Exam 2
The retrieval pathway (retrograde delivery) for returning escaped resident ER membrane proteins back to the ER utilizes __________________________________________.
COPI-coated transport vesicles
major function of diacylglycerol (DAG)
activates protein kinase C (PKC) - serine/threonine kinase - activation requires calcium and DAG (conventional PKC enzymes require both DAG and calcium) - conventional PKC enzymes; alpha, beta, and gamma - knockouts in mice reveal unique phenotypes
the 2 states of GTPases
active: Ga bound to GTP inactive: Ga bound to GDP - trimer of alpha, beta, and gamma - trimer may dissociate/change, but subunits stay together
second messenger/enzymatic cascade that amplifies signals
adenyl cyclase --> cAMP --> PKA --> activated enzyme --> product
signal integration
allows for complex cell behaviors to be regulated by specific combinations of signals rather than by a single signal acting alone
diacylglycerol (DAG) can be cleaved to release ______________________
arachidonic acid - messenger - used to synthesize eicosanoids prostaglandins-pain, inflammation
PKA in the nucleus
can modify gene regulatory proteins to alter gene expression - example: phosphorylation of CREB
What 2 molecules are made from phosphatidylinositol 4,5-bisphosphate?
diacylglycerol (DAG) - activates protein kinase C inositol (IP3) - released Ca2+ from ER
______________________________________ are delivered to lysosomes by a route from the ER via the Golgi apparatus
digestic enzymes
desensitization
enables cells to respond to changes in the concentration of an extracellular signal molecule (rather than to the absolute concentration of the signal) over a wide range of signal concentrations *inhibitory protein*
Endosome to lysosome pathway
endocytic vesicle --> early endosome --> multivesicular body --> late endosome --> lysosome
digestion process in the lysosome
hydrolytic enzymes + low pH degrade macromolecules, then release them into the cytoplasm as nutrients
endocytic pathway
*endocytosis* takes up macromolecules from the cell exterior and ingests them to produce metabolites as nutrients ER ---> Golgi ---> secretory vesicles ---> cell exterior | late endosome ---> lysosomes
Constitutive secretory (default) pathway
*exocytosis* - In all cells, transport vesicles destined for the plasma membrane leave the trans Golgi network in a steady stream - the membrane proteins and the lipids in these vesicles provide new components for the cell's plasma membrane - soluble proteins inside the vesicles are secreted to the extracellular space
Regulated secretory pathway
*exocytosis* - soluble proteins and other substances are initially stored in secretory vesicles for later release - this pathway is found in cells specialized for secreting products rapidly on demand—such as hormones, neurotransmitters, or digestive enzymes
biosynthetic-secretory pathway
*exocytosis* regulates the delivery of newly synthesized proteins, carbohydrates, and lipids to the cell exterior cell exterior ---> early endosome | late endosome ---> Golgi ---> ER | lysosome
structure of G-protein-coupled-receptors
- 7 short hydrophobic stretches form transmembrane alpha helices - "7-pass" transmembrane receptors
Process by which proteins go from ER to cis Golgi network
- COPII-coated transport vesicles - must be properly folded and, if they are subunits of multimeric protein complexes, may need to be completely assembled
cisternal naturation model
- Golgi is dynamic; the cisternae themselves move through the Golgi stack - vesicles that arrive from the ER fuse with one another to become a cis Golgi network - this network then progressively matures to become a cis cisterna, then a medial cisterna, and so on.
vesicular transport model
- Golgi is relatively static - processing enzymes held in place - molecules in transit are moved through the cisternae in sequence, carried by transport vesicles - retrograde flow retrieves escaped proteins and returns them to preceding compartments
Lysosomal pathway
- Hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases, and sulfatases (acid hydrolases) that are used for the controlled intracellular digestion of macromolecules are transported from the trans Golgi network to lysosomes. - carry a unique sorting marker in the form of mannose 6-phosphate (M6P) groups, which are added exclusively to the N-linked oligosaccharides of soluble lysosomal enzymes in the lumen of the cis Golgi network
the 3 mechanisms that terminate the initial calcium response
- IP3 is rapidly dephosphorylated and inactivated (its life is short) - some IP3 is phosphorylated (forms inositol 1,3,4,5 tetraphosphate (IP4) - promotes refilling of the intracellular calcium stores - the calcium that enters the cytosol is rapidly pumped out of the cell
cAMP response element binding protein (CREB)
- PKA catalytic subunits move into the nucleus - PKA phosphorylates CREB - phosphorylated CREB recruits histone acetyl transferases (HAT) - transcription is initiated - regulation of fuel metabolism, spermatogenesis, memory, and steroidogenesis
Gq protein
- a heterotrimeric G protein subunit that activates phospholipase C (PLC), which in turn activates Phosphatidylinositol 4,5-bisphosphate - distinct from the Trimeric G proteins that activate adenylyl cyclase
Caveolae-mediated endocytosis
- caveolae are flask shaped pits in the plasma membrane - they are thought to form from lipid rafts - Caveolae are thought to form vesicles.
