biology exam number 2

dynamin

(a large GTPase that assembles into helical structures) assembles at the neck of the clathrin coated vesicle and is required to pinch off the vesicle from the plasma membrane

How do proteins travel through the golgi?

- 2 models - vesicle transport and cisternal maturation model, and the cisternal maturation model is correct

Nonselective autophagy

- A bulk portion of the cytoplasm is sequestered in the autophagosomes - occurs during starvation conditions -- when external nutrients are limiting, metabolites derived from the digestion of the captured cytosol can help the cell survive. Begins to degrade parts of itself to survive

Selective autophagy

- A selective cargo is packed into autophagosomes that tend to contain little cytosol - Mediates the degradation of worn out, damaged, or otherwise unwanted mitochondria, peroxisomes, ribosomes, and ER - The selective autophagy of mitochondria - mitophagy... marked for degradation and it initiates the formation of the autophagosomes

How do vesicles recognize the right target compartment?

- A sequential targeting system is used to increase the probability of a vesicle fusing with the correct target □ 1st targeting system: specific Rab proteins on the surface of each type of vesicle are recognized by corresponding tethering proteins on the cytosolic surface oft eh target membrane □ 2nd targeting system: SNAREs on the vesicles (v-SNAREs) interact with complementary SNAREs on the target membrane (t-SNAREs) ® Bring the vesicle closer to the target membrane and dock the vesicle at the membrane surface ® Each transport vesicle target membrane pair has unique rab and v/t pairs -- golgi is found only in golgi for both rab and t/v snare

How are signals sensed in cell signaling?

- After a signal is produced by a signaling cell, it then needs to be sensed by the target cell. once the signal Is sensed, the signal needs to be transmitted from the outside of the cell to the inside of the cell, as well as amplified to the cell • This results in some sort of action • Receptor proteins sense the signal, transducer proteins transmit the signal, this results in the activation of an effector protein which them performs an action

autophagy

- Another pathway for materials to end up in the lysosome ◊ Components of he cytosol being enveloped in autophagosome and trafficked to the lysosome ◊ Formation of an auphagosomal membrane is induced by a particular signal sequence - Grows by the fusion of vesicles with the membrane and fully encapsulates the material with a double membrane - Sequester portions of the cytoplasm so they can engulf the cytosol as well as organelles -- fuses with a lysosome, the hydrolases break down the contents and ae released into the cytosol for the cell to use - Can contain cytosol and organelles Can be nonselective of selective

How do vesicles bud?

- Assembly of coat proteins on the surface helps to drive the budding process - Coat assembles on the outer membrane and completely surrounds the fully formed vesicle that is pinched off the membrane □ Inside vesicle is protein cargo □ Different types of coats (clathrin, COP1 and COP2)

How do dynamin and other proteins function to help pinch the vesicle off the plasma membrane?

- Because Dynamin is a GTPase that assembles into helix structures, GTP by hydrolysis of dynamin drives the conformational changes in the dynamin helix that constrict the membrane neck - Hydrolyzing GTP to GDP causes a conformational change in the helix that causes it to constrict. The other proteins function to help pinch the vesicle off the plasma membrane even further by associating with the neck and driving further constriction ◊ She talks about dynamin mutants for a while which cannot hydrolyze GTP and therefore cannot pinch off the vesicle even though they can bind GTP and does not go into a confirmational change in a helix

Binding of signals to receptors (often in complex combinations) can alter

- Cell shape, movement, metabolism, gene expression, proliferation, survival, differentiation

How does a vesicle fuse with the target compartment?

- In addition to aiding the docking of a vesicle to the correct target membrane, SNARE proteins catalyze the fusion of the donor vesicle with the target membrane - Snare proteins undergo a dramatic confirmation change that draws the two lipid bilayers into close opposition -- 2 membranes are brought so close (within 1.5 nm) that any water that remains trapped is squeezed away, which allows the lipids to flow together to form a continuous bilayer

3 main classes into which surface receptors fall

- Ion channel coupled receptors - G protein coupled receptors (GPCRs) - enzyme coupled receptors

Endocrine signaling

- Long distance ▪ The most public type of signaling ▪ Long distance signaling that is widely broadcast ▪ Hormone: extracellular signal that is secreted and transported via the bloodstream to target tissues on which it exerts a specific effect • Hormones are produced in endocrine glands and are secreted into the bloodstream

How are the signals transmitted from one protein to the next?

