MBB 201 Week 11,12, 13

Membrane Enclosed Organelles

• Membranes form compartments that are important for creating distinct environments with different metabolic functions

Evolution of Membrane-Enclosed Organelles

• Different organelles may have evolved in different ways:

  • Nuclear membranes and membranes of the endomembrane system (ER, Golgi, peroxisomes, endosomes, lysosomes) may have arisen through invaginations of the plasma membrane

  • The interiors of the endomembrane system communicate with each other extensively

Protein Sorting

• Almost all proteins begin their synthesis in the cytosol

• Proteins are transported into organelles by three mechanisms

  • Transport through nuclear pore (to the nucleus)

  • Transport across organelle membranes (protein translocators)

  • Transport by vesicles (endomembrane system)

• Proteins in the cytosol destined for other organelles must be directed by a signal sequence which is directed by the amino acid sequence

Signal Sequences direct proteins to the correct compartments

• Typically 15-60 amino acids long

  • Function of the signals is dependent on the properties of the amino acids

• Signal sequences are necessary and sufficient to direct a protein to a particular destination

• Often (but not always) removed from the finished protein once it has been sorted

Transport Through Nuclear Pores

• The nucleus contains a Nuclear envelope with 2 membranes

  • Inner nuclear membrane

  • Outer nuclear membrane: is contiguous with the ER

• Has nuclear pores to allow passage of molecules in and out of the nucleus

The nuclear pore complex forms a gate through which molecules enter or leave the nucleus

• Spans across the inner and outer membrane

• Composed of ~30 proteins, many of which are largely disordered and unstructured - creates a fibril mesh that fills the center of the channel and prevents the passage of molecules through it

• Small, water-molecules can pass non-selectively

Protein transport through nuclear pores

• Cytosolic proteins that are bound for the nucleus must contain a nuclear localization signal (NLS)

• The NLS is recognized by proteins known as nuclear import receptors

  • Help direct protein to the pore by interacting with cytosolic fibrils

  • Helps direct the protein through the pore by disrupting the interactions between the nuclear fibrils

• Proteins are transported in the fully folded conformation

Energy From GTP hydrolysis drives nuclear transport

• Nuclear import receptors are returned to the cytosol but requires the GTPase known as Ran

• Binding of Ran-GTP causes the dissociation of the imported protein from the receptor

• The receptor bound to Ran-GTP can be transported back into the cytosol where GTP is hydrolyzed to GDP

  • The hydrolysis frees Ran-GDP from the receptor so that it can bind to another NLS

Nucelar export?

• A similar process occurs with the export of proteins. Both import and export depend on Ran-GTP

• NES: Nuclear export signal

Protein Sorting: Mitochondria & Chloroplast 1

• Almost all proteins begin their synthesis in the cytosol

• Proteins are transported into organelles by three mechanisms

  • Transport through nuclear pore (to the nucleus)

  • Transport across organelle membranes (protein translocators)

  • Transport by vesicles (endomembrane system)

• Proteins in the cytosol destined for other organelles must be directed there by a signal sequence which is dictated by the amino acid sequence 1

Mitochondria and Chloroplasts

• Most mitochondrial and chloroplast proteins are synthesized in the cytosol

• Proteins will contain a signal sequence at their N-terminal to allow their import

• Proteins are unfolded as it is transported by a translocator

• Signal sequence is removed after translocation

• Chaperone proteins help proteins to fold

Transport to the Endoplasmic Reticulum

• Proteins in the cytosol bound for the ER have an ER signal sequence

• Soluble proteins in the lumen of the ER:

  • These are either found in the lumen of an organelle in the endomembrane system of get secreted out of cell

• Transmembrane proteins

  • End up in the membranes of the organelles of the endomembrane system or on the plasma membrane

Soluble proteins made on the ER are released into the ER lumen

• Most proteins that enter the ER are threaded across the ER membrane BEFORE the polypeptide chain is fully synthesized

  • Ribosomes synthesized proteins and attach to the ER membrane so that the protein can be threaded into the ER lumen as it is being synthesized