autophagy
- cell eats own obsolete organelles - enclosure of an organelle by membranes of unknown origin, creating an autophagosome, which then fuses with a lysosome (or a late endosome)
lysosomal storage diseases
- diseases that are caused by genetic defects that affect one or more of the lysosomal hydrolases - defect results in the accumulation of the undigested macromolecules inlysosomes, with severe pathological consequences, most often in the nervous system - 40+ disorders in humans (Tay-Sachs)
the 3 pathways by which cells obtain food
- endocytosis - autophagy - phagocytosis
signal termination is accomplished by:
- enzymes that remove the modifications (phosphatases) - enzymes which degrade the intracellular signaling molecule - enforced conformation changes in the intracellular signaling molecule, which inactivates it
enzyme-coupled receptors
- function as enzymes or linked with enzymes - heterogeneous class - most are protein kinases
G-protein-coupled receptors
- indirectly regulate activity of membrane-bound target proteins - mediated by a trimeric G-protein
the 3 major classes of cell-surface receptor proteins
- ion-channel-coupled receptors - G-protein-coupled-receptors - enzyme-coupled receptors
endocytic pathway: late endosome
- late endosome has a mildly acidic interior - endocytosed materials first meet the lysosomal hydrolases - fuse w/ hydrolase-bearing transport vesicles from the trans Golgi network - pH becomes more acidic
Cyclic-AMP-dependent protein kinase (PKA)
- mediates most of the effects of cyclic AMP
Phosphatidylinositol 4,5-bisphosphate (PIP2; PI 4,5-bisphosphate)
- minor phospholipid in the inner half of the cell membrane (remains membrane bound) - least abundant (less than 10% of total inositol lipids) - formed by the phosphorylation of PI and PI(4)P, respectively - enzymes required; PI 4 kinase and PI 4 phosphate 5 kinase
intracellular signaling proteins
- proteins that become phosphorylated by protein kinases - proteins that are induced to bind GTP when the signal arrives
ion-channel-coupled receptors
- rapidly synaptic signaling between electrically excitable cells - mediated by neurotransmitters
trimeric G proteins
- relay signals from GPCRs - 3 subunits (alpha, beta, gamma) - attached to plasma membrane - G alhpa binds a guanyl nucleotide - various types, each specific for a set of GPCRs and for a particular downstream target, but they all operate the same way
cyclic AMP
- second messenger - activates proteins (including protein kinases)
endocytic pathway: early endosome
- some ingested molecules are sent to plasma membrane, some are degraded - early endosome = main SORTING compartment in endocytic pathway
clathrin-coated pits
- starting point of pinocytosis - 2% of plasma membrane - pits bud into the cell, form clathrin-coated vesicles - bring in small portion of membrane + fluid - coat is shed, remaining vesicle fuses with endosomes and proceeds down the endocytic pathway - about 2500 clathrin-coated vesicles leave the plasma membrane of a cultured fibroblast every minute
phosphodiesterases (PDEs)
- the only means of degrading cAMP PDE inhibitors: theophylline, caffeine, xanthines
endocytic pathway: multivesicular body
- while endosomes migrate deeper into the cytoplasm, regions of endosomal membrane invaginate to form internal vesicles - the membrane proteins that enter the internal vesicles are sequestered from the cytoplasm
lysosome production process
-gradual maturation process - endosomal membrane proteins are selectively retrieved from the developing lysosome by transport vesicles that deliver these proteins back to endosomes or the trans Golgi network.