- Many proteins act as molecular switches, which means these proteins can be activated or inhibited - In one class of protein that acts as a molecular switch, the activity of the protein is regular by the covalent attachment of a phosphate group by a protein kinase and the removal of a phosphate group by a protein phosphatase ▪ Phosphorylation can turn some proteins on and others off - Monomeric GTPases are on when bound to GTP and off when bound to GDP ○ The switch between their two forms with the help of GAPs and GEFs

Membrane topology

- Membrane starting to bud from a membrane enclosed organelle such as the ER or Golgi - Outer leaflet: monolayer facing the outside of the organelle (dark orange) - Inner leaflet: monolayer facing the inside/lumin (light orange) □ The leaflet on the inside of the golgi and vesicles is on the outside of the cell in the extracellular fluid (see diagram) ® Another way to think about it is that the leaflet that faces the cytoplasm in the ER or Golgi faces the cytoplasm in the plasma membrane

Ion channel coupled receptors

- Open in response to binding and extracellular signaling molecule ▪ Once the signal molecule binds, the channel opens and ions of the correct size and charge then pass through the channel - the ions move with the concentration gradient ( move from high to low concentration) ▪ Can alter the potential of the plasma membrane - if conditions are right, this can result in an electrical current that can be used such as in neural signaling

Signaling by phosphorylation (molecular switch)

- Protein can be phosphorylated, meaning a phosphate is covalently added to the proteins - Phosphorylation state of a protein is regulated by protein kinases and proteins phosphatases.. Signal is received that causes the kinase to phosphorylate the blue protein, which activates the protein and allows the protein to relay the signal onward, and it will continue to do this until the phosphate is removed by a phosphatase, which turns the protein off - note: phosphorylation can turn some proteins on as shown in the picture, and others off

Possible fates of a receptor protein following endocytosis

- Recycled back into the plasma membrane - Trafficked to and degraded in the lysosome - Trafficked to a different domain of the plasma membrane -- a process called transcytosis (Especially when the cell is not symmetrical) - Fate of a receptor can be changed because receptors can be modified (oxidative phosphorylation) and a different mark can be recognized in a different way.. Reduces the number of receptors on the cells surface

How do cells communicate?

- Signal transduction is the process by which one type of signal is converted into another. when a target cell receives an extracellular signal, it converts that signal into an intracellular signal, which leads to some sort of response by the cell - Cells use extracellular signal molecules to communicate with one another in various ways (endocrine, paracrine, neuronal, and contact dependent)

How is cargo selected for vesicular transport?

- Similar to signal sequences, proteins for vesicular transport have exit sequences/signals that associate with cargo receptors that also have ER exit signals □ Commonly an amino acid sequence on the protein □ Both soluble and membrane bound cargo proteins have exit signals □ Membrane bound proteins with exit signals are either... ® Destined to reside in the membrane of another organelle in the endomembrane system ® Act as cargo receptors for soluble proteins and are recycled back to the ER ® The er exit signal on the cargo receptor gets recognized by the inner coat protein which brings the receptor and its bound soluble cargo into the forming vesicle ® Sometimes, a protein that is supposed to be in the ER (ER-resident) gets trapped in a vesicle and shipped off with the rest of the cargo

which mechanisms are required for traffic proteins into the golgi and beyond?

- The ER serves as a gateway for all proteins that are trafficked through the endomembrane system ○ So, both mechanisms for 2 and 3 are required for proteins trafficked into the golgi and beyond - mechanism 2: Moved by protein translocators generally as an unfolded polypeptide chain - mechanism 3: vesicular transport

How does a signal get translated into action?