• Regions of the ER with ribosomes attached are called rough ER

A common pool of ribosomes is used to synthesize most proteins

• Membrane-bound ribosomes:

  • Attach to the cytosolic side of the ER

• Free ribosomes:

  • Are not attached to any membrane

• Membrane-bound and free ribosomes are identical to one another

• Polyribosomes: many ribosomes bound to one mRNA molecule

Two protein components help guide ribosomes to the ER

• This works by guiding the ER signal sequence to the ER membrane

• Signal recognition particle (SRP): Present in the cytosol. Binds to the ER signal sequence and the ribosome

• SRP Receptor: embedded in the ER membrane. Binds to SRP. Passes ribosome to a protein translocator. SRP is released

• Protein synthesis occurs, passing the protein through the channel in the protein translocator

Soluble proteins cross the ER membrane and enter the lumen via the translocator channel

• The ER signal sequence causes the opening of the channel

• The signal sequence remains bound to the channel as the rest of the protein is threaded through

• Once the C-terminus has passed through, the signal sequence is removed by a signal peptidase on the luminal side of the ER and the protein is released into the lumen

• Cleaved signal sequences is then rapidly degraded

Transmembrane Proteins in the ER

• Translocation process is a little more complicated

  • Some of the polypeptide chain must be translocated completely, while other parts must be fixed in the membrane

• For a single-pass transmembrane protein, translocation is initiated by a start-transfer sequence

• Translocation continues until a stop transfer sequence is reached, preventing further translocation

  • The translocator channel releases the growing polypeptide chain. The stop-transfer sequence forms an alpha helix and remains embedded in the membrane

• Start-transfer sequence is cleaved

• Orientation: of the N- and C- will not change from one side of the membrane to the other. (above - N-term: lumen; C-term: cytosol)

Transmembrane Proteins in the ER

• For some transmembrane proteins, the start-transfer sequence used to start protein translocation is internal

  • The start-transfer sequence does not get removed like N-terminal ones do

• Start-transfer sequences can work in conjunction with stop-transfer sequences to create multi-pass transmembrane proteins

Entry into the ER lumen or membrane is usually only the first step on a pathway to another destination

• What is the destination? Generally is the Golgi Apparatus (at least, initially)

• In the Golgi, proteins are modified and sorted for shipment to other sites

• Each intracellular compartment has a unique composition

• If vesicles from one compartment fuse with another during vesicular transport, how does the cell ensure that the composition remains unique?

  • i.e. ER proteins stay in the ER, Golgi proteins stay in the Golgi

Vesicular Transport

• The continual budding and fusion of transport vesicles from ER → Golgi and from Golgi → other compartments of the endomembrane system

• The movement of material between organelles in the eukaryotic cell via membrane-enclosed vesicles

  • Allows the transport of lipids and both soluble and transmembrane proteins to various parts of the cell including the endomembrane system and the plasma membrane

  • Each organelle must maintain its own distinct identity

Coated vesicles

• Membrane-enclosed sacs that wear a distinctive layer of proteins on its cytosolic surface

• Helps shape the membrane into a bud and captures molecules for onward transport

• There are several types of coated vesicles, each involved in the transport of vesicles from specific origins and destinations

The type of coat protein present provides information about the origin and destination of a given vesicle

• Clathrin-coated vesicles: Found budding from the Golgi to endosomes as well as from the plasma membrane on the inward endocytic pathway

• COP-coated vesicles: Found in vesicles between the ER and Golgi, as well as from one part of the Golgi to another part of the Golgi

Clathrin-Coated Vesicles

• Vesicle begins as a clathrin-coated pit

• Clathrin is a protein that creates a basketlike network on the cytosolic surface of the membrane

  • Helps to shape he membrane into a vesicle

• The small GTP-binding protein dynamin functions to pinch off the vesicle

  • Assembles as a ring around the neck of each invaginated coated pit

• Adaptins secure the clathrin to the vesicle and help select cargo molecules by binding to cargo receptors