mechanism of protein kinase C (PKC) activation by DAG and calcium
1. IP3-induced rise in cytosolic calcium (from ER) 2. Calcium binds to PKC-altered confirmation 3. PKC translocates from the cytosol to the cytoplasmic face of the plasma membrane 4. PKC binds to DAG (which stays on the plasma membrane) and is activated
the 6 steps for communication within a cell
1. Synthesis of signaling molecule 2. Release of signaling molecule 3. Transport the signal to target cell 4. Detection of the signal in target cell 5. Transmission of signal within the cell 6. Removal of signal
synaptic signaling
A nerve cell releases a neurotransmitter into the synapse. A synapse is the space or gap between two nerve cells. The neurotransmitter diffuses across the synapse to reach the target cell. The neurotransmitter binds to receptor proteins at the surface of the target cell. *neurotransmitter* "phone conversation"
Ca2+ sensitive processes
Contraction Proliferation Fertilization Learning and memory Secretion Vesicle trafficking Cell proliferation Metabolism
ER to Golgi pathway
ER --> cis Golgi network --> cis cisterna --> medial cisterna --> trans cisterna --> trans Golgi network --> Golgi apparatus
Gs/Gi
G stimulatory: stimulates target enzyme G inhibitory: inhibits target enzyme
A third of all drugs work through ____________________ receptors
G-protein-coupled
Receptor-mediated endocytosis
Macromolecules bind to specific transmembrane receptor proteins, accumulate in coated pits, and then enter the cell as receptor-macromolecule complexes in clathrin-coated vesicles. thousands of times more efficient than pinocytosis 25+ receptors Examples: cholesterol import by the low-density lipoprotein (LDL) receptor and iron import by the transferrin receptor
the 2 classes of proteins that control the recognition step of vesicular transport
Rabs—initial docking and tethering SNAREs—provide specificity & catalyze fusion
major biological functions of PKC
Regulation of Proliferation Regulation of Apoptosis Regulation of Differentiation Regulation of Cell migration and Adhesion
contact-dependent signaling
The signal molecule is bound to the plasma membrane rather than secreted; it interacts directly with the receptor on the surface of the target cell example: immune cells (antibody/antigen)
paracrine signaling
The transmitting cell secretes the local regulator (chemical signal) into the fluid surrounding the cells of a tissue. The local regulator diffuses across the liquid. The local regulator binds to protein receptors at the cell surface of the target cells. The target cells respond. *local mediator/regulator* "conversation at a party"
Golgi apparatus
a complex system of membrane-limited sacs and vesicles that is mainly devoted to the modification and packaging of proteins and protein-polysaccharide complexes synthesized in the ER -the stack of membranes has a definite polarity with those near the ER (the cis face) having a different shape and enzyme content than those at the opposite end (the trans or maturing face) -contains glycosylation enzymes that add sugars to proteins while other sugars are removed as they move through its lumen -the type of glycosylation that takes place is dependent upon signals contained within the protein sequence -also processes proteins using proteases, which clip at specific amino acid sequences to form mature proteins and hormones -Golgi stacks also sort proteins for secretion -after sorting, the membrane of the Golgi buds off, forming secretory vesicles that transport proteins to their specific destination -a protein's destination is often signaled with a specific amino acid sequence at its end
synapse
a junction between two nerve cells, consisting of a minute gap across which impulses pass by diffusion of a neurotransmitter
protein kinase
a kinase enzyme that modifies other proteins by chemically adding phosphate groups (from ATP) to them (phosphorylation)
Endocytosis
a process whereby cells absorb material from the outside by engulfing it with their cell membrane big, polar molecules that can't pass through the hydrophobic membrane
internal structure of endosomes
a set of heterogeneous, membrane-enclosed tubes/vesicles extending from the periphery of the cell to the perinuclear region pH = 5 (cytosomal pH = 7)
second messengers
a substance whose release within a cell is promoted by a hormone and that brings about a response by the cell examples: calcium, cAMP
phagosome
a vacuole in the cytoplasm of a cell, containing a phagocytosed particle enclosed within a part of the cell membrane forms lysosome
autocrine signaling
cell secrets a chemical messenger (autocrine agent) that binds to autocrine receptors in the same cell, leading to changes within the cell example: cytokine interleukin-1 in monocytes
Where does sorting occur in the Golgi apparatus?
cis & trans Golgi networks
Golgi stack
cis cisterna + medial cisterna + trans cisterna
coated vesicles
coat proteins are removed from the vesicle after it is formed and used to help coat newly formed vesicles from the membrane examples: clathrin, COPI (coatmer I), COPII (coatmer II)
COPI (coatmer I)
coated vesicle mediates both biosynthetic (anterograde) and retrograde transport within the Golgi apparatus AND cis Golgi ---> rough ER.