- The length of time it takes a cell to respond to a signal depends on what needs to happen once the message is received • Some responses involve changes in gene expression and the synthesis of new proteins --- for new proteins to be synthesized, the gene must first be transcribed into Messenger RNA and the Messenger RNA is then exported into the cytosol and translated into protein by ribosomes - therefore, the responses that require regulation of gene expression and the synthesis of new proteins are therefore often relatively slow • Other responses involve altering the activity of proteins that are already synthesized, the activity of proteins that already exist in the cell tend to switch on or off - This is a faster process and occurs more quickly than that before

How does the neutrophil 'sense' the bacterium?

- The small clump of bacteria releases a chemoattractant that is sensed by the neutrophil... becomes polarized and starts chasing the bacteria. it moves around in a random path as it is bounced around by thermal energy - the neutrophil catches up with the bacteria and engulfs them by phagocytosis • Require cell signaling pathways that allow the neutrophil to sense the bacterium and modify its behavior in response to the cues it is getting from the bacterium

Vesicular transport

- The third mechanism by which membrane enclosed organelles import proteins - A process in which transport vesicles bud off one membrane and fuse with another. The vesicles carry membrane components and soluble proteins between the compartments of the endomembrane system and the plasma membrane' ○ The extra cellular space and each of the membrane enclosed compartments in the ER, Golgi, lysosome, endosomes, communicate with one another by means of transport vesicles. These membranes are collectively known as the endomembrane system ○ Carry soluble proteins, membrane proteins, and lipids between compartments - Vesicles that bud off the ER are trafficked to the golgi, bud off the plasma membrane are trafficked to the endosomes -- even if ER vesicles fuse to the golgi, the golgi stays the golgi and does not become more like the ER over time because it fuses (maintains its identity)

enzyme coupled receptors

- When an enzyme coupled receptor binds its extracellular signal molecule, an enzyme activity is switched on - Enzyme coupled receptors can either act as enzymes or associate with enzymes in the cell ▪ when stimulated, these enzymes can activate a wide variety of intracellular signaling pathways - Largest class of enzyme coupled receptors are the receptor tyrosine kinases (RTKs) ▪ The cytosolic domain of RTK's functions as a tyrosine kinase, so it covalently attaches a phosphate group to the amino acid tyrosine on a protein • Activation of RTKs ○ Binding of a signal molecule causes 2 RTK's to associate into a dimer ○ dimerization brings the kinase domain of 1 receptor in contact with the other ○ The kinase domain of 1 receptor phosphorylates the kinase domain of the other and vice versa ○ Each phosphorylated tyrosine serves as a docking site for a different intracellular signaling protein, which helps to relay the signal • MOST RECEPTOR TYROSINE KINASES ACTIVATE THE MONOMERIC GTP

4 different types of cell to cell communication

- endocrine, paracrine, neuronal, and contact dependent

cholesterol uptake by cells

- example of receptor mediated endocytosis ® To understand cholesterol uptake, we need to talk about low-density lipoproteins (LDLs) ◊ Cholesterol is transported in the bloodstream in the form of lipid protein particles known as LDLs - This is because cholesterol is extremely insoluble in H2O bc of hydrophobic cells ◊ Each LDL contains ~1500 cholesterol molecules ◊ LDLs are secreted by the liver ◊ When a cell needs cholesterol for membrane synthesis, it expresses an LDL receptor that gets inserted into the plasma membrane via exocytosis - LDLs then enter the cell via receptor mediated endocytosis

Once G protein signaling is activated, how is signaling then deactivated?

- for G proteins, the Alpha subunit has an intrinsic GTPase activity, and it eventually hydrolyzes bound GTP to GDP ▪ GTP hydrolysis usually occurs within seconds of activation ▪ following GTP hydrolysis, the Alpha subunit reassociates with the beta gamma complex to reform the inactive G protein • no GAP is needed in this process, which is unlike the hydrolysis of monomeric G proteins

BIG questions of cell signaling

- how do cells communicate? - how are signals sensed? - how does a signal get translated into action?