• Appropriate cargo proteins will have transport signals that can be recognized by the cargo receptors (clathrin plays no part in the selection process)

Clathrin-Coated Vesicles transport selected cargo molecules

• Different types of adaptins exist for the transport of different cargo (they each recognize different cargo receptors)

• Once budding is complete, the coat proteins are removed and the vesicle can fuse with its target membrane

Upon arrival, recognition, docking and fusion of vesicles with its specific target organelle occurs

• Each type of transport vesicle must display molecular markers on its surface that identify its origin and cargo

  • These markers must be recognized by complementary receptors on the appropriate target membranes

  • The identification process depends on:

    • A diverse family of GTPases called Rab proteins (vesicles have a unique combination of Rab GTPases on the cytosolic surface) and cognate tethering proteins

    • The transmembrane protein v-SNARE (“v” for vesicle) and t-SNARE (“t'“ for target)

Tethering

• The Rab proteins are recognized and bound by tethering proteins found on the target membrane bringing the two into close proximity

Docking

• The v-SNARE on the vesicle interact with complementary t-SNAREs (“t” for target) which firmly docks the vesicle in place

Fusion

• The vesicle fuses with the target membrane and the cargo protein is delivered to the interior of the organelle (or secreted if at the plasma membrane)

Role in SNAREs

• The fusion of membranes is energetically unfavourable

• Fusion occurs when the v-SNARE and t-SNARE wrap tightly around each other, winching the vesicles closer to the membrane such that the two membranes are close enough for their lipids to intermix

Formation of disulfide bonds

• Covalent bond that links pairs of cysteine side chains

  • Stabilize protein structure

Glycosylation

• Covalent attachment of short branched oligosaccharides (i.e. glycoproteins)

  • Protect proteins from degradation

  • Hold protein in the ER

  • Recognition by proteins for packaging or cell-cell interactions

  • Glycosylation is rare on the cytosolic side

Protein glycosylation in the ER

• Oligosaccharides are not added one at a time - instead they are attached en bloc (all together)

• A 14 sugar oligosaccharides is originally attached to a specialized lipid, dolichol, in the ER membrane

• Is then transferred onto the amino group of an asparagine side chain as the peptide is translocated

• Because they are attached to an amino, they are said to be N-linked

Exit from the ER is controlled to ensure protein quality

• Some proteins are destined to stay in ER and will contain an appropriate retention signal sequence

• If they escape to the Golgi, they will be recognized by receptors and sent back to the ER

• Movement from the ER → Golgi occurs along microtubule “tracks”

Exit from the ER is highly selective

• Misfolded proteins or multimeric proteins that do not assemble properly are retained in the ER by the binding of chaperone proteins

  • The chaperones assist in the folding process and prevent misfolded proteins from aggregating

• The N-glycosylation we just saw is a sensor for whether a protein is properly folded

• If the protein still fails to fold, it will be exported to the cytosol where it will be degraded

Unfolded Protein Response

• If too many unfolded proteins accumulate in the ER, the unfolded protein response (UPR) is triggered:

  • More chaperone and quality-control related proteins are produced

  • May inhibit protein synthesis

• The size of the ER can be expanded to cope with the load, but if this limit is exceeded, the cell can be programmed to die

Cisternae

• Flattened membrane-enclosed sacs

• Has distinct faces:

  • The cis face points to the ER

  • The trans face points toward the plasma membrane

  • The medical cisterna is in the middle

• Vesicles from the ER enter the Golgi at the cis Golgi network

Further modification in the Golgi

• Transport vesicles bud from one cisterna and fuse with the next

• Proteins exit from the trans Golgi network

• Proteins are further modified in the Golgi:

  • Oligosaccharide chains are added, removed and modified

• The trans Golgi network is the main sorting station for the exocytic pathway

Exocytosis

• Vesicles from Golgi fuse with the plasma membrane

Constitutive exocytosis pathway

• Supplies plasma membrane with lipids and proteins

  • Some proteins are secreted

    • Entry to this pathway does not need a particular signal sequence (other than to enter the ER)

