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105 Cards in this Set

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Signal transduction causes:
1. amplification of the response
2. is limited in duration and therefore highly controlled
3. has a phosphorylation switch
4. has regulatory control
Endocrine signals
are produced at great distances from their target tissue, and are carried by the circulatory system to various sites in the body.
Paracrine signals
are releases locally where they diffuse short distances to the target tissues. Growth factors are an example. Short distance messages
Autocrine signals
- act on the same cell that produced them. Short distance messages
Ligand
is the primary messenger that binds with the receptor.
Signal transduction
the ability of a cell to translate a receptor-ligand interaction to changes in its behavior or gene expression.
Messages can be in the form of hormones, neurotransmitters, ions, and growth factors.
Messenger molecules are amino acids, peptides, proteins, fatty acids, lipids, nucleosides, or nucleotides.
Hydrophillic compounds usually bind to receptors on a target cell while hydrophobic compounds act on receptors in the nucleus or cytosol and generally regulate the transcription of particular genes
Signal transduction pathways allow for strong amplification of a signal because at each step in the signal cascade, a signaling intermediate persists long enough to stimulate the production of many molecules required for the next step in the cascade, thereby multiplying the effects of a single initial ligand
G-protein couples receptors
G protein-linked receptors have 7 transmembrane α helicies. The N-terminus of the protein is extracellular. The C-terminus is in the cystol. The extracellular portion of the protein has the messemger-binding site. There is a cystolic loop that connects it to 5th and 6th α helicies that is specific for a particular G protein.
Large heterotrimeric G-proteins have three different subunits and mediate signal transduction through G-protein linked receptors:
Gα is the largest subunit. It binds to either GDP or GTP. When Gα is bound to GTP it detaches from Gβγ.
Gβ is always bound to Gγ
Gγ is always bound to Gβ
Some G proteins stimulate signal transduction
and are called Gs
Some G proteins inhibit transduction and are called
Gi.
steps in heterotrimetric G protein couples receptors
1. messenger binds to G protein-linked receptor on the surface of the cell
2. a conformational change of the receptor causes the Gα subunit to release the GDP to which it is bound.
3. The Gα subunit acquires a GTP and detaches from the complex.
4. Either the free Gα subunit or the stationary Gβγ can initiate the signal transduction events in the cell.
5. Either subunit binds to a target protein on the inside of the cell and inhibits or activates it depending on the type of G protein it is. In some cases, both the Gβγ and the Gα subunits simultaneously regulate different processes in the cytosol.
6. The activity of the G protein persists only as long as the GTP is bound to the Gα subunit, and the subunit remains separated.
7. The G protein pathway can be shut of rapidly by changing GTP back into GDP.
Cyclic AMP (cAMP):
ATP is used to make cAMP by enzyme adenylyl cyclase.
Adenylyl cyclase is anchored to the plasma membrane and is inactive until the Gα subunit binds to it. Binding of the alpha subunit (from a stimulatory G couples protein receptor) turns on adenylyl cyclase and allows it to turn ATP into cAMP. The cell remains very sensitive to changes in messenger concentration because each step in the signal pathway has a very limited time window. For example, cAMP is quickly degraded by phosphodiesterase. cAMP acts on the enzyme protein kinase A (PKA).
Protein kinase A
phosphorylates a wide variety of cellular proteins by transferring a phosphate from ATP to a serine or threonine on the target protein. cAMP regulates PKA by causing the two regulatory subunits of PKA to detach from the two Catalytic subunits. Once the catalytic subunits are free, they are active.