Neuronal signaling

- long distance through networks of neurons but not broadcast widely ▪ Electrical signals/impulses travel down the axon of the neuron and are converted to chemical form at the nerve terminal and released into the adjacent target cells receptors

contact dependent signaling

- most intimate, short range ▪ does Not require the release of a secreted molecule ▪ Cell surface bound signaling molecule binds to receptor on an adjacent cell, therefore, there is direct physical contact between the signaling molecules that is associated with the plasma membrane and the receptor protein that is embedded in the membrane of the target cell

A neutrophil phagocytosing a bacterium

- neutrophil is a type of white blood cell that uses phagocytosis to defend against infection by ingesting invading microorganisms ® Antibodies bound to the bacterium interact with specific receptors on the surface of he neutrophil. this induces the neutrophil to extend sheet like projections of the plasma membrane called pseudopods , that engulf the bacterium and fuse at their tips to form a phagosome ® The phagosome then fuses with the lysosome and leads to degradation of the bacteria

○ 3 types of endocytosis

- pinocytosis (cellular drinking) - receptor mediated endocytosis (specific form of pinocytosis) - phagocytosis (cellular eating)

signaling by GTP binding (molecular switch)

- small monomeric GTPases can also act as switches - we should be familiar with this and confirmation changes for the midterm! GDP is off, GTP is on in a signaling pathway

What happens when the COP 2 vesicle going to the golgi is released from the ER?

- the vesicle must uncoat, and the uncoating of COP 2 vesicles involves the GTPase SAR1 (initiates the formation of COP 2 encoded vesicles when it binds to GTP and associates with the ER membrane) ▪ At some point after the vesicle is released, SAR1 hydrolyzes its GTP to GDP, causing the coat to disassemble. Once the coat disassembles the RAB protein in the V-snare that were packaged into the vesicle are now exposed and can interact with the appropriate t-SNARE and tether protein on the target membrane, which in this case is the golgi

How does the cargo receptor bind to cargo in the ER but release cargo in the golgi?

- this has to do with PH differences between various organelles! • PH of the golgi is lower than the PH of the ER ○ Changes in PH drive changes in the confirmation of the cargo receptor ○ At the PH of the ER, the cargo receptor adopts a specific confirmation that allows it to bind cargo, however, once the cargo receptor hits the lower PH of the golgi, the receptor's confirmation has changes such that it can no longer bind its cargo and releases its cargo into the golgi lumen - Mitochondria have the highest PH, lysosomes have the lowest

clathrin-coated vesicles

-Clathrin forms a basket like network on the cytosolic surface of the membrane ® Made up of individual subunits called clathrin triskelion that assemble into basket like cages ® Forms on the surface of the membrane facing the cytosol and buds inward towards the cytosol

How does sorting of receptors and LDLs occur?

2 domains - vacuolar domain - Very large lumin - Tubular domain - Very small lumin which excludes larger soluble molecules like LDL from entering the tubular domain

In what cases are COPI and COPII coats used?

COP1 is from the golgi to the ER (retrieval pathway), COP2 is from the ER to the golgi

Paracrine signaling

Local and less public ▪ A cell produces and excretes a signal and that signaling molecule diffuses locally through the extracellular fluid, thus the signaling molecule acts as a local mediator, acting only on nearby cells ▪ Sometimes can be autocrine! - Autocrine signaling: when a cell responds to a signal it produces

Positive feedback loop

a component that lies downstream in the pathway act on an earlier (upstream) component in the same pathway to enhance the response to the initial signal

Negative feedback loop

a component that lies downstream in the pathway acts to inhibit an earlier (upstream) component in the same pathway to diminish the response to the initial signal

Phosphatase

a protein that removes the phosphatase from a protein

2 ways to regulate signaling

from the side of the cell and the side of the receptor - Cell: is the signal present? If not, there will be no response, even if the receptor is present - Receptor: is the receptor present? If not, there will be no response, even if the signal is present

Acid hydrolases

hydrolases that work best at an acidic pH

Many macromolecules that get endocytosed end up....