    • Operates continually in all eukaryotic cells

Regulated exocytosis pathway

• Only operates in cells specialized for secretion

  • Hormones, mucus, digestive enzymes

  • Proteins are sorted and packed in the trans Golgi network which has conditions that cause proteins to aggregate (low pH, high Ca2+)

  • Proteins are stored in secretory vesicles which accumulate near the PM and wait for a signal to stimulate their fusion with the PM

  • Aggregation allows secretory proteins to be at very high concentrations

Endocytosis/ Endocytic pathways

• Exocytosis delivers phospholipids to the plasma membrane, but this balanced by removal from endocytosis

• Endocytosis: The uptake of material through the invagination of the plasma membrane. Can be broken down into two types based on size:

1. Phagocytosis: involves the ingestion of large particles

• Mainly performed by specialized phagocytic cells

2. Pinocytosis: ingestion of fluid and molecules via small vesicles

• Performed by all cells

• Marcophages removes the equivalent to 100% of its plasma membrane every 0.5 hours

Phagocytosis

• Important for the uptake of food as well as for defence against infection

• After particles are engulfed, they are enclosed in vesicles called phagosomes

  • Phagosomes fuse with lysosomes, digesting the engulfed particle

• Example: Macrophage engulfing a pair of RBCs, white blood cell ingesting a bacterium, amoeba ingesting two paramecia

Pinocytosis

• Occurs continuously

  • Plasma membrane forms pinocytic vesicles

  • Mainly carried out by clathrin-coated vesicles that pinch off and fuse with endosomes

  • Indiscriminate. Budding vesicles trap whatever happens to be inside

Receptor-mediated endocytosis

• Pinocytosis that allows the selective uptake of macromolecules using specific receptors

  • Example: receptor-mediated uptake of LDL

    • LDL receptors bind to LDL and are internalized as clathrin-coated vesicles

    • Fuse with endosomes, and are delivered by lysosomes for breakdown

    • LDL-receptor recycled to PM

Endosomes: the sorting station for the endocytic pathway

• Endocytic vesicles deliver material to and sorted by endosomes

• Early endosomes are located near the PM mature into late endosomes by fusing with other late endosomes and are found near the nucleus

• Endosomes maintain an acidic environment with a proton pump

• Late endosomes eventually merge with lysosomes

• Possible paths:

  • Recycling: returned to the PM

  • Degradation: sent to lysosomes

  • Transcytosis: move to a different domain of the PM

Lysosomes are the principal site of intracellular digestion

• Lysosomes are acidic and contain many hydrolytic enzymes involved in the degradation of macromolecules

• Lysosomal membrane proteins are highly glycosylated on the luminal side - protects from degradation

  • Contains a proton pump as well as transporters for macromolecule subunits to enter the cytosol

• Lysosome destined proteins receive a mannose 6-phosphate tag in the ER and Golgi

Autophagy

• Process by which a cell digests molecules and organelles that are damaged or obsolete

  • The cell eats itself

• Process: organelle is enclosed by a double membrane, creating a autophagosome which then fuses with a lysosome for destruction

Signal transduction

• Conversion of a signal or impulse from one form to another

• Signalling cells create extracellular signal molecules which are received by a target cell

  • The extracellular signal is converted into an intracellular one

Cell communication

• Cell communication can vary in terms of how “public” a message is made

• The signalling molecule can take on large variety of forms including: proteins, peptides, amino acids, nucleotides, steroids, fatty acid derivatives and gases

Endocrine signalling

• The most “public” signalling system

  • Endocrine cells produce signal molecules known as hormones which are delivered through the bloodstream

  • Signal can be broadcast to the entire body; long range

  • Example: Insulin and glucagon are hormones secreted to regulate blood sugar levels

Paracrine signalling

• Signalling cells produce signal molecules known as local mediators which diffuse locally through the extracellular fluid

• Signal is limited and can only be delivered to nearby cells

• If the signalling cell respond to their own signal, this is a form of paracrine signalling known as autocrine signalling