An example of cAMP as a second messenger:
1. initial signal is epinerpherine from muscles or liver
a. cells break down glycogen
2. Epinepherine acts on cardiac muscle to increase heart rate and have stronger muscle contractions
3. It causes smooth muscles to relax
4. In lung epithelia, cAMP causes secretion of salt and water into the leumen
Inositol Triphosphate and Diacylglycerol as second messengers
Functions that are regulated by IP3 and DAG:
1. acetycholine:
a. stimulates contraction of smooth musces
b. secretion of insulin
c. secretion of amylase
Inositol-1,4,5-triphosphate (IP3
is a second messenger. It is generated from PIP2, a relatively uncommon membrane phospholipid. The enzyme phospholipase C cleaves PIP2 into two molecules; inositol triphosphate and diaglycerol (DAG).
steps in IP3 pathway
1. a messenger binds to a receptor on the surface of a cell
2. there is activation of a specific G protein, Gp
3. Gp activates phospholipase C-β
4. Phospholipase C-β cleaves PIP2 into IP3 and DAG
a. IP3 is water soluble and quickly diffuses through the cytosol. It binds to a ligand-gated calcium channel known as the IP3 receptor in the endoplasmic reticulum.
b. Binding causes the channel to open and release calcium into to cytosol.
c. Calcium binds to a protein called calmodulin

a. DAG remains in the membrane. It activates protein kinase C’s (PKC)
b. PKC phosphorylates specific residues on specific proteins
5. It has been shown experimentally that both IP3 and DAG are needed for a full response.
Protein Kinase-Associated Receptors
A different strategy for transmitting signals to and within a cell. Protein kinase receptors are proteins that, when bound to a ligand, they phosphorylate certain residues on amino acids and transmit the signal.
Tyrosine kinases-
phosphorylate tyrosine residues
Serine/threonine kinases
phosphorylate serine and threonine residues
RTK-
receptor thyrosine kinases, usually trigger cell transduction pathways that lead to cell growth , proliferation, and differentiation.
structure of RTK
Structure:
· Usually a single polypeptide chain with only one transmembrane segment
· Extracellular portion of the receptor has the ligand-binding domain
· On the cystolic side, one portion forms they tryrosine kinase, the rest forms the cystolic tail
· The cystolic tail includes tyrosine residues that will be the target of a tyrosine kinase.
· When the tyrosine kinase is not a direct part of the receptor it is called a nonreceptor tyrosine kinase. (either way, it binds to the receptor and has the same function)
activation of RTK
1. a ligand binds to the receptor
2. receptor tyrosine kinases aggregate, meaning that usually, two receptors cluster together within the plasma membrane.
3. the tyrosine kinases on each receptor phosphorylate the tyrosine residues on the other. This is called autophosphorylation.
4. The receptor is now active and can recruit several different cystolic proteins
Cystolic Proteins used with RTKs
· Proteins that bind with RTK must have a SH2 domain that recognizes and binds to phosphorylated tyrosine (plus some other aa)
· Different SH2 domain-containing proteins activate different pathways, so RTK’s can activate several transduction pathways at the same time
· There are two main pathways, IP3 (Cγ) (same as above except with SH2 domain) and Ras
Ras-
a second messenger important in regulating growth of cells. Ras is a single subunit and is a small monomeric G protein. It can be bound to either GTP or GDP but is only active when bound to GTP. Ras is deactivated by GTPase activating protein (GAP)
Guanine-nucleotide exchange factor (GEF)-
protein that help exchange GDP for GTP
Sos
– a GEP specific to Ras. It can only become active it must bind indirectly (with the help of GRB2 which has an SH2 domain) to an active RTK.
Ras pathway
: a cascade of events that ultimately results in the formation of a transcription factor that stimulates cell growth gene expression. An important step in the pathway is the activation of mitogen-activated protein kinases (MAPKs).
MAPK
phosphorylates Jun
Jun
one of the proteins that is a part of transcription factor AP-1, responsible for gene transcription of genes having to do with cell growth.
Platlet-derived growth factor (PDGF)-
)- factors that are found in blood serum after a clotting reaction. They stimulate growth of fibroblasts, a part of connective tissue. The receptor for PDGF is a tyrosine kinase.
Growth factor
secreted molecules that act at short range and have specific effects on cells processing the appropriate receptor.
Fibroblast growth factors and fibroblast growth factor receptors (FGFs and FGFRs)
play an important part in the development of specialized tissue in the mesoderm. A mutation in an FGFR is dominant and negative. It is dominant because, since the receptor has to form a dimmer to work, and mutation in one of the dimmers results in loss of function.