in lysosomes

SAR1 and COPII mediated ER to golgi vesicle traffic

monomeric GTPase that is responsible for the assembly of COPII coats at the ER membrane ◊ Activity of SAR1 is spatially regulated by an ER associated Sar-GEF --- can exist in gtp or gdp bound form! - Sar1-gef is an integral membrane protein, so its domain is restricted to the ER ◊ When SAr1 is in its GDP bound form, its soluble and in the cytosol. When it comes near the surface of the ER, it interacts with SAR1GEF which promotes the SAR1 to exchange GDP for GTP -- converting to GTP bound form - Sar1 changes its confirmation such that an amphiphilic helix is exposed which anchors SAR1 into the ER membrane... helix is hidden in the GDP bound form of Sar1 - Sar1 can only be activated near the surface of the ER, which means the anchor is only exposed near the surface of the ER ◊ When it is anchored into the membrane, Sar1-GTP recruits COPII adaptor proteins that function to... - Select cargo proteins to be packaged - initiate deformation of the ER membrane - Recruit outer coat proteins which help to form a bud

Cisternal maturation model

one model for which proteins travel through the golgi - Golgi cisternae are dynamic structures that mature from early to late by acquiring and then losing specific Golgi resident proteins - Proteins stay in the cisterna, and takes on molecules and lose other molecules that allow that cisternae to transition from one to the next (cis--medial---trans)

Vesicle transport model

one model for which proteins travel through the golgi - Golgi cisternae are long lived structures that retain their characteristic set of Golgi resident proteins firmly in place, and cargo proteins are transported from one cisternae to the next by transport vesicles - Cisternae are stable and stay the same

Kinases

proteins that covalently attach a phosphate group to a protein

Adaptins (adaptor proteins)

proteins that help select cargo molecules in the plasma membrane for endocytosed transport and secure the clathrin coat to the cytosolic face of the budding membrane

2 classes of molecular switches

signaling by phosphorylation signaling by GTP binding

The fate of a receptor protein following its endocytosis depends on...

the type of receptor it is

Unfolded protein response (UPR)

when chaperones in the ER get overwhelmed, misfolded proteins can accumulate in the ER. when this happens, the misfolded proteins are recognized by several types of transmembrane sensor proteins in the ER membrane which activate the UPR. ○ When these sensor proteins bind to misfolded proteins, the sensor proteins get activated and their activation initiates the signaling cascade that results in the activation of transcription factors for transcription regulators that enter the nucleus and drive the expression of chaperone gene and other genes that increase the protein folding capacity of the ER ○ The can result in the activation of a protein inhibitor which halts the translation of all proteins except those that increase the folding capacity of the ER, giving the ER some time to catch up on getting all of its proteins folded

a macrophage phagocytosing two damaged red blood cells

® Phagocytic cells, such as macrophages, play an important role In scavenging dead and damaged cells and cell debris ® Macrophages ingest more than 10^11 of your worn our blood cells each day

The basics of vesicle transport

® Transport vesicles bud off a donor compartment and fuse with a target compartment ® Vesicles carry cargo from the donor compartment to the target compartment ® Cargo can be soluble proteins, membrane proteins, and lipids

Untangling cell signaling pathways

• A signaling pathway involves three proteins: Ras ( a monomeric GTPase), protein X, and protein Y... how can you make an overactive ( always active) form of Ras? □ Could inhibit activity of Ras GAP, keeping it in its GTP bound state ® Could make a mutation that no longer allows Ras to interact with RAS gap □ Could make a mutation that no longer allows ras to hydrolyze GTP to GDP

intracellular signaling molecules can be recycled

• Cellular signaling protein served to integrate incoming signals - a way to fine tune a cell's response different extracellular signals can activate the same kinases and target proteins - Higher concentrations are more likely to interact with receptors than signaling molecules present at lower concentrations □ Net outcome depends on what signaling molecules are present and how much is present in the extracellular space

Intracellular signaling cascades use a combination of mechanisms to transmit the signal

• Here, there's' a combination of GTPases and phosphorylation • Here, when the protein is in its GTP bound form, its active, and when its active, it interacts with a green kinase and turns that kinase on... then this kinase phosphorylates the blue kinase, which is now active, then phosphorylates the pink kinase, which goes on to phosphorylate effector proteins which then go on to change the behavior of the cell

Terms to describe relative location in the pathway

• Upstream: against the flow of information • Downstream: with the flow of information

Phagocytosis (cellular eating)

□ A specialized form of endocytosis in which a cell uses large endocytic vesicles called phagosomes to ingest large particles such as bacteria and dead cells ® Carried out by specialized cells -- referred to as professional phagocytes ® The diameter of phagosome is determined by the size of its ingested particles, and those can almost be the size of the phagocytic cell itself ® In mammals, macrophages and neutrophils are capable of phagocytosis