• Example: cancer cells secrete local mediators that promote their own survival

Neuronal signalling

• Signals can be delivered very quickly over long distances (>1m) however instead of broadcasting a signal widely, the signal is sent to specific target cells

• Signal is transmitted along a neuron in the form of an action potential. The electrical signal is converted into a chemical signal in the form of neurotransmitter at the nerve terminals

• The neurotransmitter binds to the receptors on the target cell which can be converted back into an electrical signal

Contact-dependent signalling

• Most intimate and short range of all

• No signalling molecule is secreted. Instead, physical contact is made between molecules embedded in the plasma membrane of the signalling cell and receptors on the target cell

The same signal molecule can cause different responses in different target cells

• Cells respond selectively to a mixture of signals

  • The ability of a cell to respond is dependent on whether or not it has an appropriate receptor

• Even if two cells have the same receptor, they may respond in different ways

• The extracellular signal alone is not the message: the information conveyed is also dependent on how the target cell receives and interprets the signal

Multiple extracellular signals can dictate how a cell behaves

• Cells will contain a limited set of receptor proteins that will respond to different extracellular signals

• A combination of signals can evoke a response that is different from the sum of the effects of each individual signal

• Cells are programmed to kill themselves in the absence of signals

Extracellular signals can act slowly or rapidly

• The length of time a cell takes to respond to an extracellular signal can vary greatly

• Responses to extracellular signals can be (relatively) fast or slow depending on what needs to happen

  • Responses range in the millisecond range to several hours

Extracellular signal molecules bind either to cell surface receptors or intracellular receptors

• Can fall into two general classes:

1. Molecules that do not cross the plasma membrane and bind to surface receptors

• Usually large and/or hydrophilic

2. Molecules that cross the plasma membrane and enter the cytosol and bind to intracellular receptors

• Usually smaller and/or hydrophobic

Steroid Hormones

• One important category of signal molecules that rely on intracellular receptor proteins is the family of steroid hormones

  • Hydrophobic molecules that can cross the plasma membrane

• Bind to nuclear receptors: receptors that when bound to ligand can enter the nucleus and initiate transcription

  • Receptor can initially be found in the cytosol or nucleus

• Example: Cortisol

Nitric Oxide

• Nitric Oxide (NO) is a gas that can diffuse across the plasma membrane and bind to proteins like guanylyl cyclase forming cyclic GMP (cGMP)

• Only works locally because it is quickly converted into nitrates and nitrites

• NO produced in endothelial cells cause a smooth muscle cells to relax, causing blood vessels to dilate

• Examples:

  • Nitroglycerine is used to treat angina because it is converted into NO

  • VIAGRA work by blocking the enzyme that degrades cyclic GMP prolonging the NO signal (i.e. more blood flow)

Cell Surface Receptors

• Most extracellular signal molecules bind to cell surface receptors

• Binding of the extracellular signal to the receptor generates an intracellular signalling response using intracellular signalling molecules

• Intracellular molecules activate effector proteins which cause a cellular response

Cell Signalling Pathways

• Intracellular signalling pathways perform one or more crucial functions:

1. Relay signals onwards - spread throughout the cell

2. Amplify signals - making it stronger

3. Detect signals from more than one intracellular signalling pathway and integrate them

4. They can distribute the signal to one or more effector proteins causing a complex response

Molecular Switches

• Some intracellular signalling proteins act as molecular switches

• Molecular switches: signalling protein that toggles between active and inactive states in response to a signal

  • It is important to be able to control both the activation and inactivation

• Two classes of molecular switches:

  • Proteins activated or inactivated by phosphorylation

  • GTP-binding proteins

Signalling by Protein Phosphorylation

• Largest class of molecular switches

• Involves protein kinases, which phosphorylate proteins, and protein phosphates, which desphosphorylate proteins

• Phosphorylation can either activate or inactivate a protein

• Two main types:

  • Serine/ Threonine kinases

  • Tyrosine kinases

Phosphorylation Cascades

• Many molecular switches controlled by phosphorylation are also protein kinases

• The phosphorylation of one molecular switch causes it to phosphorylate another molecular switch allowing the transmission, amplification, distribution and regulation of signals