Receptor seri
Transforming growth factor-β (TGF-β)
a type of growth factors that binds with a family of serine/threonine kinase receptors. These growth factors can control cell proliferation, programmed cell death, cell specialization, and parts of embryonic development.
Steps in binding TGF
1) The growth factor binds to the transmembrane receptor.
a. TGF β family members bind to two types of receptors, type 1 and type II.
b. Two type II receptor dimmers flank and phosphorylate two Type I receptor dimmers initiate signal transduction inside the cell.
2) Type I receptor phosphorylate a class of protein called Smads
3) Phosphorylated smad binds with co-smad and move into the nucleus.
4) The smad complex can associate with DNA binding proteins ti regulate gene expression.
Epidermal Growth factor (EFG
a 53 amino acid peptide, it acts on nearby cells in the paracrine autocrine system. The ultimate result of the peptide it to cause cell growth and division.
1. Binding of EGF to the receptor causes phosphorylation of the receptor.
2. a target protein with an SH2 domain binds to the receptor
3. the target protein gets phosphorylated. The target protein in this case is phosplipase C-γ. (it has an SH2 domain)
4. PLC-γ results in the production of IP3 and DAG\

3b. activation of the receptor also causes phosphorylation of Ras, a lipid linked protein that is active when bound to GTP
4b. The Ras-GTP complex causes a cascade that eventually results in cell growth and division.
Experimental approach to determining the function of protein domains: cut and paste experiment à hybrid
Take different parts of two membrane receptor proteins and fuse them together. The ligan binding secton of the protein should still respond to the original ligand but cause the activity of the cystolic protein. Can show what is ligand, what is not.
Hormones
chemical messengers secreted bu one tissue that regulate the function of other cells to tissues in the same organism. Hormones can be cholesterol derives steroids, peptides, proteins, amino acid derivatives. The are long distance messages.
Endocrine hormones
travel by means of the circulatory system from the secreted tissue to the target tissue. Time range varies. Can travel over large distances
Paracrine hormone
a more loca signal that is taken up, destroyed, or immobilized so quickly that it can only act on cells in the immediate environment.
Adenergic hormones
(epinephrine and norepinepherine) trigger increased cardiac output, shunting blood from the visceral organs to the muscles and heart, dilate aterioles to facilitate oxygenation, stimulate the breakdown of glycogen to increase the glucose supply to muscles.
Adenergic receptors
a family of g protein liked receptors.
α-adenergic receptors
bind to both epineperine and norepinepherine. Located on the smooth muscles that regulate blood flow to the visceral organs. Linked to Gp proteins. (Phospholipidace C (IP3 and DAG ) pathway)
β-adenergic receptor
bind to epinephrine much more than to norepinephrine. Found on smooth muscles associated with the arterioles that feed the heart, smooth muschles of the bronchioles in the lungs and skeletal muscles. Linked to Gs proteins. (cAMP pathway)
Glycogen phosphorylase
catylzes the breakdown of glycogen by cleaving glucose units from plycogen as glucose-1-phosphate by adding inorganic phosphate.
Stress response
1) Epinephrine molecule binds to a β-adenergic receptor on the plasma membrane of a liver or muscle cell.
2) The receptor activates a Gs protein
3) Gs stimulates adenyl cyclase
4) Adenyl cyclase tuns ATP into cAMP
5) cAMP activated protein kinase A (PKA)
6) PKA phosphorylates and activates phosphorylase kinase
7) Phosphorylase kinase changes glycogen phosphorylase from phosphorylase b (less active) into the more active phosphorylase a
8) Glycogen breakdown increases.

5b) cAMP activates PKA
6b) PKA phosphorylates and deactivates glycogen synthase
7b) glucose is not converted into glycogen
α1-adenergic receptor response:
these receptors are located on smooth muscles on the blood vessels surrounding the intestines (etc). when stimulated they set off the IP3 response, increasing the calcium concentration inside the cell. The increase of calcium causes smooth muscle to contract, constricting the blood flow to the Gi tract and shunting it to where it is needed.