Process of clathrin-mediated endocytosis

□ Adaptins (adaptor proteins) help select cargo molecules in the plasma membrane for transport and secure the clathrin coat to the cytosolic face of the budding membrane □ Clathrin itself plays no part in choosing specific molecules for transport... adaptins do this! ® As more clathrin is recruited by the adaptins, it forms a basket-like cage that helps to drive vesicle formation ® dynamin protein (a large GTPase that assembles into helical structures) assembles at the neck of the clathrin coated vesicle and is required to pinch off the vesicle from the plasma membrane ◊ Once this happens, the vesicle eventually uncoats in the cytosol and is able to fuse with a membrane ® Other membrane bending proteins are recruited to the neck of the vesicle to help it pinch off

Pinocytosis (cellular drinking)

□ Involves the ingestion of fluid molecules via small pinocytic vesicles (<150 nm in diameter) □ Occurs continuously and is indiscriminate -- any small molecules that happen to be present in the extracellular space get trapped and carried into the cell □ Rates vary by cell type ® Macrophages endocytose 3% of their plasma membrane per minute via pinocytosis ® Fibroblasts endocytose 1% of their plasma membrane per minute via pinocytosis □ About 2500 clathrin coated vesicles pinch off from the plasma membrane of a fibroblast every minute ® Despite a high rate of pinocytosis, the cell's total surface area remains unchanged

The lysosome

□ Membrane enclosed organelles filled with soluble hydrolytic enzymes that digest macromolecules (pH of 5) □ Contain around 40 different types of hydrolytic enzymes □ V-ATPase pumps in H+ to maintain acidic pH □ After macromolecules are digested, their building blocks (amino acids, sugars, nucleotides, etc...) are transferred to the cytosol via transporters and can be utilized by the cell or excreted

Receptor mediated endocytosis (specific form of pinocytosis)

□ Provides an efficient pathway to take up specific macromolecules from the extracellular fluid □ Specific macromolecules bind to complementary transmembrane receptor proteins which accumulate in coated pits, and then enter the cell as receptor-macromolecule complexes □ Provides a selective concentrating mechanism that increases the efficiency of internalization of specific macromolecules more than 1000 fold compared to ordinary pinocytosis - ex - cholesterol uptake by cells

Coat serves 2 main functions

□ The inner coat serves to concentrate cargo at the site of vesicle formation □ The outer coat, which is curved in structure, deforms the membrane and shapes the vesicle

The golgi apparatus (1897)

▪ A series of cisternae - flattened membrane-enclosed compartments • The face of the golgi onto which COP2 coated vesicles fuse is called the cis face, the other side is the trans face • Can be thought of as a sorting station ○ Receives proteins from the ER and then must sort the proteins into ones that stay in the golgi and ones that leave the golgi • Proteins are modified as they travel through the golgi ○ protein enters the cis golgi when the vesicle its in fuses and then it travels through to the medial golgi and the trans golgi, and it is modified along the way

What happens when a resident ER protein gets trapped inside of the vesicle for packaging?

▪ Escaped resident ER proteins can be returned to the ER -- COP1 is ER retrieval pathway • Resident ER proteins have an ER retrieval signal (KDEL) • KDEL (lysine, kaspartic acid, eutamic acid, lucene) = ER retrieval signal ○ The ER retrieval signal is recognized by the KDEL receptor in the golgi ○ The KDEL receptor with its bound resident ER protein gets incorporated into COP1 coated vesicles ▪ These vesicles bud off the golgi and fuse with the ER ▪ This is known as the ER retrieval pathway

How does the G protein coupled receptor then activate a heterotrimeric G protein?

▪ Proteins are trimeric. They have Alpha, beta, and gamma protein subunits ▪ in an unstimulated state, the receptor and G protein are both inactive ▪ binding of an extracellular signal to the receptor causes a conformational change of the receptor, which in turn alters the confirmation of the bound G protein • the confirmational change of the G protein allows it to exchange its GDP for GTP • when bound to GTP, the beta and gamma subunits dissociate from the Alpha subunit ○ the Alpha subunit and the beta gamma complex are now active and activate downstream components in the signaling pathway

What are the signals that get sensed in cell signaling?