• Example: MAP kinase (MAPK)

GTP-Binding Proteins

• Toggles between active and inactive depending on whether they have GTP or GDP bound

• GTP-binding proteins posses GTP-hydrolyzing (GTPase) activity

• Two main types of GTP-binding proteins:

  • Large, trimeric GTP-binding proteins (Aka: G-Proteins)

  • Monomeric GTPases

Monomeric GTPases

• Small GTP-binding proteins that are aided by two sets of regulatory proteins:

1. Guanine nucleotide exchange factors (GEFs): which activate proteins by exchanging GDP for GTP

2. GTPase-activating proteins (GAPs): which inactivate proteins by promoting GTP hydrolysis

Cell Surface Receptors

• All cell-surface receptors proteins bind to an extracellular signal molecule and transduce its message into one or more intracellular signal molecule that alter cell’s behaviour

• Three major classes:

1. Ion-channel couple receptors

2. G protein coupled receptors

3. Enzyme coupled receptors

Ion Channel Coupled Receptors

• Important in neurons and electrically excitable cells like muscle cells

• We’ve seen example of this while exploring how a postsynaptic cell can receive a chemical signal (a neurotransmitter) and transduce it into an electrical signal by opening ion channel and causing a change in the membrane potential

G Protein Coupled Receptors

• G Protein coupled receptors activate membrane bound, trimeric GTP-binding proteins causing the activation (or inactivation) of an enzyme or an ion channel in the plasma membrane

Enzyme Coupled Receptors

• The receptor itself can act as an enzyme or associate with enzymes in the cell

GPCRs

• The largest family of cell surface receptors

• Mediate responses to an enormous diversity of extracellular signalling molecules including hormones, local mediators and neurotransmitters

• ~1/3 of all drugs used today work through GPCRs

• All are composed of a single polypeptide chain that spans the lipid bilayer 7 times

Activation of GPCR

• Binding of an extracellular signal molecule to a GPCR causes it to change conformation

• This in turn activates a trimeric G-protein which results in the transmission of a signal

• There are several G-protein - each is specific for a set of receptors and target enzymes or ion channels

Trimeric G proteins

• made up of an a,B and y subunit

  • a and y are tethered to the PM by a short lipid tail

• When unstimulated, a is bound to GDP

Activation of a GPCR (affinity)

• Upon stimulation by a signal molecule, the GPCR changes conformation that facilitates binding of the trimeric G protein complex. This leads to a decreased affinity of the Ga for GDP and increased affinity for GTP

• GDP dissociates and is exchanged for GTP

• Usually, the G protein subunits dissociate and are switched on

• G proteins interact with target enzymes or ion channels

Switching off

• The amount of time that the G-protein subunits are “switched on” dictates the length of the response

• The subunits will remain on when GTP is bound to the a subunit

• The a subunit contains a GTPase activity which can hydrolyze the GTP to form GDP

• The a subunit reassembles with the By, complex

• The protein is returned to its original, inactive state

Some G-Proteins directly regulate ion channels

• Example: Regulation of heart rate - slowing down heart rate

• Acetylcholine binding to GPCRs of heart pacemaker cells activates the G-protein Gi

• The By subunit binds to K+ channel, causing it to open (slowing heart rate by increasing the membranes permeability to K+)

• Channel closes when GTP is cleaved and the subunits re-associate with one another

Many G-Proteins activate membrane bound enzymes that produce small messengers molecules

• The two most common enzyme targets include:

1. Adenylyl cyclase:

• Produces cyclic AMP (cAMP)

2. Phospholipase C

• Produces inositol triphosphate (IP3) and diacyglycerol (DAG)

  • AC and Phospholipase C are activated by different G proteins

  • cAMP, IP3 and DAG are examples of second messengers

The cAMP signalling pathway can activate enzymes and turn on genes

• Adenylyl cyclase synthesizes cAMP:

  • Generates cAMP from ATP, releasing PPi

  • The a subunit of the G-protein Gs is responsible for the activation of adenylyl cyclase

    • S is for stimulate

• cAMP phosphodiesterase degrades cAMP:

  • Converts cAMP to AMP using water

cAMP-Glycogen Breakdown

• cAMP exerts many of its effect by activating cAMP-dependent protein kinase (PKA)

  • PKA is normally inactivated by binding to a regulatory protein

  • Binding of cAMP to PKA releases the regulatory protein

  • PKA can then phosphorylate other proteins (like glycogen phosphorylase in skeletal muscle)

    • Glycogen breakdown is an example of a relatively fast response

cAMP - slow responses

• cAMP can also cause the activation of gene expression - a relatively slow response

• PKA phosphorylates transcriptional regulators which can initiate transcription

Inositol phospholipid pathway

• Some GPCRs activate the membrane bound enzyme phospholipase C (instead of adenylyl cyclase)

• Phospholipase cleaves an inositol phospholipid (found in the plasma membrane) into:

  • Inositol 1,4,5-triphosphate (IP3): released into the cytosol

  • Diacyglycerol (DAG): remains embedded in the membrane

  • Both products are important in signalling

Phospholipase C activates two signalling pathways that trigger and need a rise in intracellular Ca2+

• IP3 binds to and opens Ca2+ channels embedded in the ER membrane

  • Free Ca2+ is released into the cytosol which can act on other proteins

• DAG recruits a cytosolic protein to the plasma membrane known as protein kinase C (PKC)

  • Activation of PKC requires the binding of Ca2+

  • PKC phosphorylates several intracellular proteins

Calmodulin: the calcium receptor

• Ca2+ binds to specific proteins in order to exert their effects. The most common of which is calmodulin

• Calmodulin binds to four Ca2+ ions, inducing a conformational change allowing it to interact with other proteins like Ca2+/ calmodulin-dependent protein kinases (CaM-Kinases)

Enzyme Coupled Receptors

• Transmembrane proteins that either

  • Act as enzymes themselves or

  • Associate with another protein that acts as an enzyme

• Responses can be fast (e.g. reconfigurations of the cytoskeleton) slow (e.g. result in changes in gene expression)

Receptor Tyrosine Kinases: largest class of enzyme-coupled receptors

• The receptor often form dimers upon binding of an extracellular signalling molecule

• Each receptor protein possesses a tyrosine kinase domain which allows each receptor to phosphorylate tyrosines on the other

  • Tyrosine residues on the cytoplasmic tail are phosphorylated which serve as docking sites for other proteins

Receptor Tyrosine Kinases

• The phosphorylated tyrosines serve as dock for many other proteins

• Some known as adaptor proteins which act as a scaffold so that other proteins can bind while others propagate the signal

  • Each contains an interaction domain which recognizes phosphorylated tyrosines on the tail

Most RTKs activate the monomeric GTPase Ras

• Ras is a small GTP-binding protein that is bound to the cytoplasmic face of the plasma membrane

• Ras-GEF encourages Ras to exchange GDP for GTP, which activates Ras

• Ras-GAP promotes the hydrolysis of GTP to GDP, which inactivates Ras

Ras activates a phosphorylation cascade: MAP-kinase signalling

• Ras activates a series of serine/ threonine protein kinases

• Example: Mitogen-activated protein kinase (MAP kinase) pathway

• Each member of the cascade is a molecular switch that when activated, phosphorylates the next member of the cascade

• Mitogen: extracellular signalling molecule that stimulates cell proliferation

Ras and Cancer

• MAPK pathway is involved in cell proliferation, survival and differentiation

• 30% of human cancers involve a mutation in Ras which inactivates the GTPase activity and so keeps Ras in its GTP-bound “ON” state ( the others have mutations in genes that encode proteins that function in the same signalling pathway as Ras)

• This prevents the signal from being turned off and results in uncontrolled cell proliferation

• Oncogene: A gene that when activated can potentially make a cell cancerous (e.g. Ras)