Human disease and GPC signaling: cholera
Cholera is a bacteria that causes dehydration. It secretes a toxin into the intestines. The toxin binds to a G-alpha protein and causes it to be perminantly activated. The G-alpha protein stimulates the secretion of water into the leumen and intestine.
Extracellular matrix (ECM
amorphous, hydrated matrix of branched molecules that provides structural support and influences tissue extensibility, cell shape and movement. ECM can be bone, carillage, or connective tissue. It is found around and inbetween cells and cells within the extracellulatr matrix produce them. The ECM always consist of three classes of molecules:
1) structural proteins give strength and flexibility
a. collagens
b. elastins
2) protein-polysaccharides complexes called proteoglycans provide the matrix
3) adhesive glycoproteins attach cells to the matrix
a. fibronectins
b. laminins
The type of structural proteins and kinds of proteoglycans as well as the ratio of structural protein (collagen) to proteoglycans determines the specific properties of the ECM
Bone
is mostly exctracellular matrix and two types of cells:
Austioblasts that are responsible for making the matrix
Austioclasts- that are responsible for breaking the matrix down
Cartilage
is found in the nose, ears and joints and is mostly ECM. Its cell types are called chondroblasts
Connective tissue
surrounds blood cessles and glands and connects tissue. Its cell types are fibroblasts.
Collagens
Most abundant component of the ECM form fibers with high tensile strength and account for much of the strength of the ECM. Collagen is secreted by several connective tissues including fibroblasts. Collagens occur as a ridgid triple helix of three intertwined polypeptide chains. They are high in glycine, hydroxylysine, and hydroxyproline.
The high glycine composition makes the triple helix possible because the spacing of glycine puts it on the inside of the helix and it is the only amino acid small enough to fit on the inside of the helix.
The hydoxylysine and hydroxoproline are involved in hydrogen bonds that cross linking both within and between the individual collagen molecules in a fibril reinforcing collagen’s extreme stability.
Different α chains combine in various ways to form different collagen molecules
Some types of collagen are striated (about every 67nm). These bands reflect the regular but offset manner in which the triple helices associate laterally into fibrils.
structure of collagen
· three polypeptide chains called α-chains are twisted together into a ridgid, right-handed triple helix make a collagen molecule
· Many collagen molecules make up a fibril
· Fibrils bundle together to form collagen fibers
· Collagen molecules are 270nm in length and 1.5 nm in diameter
constuction of collagen
1. in the lumen of the ER three α-chains assemble into a triple helix called a procollagen
2. The ends of procollagen have short nonhelical sequences at either end on the amino acid chains that prevent the formation of fibrils while still in the ER
3. Procollagen is secreted into the intercellular space
4. procollagen peptidase removes the extra amino acids from either end of the procollagen.
5. The procollagen sponstaniously self assembles into fibrils
6. Fibrils assemble laterally into fibers
Elastin
a structural protein that provides elasticity and flexibility to tissues. Elastins are rich in glycine and proline. There are no hydroxylated resuides. Elastins are cross linked to one another by covalent bonds between lysine residues. Tension of an elastin network causes the individual molecules to adopt expanded conformations that permit the overall network to stretch. When the tension is released, the individual molecules relax, retuning to their normal state and the crosslinks between molecules cause the network to return to its original state
Proteoglycans
make up the hydrated, gel-like network in which collagen and elastin fibers are enmeshed. Proteoglycans are glycoproteins in which a large number of glycosaminoglycans (GAGs) are attached to a single core protein molecule. (10% protein, 90% carbs) The combo of different core proteins and GAGs of different type and length can form many different kinds of proteoglycans. Most proteoglycans are huge and are linked directly to collagen fibers to make up the ECM. The two important functions of proteoglycans is to trap water and resist compression forces. .Proteogylcans associate with cells by different types of linkages. The protein core may be integrated into the plasma membrane or they may be covalently linked to a membrane lipid. They may also bind to a receptor at the plasma membrane or have indirect linkages
GAG- have repeating disaccharide units. The three most common types of Gag
GAG
- have repeating disaccharide units. The three most common types of Gags are chondroitin sulfate, kertan sulfate, and hyaluronate. One of the two sugars is an amino sugar, either GlcNAc or GalNAc. The other sugar is usually a sugar or sugar acid, commonly galactose or glucronate. In most cases the amino acid has one or more sulfates attatched. GAGs are hydrophilic and have many negatively charged sulfate groups and so they attract water and cations forming the hydrated, gel like matrix. Most GAGs are covalently bound to protein molecules and form proteoglycans.