▪ Signal molecules can be proteins, peptides, amino acids, nucleotides, steroids, fatty acid derivatives, or even dissolved gases - Whether a cell responds to that signal depends on whether or not they have a receptor for that signal. The receptor is usually activated by only one type of signal. Without the appropriate receptor, a cell will not be able to sense the signal and it will not respond to it

How does the process of receptor mediated endocytosis work (through LDLs)?

◊ LDLs bind to LDL receptors and is encoated in a clathrin coated vesicle ◊ Once this vesicle is formed, and is released from the plasma membrane, the vesicles lose their coat and fuse with endosomes ◊ In the acidic environment of the endosome, the LDL receptor dissociates from its receptor (another example of PH difference between endosome (6) and extracellular space (around 7)) - Receptor can bind LDL in the extracellular space but not in the endosome when the PH is lowered ◊ Receptor is returned to the plasma membrane via transport vesicles ◊ LDL is trafficked to he lysosome, where it is degraded to free cholesterol ◊ WHETHER AN LDL RECEPTOR IS OCCUPIED OR NOT, AN LDL RECEPTOR TYPICALLY MAKES ONE ROUND TRIP INTO THE CELL AND BCK TO THE PLASMA MEMBRANE EVERY 10 MINS

How do acid hydrolases get to the lysosome?

◊ Starts in ER -- Acid hydrolases are tagged with specific sugar groups in the ER and cisgolgi that allow them to be sorted at the transgolgi into transport vesicles that go on to fuse with endosomes - Had an ER signal sequence that causes the protein to be translocated into the ER... then folds correctly and is somewhat modified in the ER - Packaged into a cop2 vesicle and is trafficked to the golgi... further modified and moved to the transgolgi which sorts them for vesicles ◊ The tagged acid hydrolases are then trafficked from endosomes to lysosomes ◊ The acid hydrolase is only active once it reaches the lysosome because it works best at an acidic PH - If active everywhere, they would begin to degrade molecules in the ER and elsewhere in the system so it would cause massive damage to the cell - modifications to the cell allow it not to degrade other hydrolases

ER to golgi transport

○ Cop 2 vesicles used ▪ a soluble protein in the ER lumen that has an ER exit signal cannot interact directly with the inner coat proteins because they are on the opposite side of the membrane. Instead, the sizable proteins with ER exit signals can interact with cargo receptors • Cargo receptors are transmembrane proteins that span the membrane, they bind the exit signals on the soluble proteins in the ER lumen, and they themselves have exit signals that interact with the coat proteins for concentrated in the forming vesicle, bringing with them their soluble cargo. • The exit signals help to determine what exists in the ER in a COP2 coated vesicle ▪ Additional mechanism in the ER that helps to ensure the proteins that exit the ER are properly folded: chaperones bind and hold onto unfolded or misfolded proteins and do not allow them to be packaged into the forming vesicle • Chaperones are an ER exit quality control pathway

G protein coupled receptors (GPCRs)

○ Largest family of cell surface receptors, we have more than 700 in humans ▪ bind to a wide variety of extracellular signaling molecules, including hormones, neurotransmitters, and local mediators ▪ involved in an enormous variety of cellular processes, which makes them an attractive target for drug development • around one out of three drugs used today work on GCPRs ▪ all have a similar structure - pass through the membrane seven times (I.e. have 7 transmembrane domains) ○ Activate membrane bound, trimeric GTP binding proteins (G proteins) ▪ trimeric means a G protein is composed of three other proteins ▪ The G coupled protein binds to its extracellular signaling molecule, the activated receptor signals to a trimeric G protein on the cytosolic side of the plasma membrane. The activated G protein then activates the activity of an enzyme in the same membrane, that enzyme goes on to propagate the signal • activated G proteins then go on to activate a downstream component in the signaling cascade

exocytosis Regulated pathway

○ Mainly found in cells specialized for secreting products rapidly on demand (hormones, digestive enzymes, neurotransmitters) ○ Secretion usually occurs in response to an extracellular signal ▪ Example would be the release of insulin from a secretory vesicle of a pancreatic beta cell - Insulin is released in response to an increase in glucose levels in the blood - Insulin is stored in the secretory vesicles in a highly concentrated, aggregated form and stays there until an extracellular signal signals their release. After secretion, the insulin aggregates dissolve rapidly in the blood

What coat proteins function in the recycling of receptors back into the plasma membrane for receptor mediated endocytosis?