COPII (coatmer II)
coated vesicle mediates transfer from the rough ER to the cis Golgi network
clathrin
coated vesicle trans Golgi ---> lysosomes plasma membrane ---> endosomes
Pinocytosis is a _______________________ process
constitutive
the 3 branches of post-Golgi vesicular traffic
constitutive secretory (default) pathway (exocytosis) regulated secretory pathway (exocytosis) lysosomal pathway
During their passage through the Golgi apparatus, transported proteins undergo an ordered series of __________________________________.
covalent modifications: glycosylation (Golgi stack) phosphorylation (cis Golgi network) sulfation (trans Golgi network) cis --> medial --> trans
cAMP exerts its effects mainly by activating ___________________
cyclic‐AMP‐dependent protein kinase (PKA) - serine/threonine kinase - 2 types: in cytosol or anchored to plasma membranes
Cargo concentration _______________ as it moves from Golgi to secretory vesicles
increases
_____________________ have an important role in scavenging senescent and dead cells
macrophages
In mammals, three classes of white blood cells act as professional phagocytes:
macrophages, neutrophils, and dendritic cells defend us against infection by ingesting invading microorganisms
budding
occurs first from the lumen of the ER to form a vesicle containing materials to be transported and fuse with the membrane of the target organelle/cell membrane to release contents
lipid rafts
patches of the plasma membrane that are especially rich in cholesterol and sphingolipids
In protozoa, _____________________ is a form of feeding.
phagocytosis - large particles taken up into phagosomes end up in lysosomes - the products of the subsequent digestive processes pass into the cytosol to be utilized as nutrients
the 2 types of intracellular signaling processes that act as molecular on/off switches
phosphorylation GTP binding
________________ is a synonym for endocytosis
pinocytosis
the 2 types of endocytosis
pinocytosis (cell drinking) phagocytosis (cell eating) type depends on size of endocytic vesicles
kinds of signaling molecules
proteins small peptides amino acids gases lipids steroids thyroid hormone, retinoic acid, vitamin D3 neurotransmitters
SNAREs
provide specificity & catalyze fusion v-SNAREs - on vesicles t-SNAREs - on target membranes both interact to aid in vesicles' fusion specificity
GPCR desensitization depends on ___________________________.
receptor phosphorylation - carboxy terminal tail of the receptor becomes phosphorylated ‐ phosphorylated tail is bound by proteins (arrestins) which sterically blocks receptor/G‐protein interaction
How do vesicles select their cargo?
specific cargo receptors, sorting signals
PKA in the cytoplasm
targets are often enzymes involved in metabolic pathways
autolysis
the destruction of cells or tissues by their own enzymes, especially those released by lysosomes *lysosome rupture*
The biosynthetic-secretory pathway and endocytic pathway form a linked and balanced process:
the endocytic-exocytic cycle
pinocytosis
the ingestion of liquid into a cell by the budding of small vesicles from the cell membrane the uptake of solutes and single molecules such as proteins via a small pinocytic vesicle (100nm) *major route for endocytosis in most cells*
phagocytosis
the process by which cells ingest large objects, such as dead cells, bacteria, or viruses via a large vesicle (250nm) In order to maintain a cell's surface area and volume, the same amount of membrane that is being removed by endocytosis is being added to the cell surface by exocytosis
Hydrolysis of GTP (leading to inactivation of trimeric G-protein) is enhanced by ________________________ and ___________________________.
the target enzyme & RGS proteins (regulator of G‐protein signaling)
GTP binding
to turn on: a GTP-binding signaling protein is induced to exchange its bound GDP for GTP to turn off: GTP hydrolysis - phosphate is removed and GDP remains
phosphorylation
to turn on: a phosphate is added covalently to the signaling protein by a protein kinase to turn off: protein phosphatase removes phosphate group from signaling molecule
Where is mannose 6-phosphate (M6P) recognized by the M6P receptor?
trans Golgi network - M6P receptor interacts with specific adaptor proteins in the TGN that recruit the clathrin coat - low pH in late endosome causes ligand-receptor dissociation: receptor is recycled to the TGN, ligand is transported to the lysosome
Phagocytosis is a ______________________ process
triggered
first messengers
typically extracellular signaling molecules such as hormones; bind to receptors on plasma membrane
exocytosis
vesicles fuse with the plasma membrane Constitutive secretory (default) pathway Regulated secretory pathway - secretory vesicles
transcytosis
vesicular transport of macromolecules from one side of a cell to the other, - strategy used by multicellular organisms to selectively move material between two environments without altering the unique compositions of those environments. - most common in epithelial cells but present elsewhere - example: antibody secretion from mammary epithelial cells, antibody absorption by gut epithelium of newborn animals. (passive immunity)