Hyaluronate is a GAG that exists as a free molecule consisting of hundreds or repeating disaccharide units and has lubricating properties.
Anchoring cells to the ECM:
Cells are anchored to the ECM by proteoglycan linkages. The proteoglycan can be a part of the plasma membrane, can be linked covalently to membrane phospholipids, or may bind to specific receptor proteins on the outer surface of the plasma membrane
Adhesive glycoproteins
: reinforce the direct link between the ECM and the plasma membrane. They bind proteoglycans and collagen molecules to each other and to receptors on the cell surface. Adhesive glycoproteins have multiple domains for binding with different molecules and receptors. Fibronectins and laminins are the most common adhesive glycoproteins. The family of receptors to which they bind are called integrins
Fibronectins
a family of closely related adhesive glycoproteins in the ECM. They are insoluble as fibrils in the ECM and as an intermediate form loosely associated with the cell surface. In blood and body fluids they are soluble. Firbonectins consist of 2 very large polypeptide subunits linked near the carboxyl ends with a paid of disulfide bonds. There are several rod like domains connected by short flexible segments of polypeptide chain. The domains bind different molecules of the ECM or cell surface or to receptors in the cell surface. The RGC (argentine-glycine-asparigine) sequence is found in the domain that binds to cell surface receptors (integrins). Fibronectins may play a role in cell movement because cells have been shown to attach and migrate along fibronectin fibers. Free firbronectins play a role in blood clotting.
Basal lamina
a thin sheet of specialized extracellular material, about 50 nm thick, that underlies epithelial cells and separates them from connective tissue. It serves as a structural support that maintains tissue organization and as a permeability barrier that regulates the movement of molecules and cells. All forms contain type IV collagen, proteoglycans, laminins and gylcoprtein enactin or nidogen.
Laminins
- adhesive glycoproteins that bind cells to the basal lamina. They are localized mainly on the surface of the lamina that faces the overlying epithelial cells. They are very large and have three long polypeptide chains, α, β, and γ. Disulfide bonds hold the polypeptide chains together in the shape of a cross. There are several domains to bind to cell receptors and the ECM.
Fibronectins are located on the side of the lamina with the connective tissue and help anchor those cells.
Integrins
- transmembrane proteins (receptors) that integrate the cytoskeleton with the ECM. The primary means by which the cell binds to the ECM. They are important in cell movement and attachment. They can also sometimes signal changes inside the cell or be altered by changes inside the cell
structure of integrins
· Two large transmembrane polypeptides: α and β subunits that are associated with each other non-covalently.
· The extracellular portion of the subunits interact to form the binding site for the ECM proteins. Most of the binding specificity is with the alpha subunit.
how integrins integrate the cytoskeleton with the ECM
· Indirectly
· Tails of integrins interact with cystolic proteins that link the integrin with the cytoskeleton.
· Two main types of connections
1) Focal adhesions – for non-epithelial cells
o Focal adhesions contain clustered integrins that interact with bundles of actin via linker proteins such as talin and alpha actine
o They are found in cells that migrate such as fibroblasts.
o Most parts of the cell are not tightly attatched to surface, the parts that are attatched are called focal adhesions
o Linkage is to actin filaments and microfilaments
o They allow fibroblases to pull and move
o Focal adhesions are made and broken
o They can activate signal transduction, movement, growth and cell shape
2) Hemidesmosomes – for epithelial cells to basal lamina
o Attatch to an intermediate filament called keratin.
o They form a plaques that connects clustered integrins to the cytoskeleton
o
o They are strong attatchements so that epithelial cells stay in a sheet.