○ No coat proteins shown for the receptor recycling pathway because this is still a matter of debate - In reality, it seems that different cells use different coat proteins for this pathway... no consensus as to which one is the primary coat protein

exocytosis Constitutive pathway

○ Operates continuously and required in all cells ○ Supplies newly made lipids and proteins to the plasma membrane ○ Secretes proteins into the extracellular space

What we know about cell signaling receptors

○ Receptors are often found in the plasma membrane ▪ Cell surface receptors are proteins that span the plasma membrane. the extracellular portion of the protein binds the extracellular signaling molecule and the intracellular portion of the receptor protein transmits that signal into the inside of the cell • These are commonly used because many signals are too large or hydrophilic to cross the plasma membrane of the target cell • Instead, the signals bind to cell surface receptors which in turn generate one or more intracellular signaling molecules in the target cell ▪ There are some small, hydrophobic extracellular signaling molecules that can pass through the target cells' plasma membrane and bind to intracellular receptors in the cytosol or the nucleus • Once bound to the signal, the intracellular receptor then regulates gene transcription or other functions ○ The same signal can induce different responses in target cells ▪ For acetylcholine, binding to heart pacemaker cells results in decreased rate of firing, binding in salivary gland results in secretion of components of saliva, in muscle it causes contraction ○ Signal molecules can work in combination to regulate behavior of the cell

Endocytosis

○ The process by which cells take in materials through invagination of the plasma membrane. The material to be ingested is progressively enclosed by a small portion of the plasma membrane, which buds inward and pinches off to form an intracellular endocytic vesicle - Endocytosed cargo includes receptor-ligand complexes, a spectrum of nutrients and their carriers, extracellular matrix components, cell debris, bacteria, viruses, and, in specialized cases, even other cells - Cells can use endocytosis to regulate the composition of their plasma membrane ○ clathrin coated vesicles used

Slightly more complicated cell signaling pathway

○ The receptor protein in the first 2 proteins that are being activated in the pathway are held close in proximity by a scaffold protein. By holding these 3 proteins in proximity, scaffolds function to allow pathways to be activated at specific locations in the cell and with greater speed, efficiency, and selectivity - Some proteins in the intracellular signaling pathway amplify the signal small intracellular Messenger molecules that go off and activate other proteins - Other proteins in the pathways can detect signals from more than one intracellular signaling pathway and integrate them before moving forward - Other proteins can distribute the signal to more than one effector, causing multiple responses in the cell - other proteins are involved in feedback: regulate activity of components upstream in the signaling pathway as its function

golgi to plasma membrane vesicle transport

○ This leads to the process of exocytosis: excretion from the cell by vesicle fusion with the plasma membrane - Can occur constitutively or be regulated

Experiment to test cisternal maturation vs vesicle transport

○ Uchicago - dr ben glick targeted GFP to one cisternae and tracked it over time to see if it was fluid or stayed that same ▪ The limit here is resolution (flourescence is only around 200 nm, which is about the size of the whole golgi itself, so you can never resolve a cis- cisternae from a trans- cisternae). One solution -- split them apart, which thy already are in budding yeast (they are not stacked), which means we would be able to visualize the individual cisternae ○ GREEN BECAME RED -- this means that it supported the cisternal maturation model because it moved from cis-cisternae to trans-cisternae!

A simple cell signaling pathway

○ we can think of this as a molecular relay race ○ a receptor recognizes an extracellular signal and generates an intracellular signal from that. the message is passed downstream from one intracellular signaling molecule to another. then we get a response to the signal, which occurs when the intracellular signaling molecules interact with specific effector proteins, altering their activity to drive changes in the behavior of a cell in a variety of ways