Some RTKs create lipid docking sites

• RTKs also work through the phosphoinositide 3-kinase (PI 3-kinase) signalling pathway involved in cell growth and survival

• PI 3-kinase phosphorylates inositol phospholipids in the plasma membrane which then serve as docking sites for other proteins

  • Same inositol phospholipid as the Phospholipase C substrate

  • Converts to Phosphatidylinositol triphosphate (PIP3)

• These other proteins are recruited to the PM from the cytosol where they can activate one another

• Example: AKt, promote growths and survival

Activated Akt promotes cell survival

• PI 3-Kinase-Akt pathway promotes cell survival:

  • Akt (Protein kinase B) is a cytosolic protein that binds to phosphorylated inositol phospholipids

  • Phosphorylated Akt phosphorylates proteins and, in this way, prevent cell death

Activated Akt also promotes cell growth

• PI 3-kinase-Akt pathway promotes cell growth:

  • Akt can also activate the serine/ threonine kinase called Tor

  • Tor enhances protein synthesis and inhibits protein degradation

  • Overactive Tor may play a role in cancer

The Cytoskeleton

• Network of protein filaments that gives the cell shape and capacity for directed movement

  • Dictates the location of organelles and allows transport between them

• Directly responsible for:

  • Cells crawling along a surface

  • Contraction of a muscle cell

  • Changes in cell shape

  • Wound healing

  • Sperm swimming

  • etc.

Intermediate Filaments (F)

• Function:

  • Great tensile strength which enables cells to withstand mechanical stress

  • Strong and durable

  • Found in most but not all eukaryotes

• Location:

  • Surrounds the nucleus

  • Often anchored to the plasma membrane at cell-cell junctions

  • Found within the nucleus forming the nuclear lamina

• Prominent in the cytoplasm of cells that are subject to mechanical stress

• IFs distribute the effects of locally applied forces preventing membranes from tearing

• Provide internal reinforcement

Intermediate Filament Structure

• There are several types of IFs, but they all share a similar structure:

  • The IF monomer has a a-helical central rod domain

  • 2 monomers wrap around each other to form a coiled-coil dimer

  • 2 coiled-coil dimers associate to form a staggered tetramer

  • Each dimer runs in opposite directions (“head to head”)

• The central rod domain of different intermediate filaments contain similar amino acids

• The AAs in the termini are generally different to distinguish one type of filament from another

• 8 tetramers associate with each other side by side

  • The 8 tetramers add to a growing, overlapping filament

  • Each end is the same. The N-termini of the dimers are on the ends

  • Noncovalent binding holds the filaments together

Intermediate Filaments are divided into four major classes

• Keratin, vimentin and neurofilaments are found in the cytoplasm

  • Form ropelike structures

• Nuclear lamins are found in the nucleus

  • Form a 2D mesh

• Each class has several subtypes

Keratin Filaments

• Found in every epithelial cell

• Specialized keratins form hair, feathers and claws

• Anchored to cell-cell (desmosomes) and spans the interior of the epithelial cell from one side to another

• Example: Epidermolysis bullosa simplex is a disease where the formation of keratin is impaired. The skin is vulnerable to mechanical injury causing skin to blister

Nuclear lamina

• Nuclear lamins are a class of IFs that form the nuclear lamina

• Strengthen the inside of the nuclear envelopes and provides attachment sites for DNA binding proteins

• Must disassemble and reassemble during mitosis - controlled by phosphorylation and dephosphorylation of lamins

• Progeria: Disease with a defect in particular nuclear lamin. Causes premature aging

Microtubules

• Crucial organizing role in all eukaryotic cells

• Long, relatively stiff hollow tubes

• Important for transporting and positioning of membrane-enclosed organelles

• Form mitotic spindle during mitosis

• Allow cell motility by forming cilia and flagella

Microtubule Structure

• Microtubules are made from many microtubule subunits known as tubulin

• Each subunit is comprised of a globular a- and B- tubulin dimer bound by noncovalent interactions

• Protofilament: linear chain of tubulin dimers

• 13 protofilaments form the hollow tube of a microtubule