Plant cell walls
1) Focal adhesions
– for non-epithelial cells
o Focal adhesions contain clustered integrins that interact with bundles of actin via linker proteins such as talin and alpha actine
o They are found in cells that migrate such as fibroblasts.
o Most parts of the cell are not tightly attatched to surface, the parts that are attatched are called focal adhesions
o Linkage is to actin filaments and microfilaments
o They allow fibroblases to pull and move
o Focal adhesions are made and broken
o They can activate signal transduction, movement, growth and cell shape
2) Hemidesmosome
for epithelial cells to basal lamina
o Attatch to an intermediate filament called keratin.
o They form a plaques that connects clustered integrins to the cytoskeleton
o
o They are strong attatchements so that epithelial cells stay in a sheet.
Plant cell walls
- have fibrous polymers embedded on a hydrated matrix
- provide resistance to mechamical stress, stress forces, compression forces
- provide cell-cell contact
- functions in cell expansion (turgor pressure)
- bind water and ions
- is important in plant defese
- important in plant shape and morphogenesis
classes of cell wall components include:
fibrous component: cellulose
hemicellulose: connects cellulose
pectins: a hydrated matrix
structureal glycoprteins
Why do cells need to adhere?
To maintain the integrity of issues and organs
Cell-cell adhesion in early vertebrate embryos:
In vivo embryos cells form clumps. To get rid of cell-cell attatchements:
1. add trypsin to cleave extracellular proteins
2. add chelators such as EGTA or EDTA to get rid of calcium and other divalent cations.
You find that cell-cell contacts are mediated by proteins and cations. When you wash away the trypsin and EDTA, the cell-cell contacts reform is a similar configuration to before the experiment
What molecules are responsible?
1. Isolate cell surface/plasma membrane proteins (by SDS)
2. separate by SDS page
3. purify proteins
4. generate antibodies against proteins
5. repeat experiment with modification
a. after trypsin and EDTA are added and cells lose adhesions, add antibodies
b. wash trypsin and EDTA
c. if the cells reassociate, the protein for which you targeted the antibody is not responsible for cell-cell adhesion. If the cells DO NOT reassociate than the protein for which you targeted the antibody play a role
5. Take the candidate proteins and do a westen blot.
a. Isolate protein
b. Separate by SDS page
c. Transfer proteinsto nylon membrane
d. Run gell
e. Probe with anibodies to see where proteins are
f. Cut out bands and isolate, purify, and sequence proteins
Cell adhesion molecules are
1. calcium dependant cadherins and selectins
2. calcium independent adhesion molecules such as the amino globulin super family.
Cell-cell adhesion is mediated by transmembrane proteins called adhesion molecules.
Especially important is the immunoglobulin superfamily (IgSF) of proteins that includes cadherins, selectins and sometimes integrins. There are 3 types of ways cells can form connections to each other:
. There are 3 types of ways cells can form connections to each other:
1) homophilic interactions are those in which cells interact with identical molecules on the surface of the cell to which they adhere. CAMs fall into this group
2) heterophilic interactions- a cell adhesion receptor on one cell interacts with a different molecule on the surface of the cell to which it attaches
3) via linker protein
CAMs
- members of the IgSF. Have well organized loops
Cadherins
a group of adhesive glycoproteins. They require Ca++ to function. Mediate cell-cell adhesions. Are characterized by a series of structurally similar subunits (repeats). Interact by homophilic interaction. Connected to the cytoskeleton the their cystolic ends. If a cadherin gene is transfected into a cell line that does not show cell-cell adhesion it will display that ahavior. (controls in this experiment could include untransfected lines, nonsense cadherin gene or another gene, empty vector, or mock transfection)
Gap junctions
are used for chemical and electrical communication. Structure: many polypeptides form an aqeous pore in between cells. Can be regulated by cystolic calcium. (high Ca closed, low Ca open) Water, ions, aminoacids, monosacherides, and nucleotides can pass through.
