring nutrients into the cell

Vesicle formation/coat assembly: coat complex

• general mechanism applies to all of the coats, but clathrin is used as an example (different coats use different proteins and are made in different locations)

• coat complex

  • cargo molecule binds to cargo receptor

  • receptor binds inside the cell to adaptor protein

  • adaptor protein binds to clathrin triskelion (outer coat protein)

Coat recruitment GTPase function

• coat recruitment GTPase clusters coat complexes

• when these cluster together it starts to bend the membrane and starts to create the vesicle

  • EX: COPII (Sar1)

    • Sar1 is a GTPase

    • the Sar1 originally is bound to GDP with an amphipathic alpha-helix hidden in the 3D structure of the protein

    • the Sar1 GEF (integral membrane protein) induces the GDP to be released

    • The Sar1 is then bound to GTP and the alpha helic is inserted in the cytosolic leaflet

  • Once it is in the GTP form, it interacts with adaptor proteins to cluster the coat complexes

vesicle release

• Dynamin (protein)

  • vesicle release

  • protein wraps around and squeezes the membrane to physically detach the vesicle

• Uncoating

  • coat proteins come off (some targeting proteins are left)

Vesicle targeting (SNARE and RabGTPase)

• SNARE

  • Targeting specificity— another protein in the membrane of a vesicle that targets the vesicle to a specific compartment

  • drives membrane fusion

  • Two types of snares: V-snare (vesicle) and T-snare (target (in the target membrane))

  • v snare on the vesicle binds to the t snare in the target membrane

• RabGTPase

  • targeting specificity

  • docks the snares together (snare needs rab to initiate the contact of the snares)

  • has a hidden lipid anchor that gets exposed

    • Rab-GEF is also in the ER membrane

      • It exposes Rab’s lipid anchor, leading to ER membrane insertion

  • Rab effector protein in the target membrane

  • Rab GAP in the target membrane (ex: Golgi)

    • releases Rab from target membrane

Rab and SNARE mechanisms

• Rab GEF creates Rab GTP, extends its lipid tail

• the Rab bound to GTP binds to rab effector

• the rab effector changes conformation and pulls the vesicle down so that the v snare can interact with the T snare

• Fuses membranes

• Rab is released

• The v snare has to go back to the ER for membrane balance, receptor proteins have to go back to the ER, goes through retrieval pathway

  • for this to happen in the golgi membrane we have the Rab GAP which induces Rab back to RabGDP and and hides teh anchor and the rab is released from the membrane and back to the ER

• NSF is an ATPase and it dissociates the V and T snares

  • uses ATP to separate the V and T snares and the V snare goes back to the ER through the retrieval pathway

ER → Golgi Transport

• The default pathway if you are not a resident protein is to get into a COPII vesicle

  • These proteins may have special signals (exit signals) that will specifically bind in the vesicle but most commonly the proteins just end up in the vesicle by default

• proteins leave the ER to function elsewhere

• default for soluble and membrane ER proteins: protein leaves the ER and goes to the Golgi (if there is no ER retention signal/retrieval signal)

• Once they reach the golgi they either go to the plasma membrane (to be a membrane protein or to be secreted) or to lysosomes

Resident ER proteins

proteins that stay in the ER and function there

tubular clusters

• vesicles form together to create tubular clusters and move across a microtubule that runs from the ER to the golgi

• The vesicular tubular clusters are made of COPII vesicles and are attached to the microtubules by motor protein (motor protein binds to the vesicle and the MT and they can move along the MTs and take the whole vesicle system over to the golgi)

Retrieval pathway (Golgi → ER)

• vesicles that form at the Golgi surface with COPI coats go back to the ER

  • Take things back to balance things out: membrane, v snares, receptors, resident ER proteins that were missorted to the Golgi by mistake

• transmembrane resident ER protein has a KKxx signal at the C terminus (2 lysines and 2 other amino acids) and has a COPI coat on it?

• KDEL receptor is also a transmembrane protein where soluble ER resident proteins with KDEL amino acid sequence binds

• the KDEL sequence (K for Lys, D for Asp, E for Glu, L for Leu (3/4 are charged)) tells the protein to return to the ER

• in the lumen of the Golgi, the pH is slightly acidic so the resident ER protein and the KDEL signal combine to the KDEL receptor and get into the vesicle and then when this vesicle fuses with the ER membrane where the pH is higher, it can no longer bind to the receptor and it falls off/is delivered back to the lumen with the neutral pH

What happens to proteins delivered to the Golgi?

• Golgi is like a stack of pancakes all connected to each other but like a stack of membrane structures

• Golgi has two faces: the Cis face and the trans face (the cis towards the ER)

• Vesicles bud from the first layer then to the next layer and so on

• While the proteins move through the layers they get different modifications

• Once proteins get to the other side of the golgi (trans face that faces the plasma membrane— where vesicles bud off and either go to PM or to lysosome)

Simple functions of Golgi

• Protein sorting

  • should the protein be sent to the PM or to a lysosome

• Protein modification

  • N-linked sugars are further modified in the Golgi

    • (e.g. for lysosome targeting)

    • Added onto asparagines in the ER

  • O-linked glycosylation

    • (e.g. sugars called GAGs get attached in a different way from the N-linked sugars, when a GAG gets attached to a protein it makes the protein a proetoglycan)

    • Happening on serines and threonines in the Golgi

Lysosome Function

• Acid hydrolases only function at really low pH

• Inside the lysosome there is a big pH difference from the rest of the cell

• The acid hydrolases cannot be active until they are in the lysosome because the pH is higher in other places

• You don’t want these enzymes to be active anywhere else in the cell because they would start to degrade things

• Things that get released from the lysosome are much simpler to be reused by the cell

• Synthesis begins in the cytoplasm then the ribosome synthesizing the hydrolase goes to the ER and is a soluble protein in the lumen then COPII to the golgi then golgi to lysosome

Golgi → Lysosome