To determine experimentally what is the maximum size particle that can pass through sugar based polymers called detrains were made in varying lengths and molecular weights. They ewr coupled with a flurescent dye and put into cells by injection. If movement was seen into the next cell, that size of particle could get through.
Plasmodemata
(plants) tubular structures that connect adjacent plant cells (cytoplasm to cytoplasm) The function of these structures is to transport ions, water, sugars (sucrose) and amino acids. They are important in phloem transport and are used by plant viruses. Developmental RNA molecules are also transported through them. Size of the molecules that gets though varies. A sixe x of dextran can get through alone but when bound to a protein a 10x can get through because movement proteins increase the size limit of plasmodemota.
Tight junctions
in intestinal epithelial cells) seal mmranes over certain junctions creating a water tight seal between cells to prevent nonspecific leakage between the gut and blood side making transport highly regulated.
Structure: claudin gene family with 24 members
Pathology: heliobactor pylori attatches to tight junctions in intestina epithelium causing leaky junctions that cause acid to leak to the blood side.
Endomembranes
are internal membranes that include the Rough er, Smooth er, Golgi
Transition vesicles, Secratory vesicles, Lysosomes and Endosomes
Secratory pathway
How do proteins ge out of cells?
It all starts in the Rough ER where proteins are synthesized. There are two types of secretion, constitutive (unregulated) and regulated secretion.
Constitutive secretion
- is most common. Vesicles fuse with the plasma membrane and proteins are released as soon as they are synthesized. Clear under microscope.
Regulated secretion
use secretory granules, a special type of secretory vesicle. They are different because they store proteins that are made and are ready for release upon receiving a certain signal. Insulin is an exmple. An increase in calcium causes secretory granules to fuse to the membrane and release their content. The interior of granules is dark in EM because the contents ar very dense and pick up the stain well
Secratory pathway in pancreatic cells:
Techniques: pulse chase, sub cellular fractionation, autoradiography
Pulse chase:
Tridiated leucine is a common lable that is non specific and labels all proteins.
35S sulfate labels proteins that are sulfated on tyrosine and is more specific
14C for glucose
In pancreatic cells pulsing with tridiated leucine is effective because most of the proteins that the cell makes are digestive proteins meant for secretion so they will have most of the label. Chase for a few hours.
Then use subcellular fractionation to find out where the proteins are
Take fractions and prep for TEM, audoradiography, silver dark stain shows places where granules are.
Rough er
side note: ribosomes ca be free or bounded to the ER) The function of the ER is to help protein folding. Chaperone proteins in the RER help fold. Imporperly folded proteins are destroyed. Another function of the RER is contanslationsal import. Proteins translated are injected into the leumen of the RER. Intial processing for soluble proteins meant for the membrane is done here.
Smooth er-
- system of connected tubules that is abundant in cells that need it. Its function is steroid synthesis (such as testosterone) and detox of drugs (in liver cells). Many drugs are hydrophobic and therefore insoluble and difficult to secrete. Smooth Er adds OH groups to make it more hydrophilic. Smooth ER is also important in carb metabolism. (remember glucose-6-phosphate is a marker enzyme for smooth ER in subcellular fractionation)
Also remember the epinephrine G protein pathway (epi à g coupled β-adeneric receptor --. Alpha subunit activated à adenyl cyclase à CAMP à PKA in liver cells à phosphorylase kinase à phosh B à break down of glycogen
Pathway for membrane proteins:
ER à Golgi à Lysosomes à plasma membrane à secreted proteins
What happends in the RER:
(* cystolic proteins are made in the cystol and are not a part of the ER pathway)
1. synthesis of protei starts on the cystolic side of the ribosomes
2. the amino terminal is synthesized (~20 aa)
3. all proteins destined for the above pathway have a signal sequence on the amino terminus
4. signal sequences are recognized by the signal recognition particle SRP (made of preotein and RNA)
5. SRP arrests translation in cytosol (so that the protein can be moved into the ER)
6. SRP binds to the SRP receptor (a transmem receptor in the rough ER membrane)
7. the ribosome of the complex binds to the ribosome receptor in the RER mem
8. translation restarts
9. signal peptide is cut off in the ER
10. protein import requires energy (GTP)
11. a pore protein opens (in the ER) and lets the protein through
12. when SRP is released protein synthesis continues
13. The ER adds oligosacharides to proteins (N linked sacerides at aperagine residues)
14. lipid biosynthesis also takes place in the ER
Golgi
– is made up of the cis Golgi network (facig the cystol), the cis, median, trans, and trans Golgi network layers. The cis side of the Golgi has shorter, fater cisternea while the trans side is longer and thiner. Transport vesicles bud off form the TRG.
The function of the Golgi is to:
1. CGN: add o-linked glycoproteins to serine ad thrionine residues added in the CGN. The CGN also start the first step in the phosphorylation of lysosomal proteins.
2. processing of both N and O linked sugars
3. proteoglycans get GAGs added to them
4. in plants, hemicellulose and pectin polysacerides are added
5. sorting of protein and polysaccharides
6. packaging into the appropriate vesicles
Lysosomes
– are animal organelles discovered by Duve (see subcellular fractionation experiment). They are bound by a single bilayer ~.5 microns in diameter, are round and oval, have a pH of 4-5. The acidic pH is maintained by H+ ATPase proton pums in the lysosome membrane. Function: Lysosomes contain digestive enzymes called acid hydrolases (ex acid phosphatate, a marker enzyme for sub cell fractionation) The acidic pH enhances digestion. Macromolecules are partially denatures at pH of Lysosomes and thus degradstion enzymes have easier acces to break them down. Lysosomes have three origins: pagocytosis, ednocytosis, autophagy (degradation of ones own organells)
Targeting soluble lysosme hydrolases to the Lysosomes:
All of these proteins have N-glycocilated attatchments that are put there in the ER. AN address tar is attatched to lysosome proteins in the Golgi. When proteins reach the TGN they bind to recerptors for M6P. The receptor ligand complex in packaged into transport vesicles targeted… endosomes. Endosomes that fuse with the TGN habe a lower pH which causes the protein to dissacociate from the receptor.
Address tag
is mannose-6-phosphate and is selective for soluble lysosome proteins
endosomes
there is a gradient ranging form early to late endosomes. Later endosomes have a lower pH
retrograde tansport
movement in the opposite direction. The tag for retrograde transport is KDEL or HDEL (amino sequences) Used to bring proteins back to ER or Golgi
ER mem proteins
- all have SRP receptor. The retrival signal is at the amno term and is two lycines (KK) and two other aa YY (KKYY)
Targeting to the Golgi
needs resident enzymes in particular compartments. Retension to a particular compartment depends purely on the length of the trransmembrane segment.short segments in the CGN longerones in the TGN. They go through the pathway until they match the thickness of the membrane.
there are two types of vesicles
coated and uncoated.
Uncoated vesicles
vesicles are just membrane and are made for constitutive secretion, the default pathway. From TGN to plasma membrane. Can id cardo by immonoelectron microscopy ( secondary antibody is bound to colloid gold and can vary in size for diff lablng)
Coated vesicles
have a protein coat on the membrane and are made for transport to endosomes, Lysosomes, vacules, from the ER to Golgi and to the ER and Golgi. There are different coats for each. The coats cause curving of the membrane and aid to sort the cargo into specific vesicles.
the three types of protein coats on vesicles are
COP1 – Golgi to ER
COPII – ER to golgi
Calathryin –golgi to endosomes, Lysosomes and plasma membrane
COP1
vesicle protein coat that is on vesicles that go from the Golgi to the Er
COPII –
vesicle protein coat that is on vesicles that go from the ER to the golgi
Calathryin
protein coat on vesicles that go from the golgi to endosomes, lysosomes, and the plasma membrane
Plant vacuoles
are like Lysosomes in that they have a single membrane and size depends on maturity of the plant cell. They have an acidic pH ad function to maintain turgor pressure, store materials such as wastes, strore flavanoid pigment, toxic chemicals to protect against herbivory and some digestive enzymes.