Chapter 8: Endomembrane Systems Flashcards
Organelles in the endomembrane system
ER Golgi Complex Endosomes Lysosomes Vacuoles
Materials are shuttled between organelles in membrane-bound __________
Materials are shuttled between organelles in membrane-bound TRANSPORT VESICLES
The membrane of a vacuole is called the ____
tonoplast
Biosynthetic pathway
synthesis, modification, and transport of proteins
Secretory pathway:
constitutive vs regulated secretion
Secretory pathway: when proteins are discharged from cell.
Constitutive Secretion: in a CONTINUOUS FASHION
Regulated Secretion: in response to STIMULI
During regulated secretion, materials to be secreted are stored in large, membrane-bound ____ _____
secretory granules
T/F: Cells remain alive in the experimental approach of autoradiography and electron microscopy
False. Cells are fixed (killed)
T/F. Cells remain alive in the experimental approach of green fluorescent protein labelling
True
Which experimental approach involves the fractionation of cells?
Biochemical analyses
Pulse-Chase experiment object and general method
Purpose: to track proteins from pancreatic mucus-secreting cells using AUTORADIOGRAPHY.
Method: A pulse of radioactive amino acids was given to the cell followed by a chase (addition of non-radioactive amino acids).
First trial = 3 min pulse. No chase.
Second Trial = 3 min pulse. 17 min chase.
Third Trial = 3 min pulse. 117 min chase.
Results of the pulse-chase experiment
NOTE: This was the first developed strategy of observing movement of proteins from ER to Golgi.
First trial = 3 min pulse. No chase.
Result: radioactive aa’s incorporated in the rough ER
Second Trial = 3 min pulse. 17 min chase.
Result: radioactive aa’s incorporated in the Golgi Complex and some in vesicles.
Third Trial = 3 min pulse. 117 min chase.
Result: radioactive aa’s incorporated in secretory vesicles and in the extracellular side.
GFP?
- stands for Green Fluorescent Protein.
- isolated from jellyfish
- GFP is fused to DNA encoding the protein to be studied
- useful for tracking protein movement and protein synthesis
Describe the experiment involving GFP and VSV virus
- VSVG gene in the VSV virus was fused with GFP causing green proteins to be synthesized in the cell.
- VSVG also contained a temp-sensitive mutation that prevented newly-synthesized VSVG to leave the ER at 40 degrees.
- mutated VSVG accumulated in the ER when left at 40 degrees.
- after a 10 minute incubation at 32 degrees, VSVG was observed to move to the Golgi Complex.
Describe the successive fractions and pellets after every centrifugation
- centrifuge homogenate:
- postnuclear supernatant
- nuclear pellet: contains whole cells and nuclei
- centrifuge postnuclear nupernatant:
- post-mitochondrial supernatant
- mitochondrial pellet
- centrifuge post-mitochondrial supernatant:
- post-microsomal supernatant
- microsomal pellet: contains microsomes
How to separate smooth and rough ER
- centrifuge homogenized smooth and rough ER in a tube with gradient of INCREASING SUCROSE CONCENTRATION.
- smooth microsomes have a low density, stop sedimenting, and will float at low sucrose concentration.
- rough microsomes have high density, stop sedimenting, and float at high sucrose concentration.
What is a cell-free system?
- method of analysis which does not contain full cells.
- provides info about the roles of the proteins involved in membrane trafficking.
ex: extraction of rough ER via diff. centrifugation after homogenization. Isolated ribosomes continue to synthesize proteins.
What occurred when the gene for vesicle formation in the ER was mutated? (Sec12 mutant)
- When Sec12 was mutated, protein build up occured in the ER due to inability of vesicles to bud off.
- ER INCREASED IN VOLUME.
What occurred when the gene for vesicle fusion in the ER was mutated? (Sec 17 mutant)
- When Sec17 was mutated, vesicles could not fuse with Golgi.
- ACCUMULATION OF UNFUSED VESICLES IN THE CYTOPLASM.
Describe RNA interference
- RNA interference is a process in which cells produce small RNAs (siRNAs) that bind to SPECIFIC mRNAs and inhibit the translation of these into proteins.
- process allows scientists to identify genes involved in a particular process by determining which siRNAs interfere with that process.
Network of flattened sacs in the RER is called ____
cistenae
T/F: the luminal or cisternal space inside ER membranes has the same composition as the surrounding cytosol
FALSE
ya ninny
The ribosomes on the rough ER are present on the ______ space
CYSTOLIC space
Functions of SER
- Synthesis of steroid hormones in endocrine cells.
- Detoxification in the liver of various organic compounds
- sequestration of calcium ions into the cytoplasm of muscle cells.
Functions of RER
-starting point of the BIOSYNTHETIC PATHWAY. Aka, it is the site of synthesis of proteins, carbohydrate chains, and phospholipids that journey through the membranous compartments of the cell.
Polypeptides synthesized by membrane-bound vs. free ribosomes.
- Polypeptides synthesized by ribosomes in the RER include secreted proteins, integral membrane proteins, and soluble proteins of organelles.
- Polypeptides synthesized by free ribosomes include 1/3 of those encoded by the human genome, cytosolic proteins, peripheral membrane proteins, nuclear proteins, and proteins incorporated into chloroplasts, mitochondria, and peroxisomes
What is the N-terminal portion of a polypeptide?
The first part to emerge from the ribosome during protein synthesis
Secretory proteins contain a ___________ at their N-terminus that directs the emerging polypeptide and ribosome to the __________
Secretory proteins contain a SIGNAL SEQUENCE at their N-terminus that directs the emerging polypeptide and ribosome to the ER MEMBRANE.
What do polypeptides move through to reach the cisternal space of the ER?
polypeptides move through a protein-lined, aqueous channel
- Cotranslational movement
2. Posttranslational movement
- Cotranslational movement
- polypeptides moving through the membrane as they are being synthesized. - Posttranslational movement
- polypeptides are synthesized fully in the cytosol and then imported into ER lumen
Secretory proteins synthesized on RER ribosomes have their signal sequence recognized by ________.
SRP (Signal Recognition Particles)
What does SRP do?
as a signal sequence emerges from a free ribosome, it binds to SRP, which stops translation until the SRP-ribosome complex can make contact with the ER membrane.
To what does the SRP-ribosome complex attach?
The SRP-ribosome complex collides with and binds to an SRP RECEPTOR situated WITHIN THE ER MEMBRANE.
What occurs upon the binding of an SRP-ribosome complex with an SRP-receptor on the ER membrane?
- The SRP is released (along with the hydrolysis of GTP)
- The ribosome associates with a TRANSLOCON of the ER membrane.
- The signal sequence binds to the interior of the translocon.
- The remainder of the polypeptide translocates through the membrane COTRANSLATIONLLY.
What kind of proteins are needed for the release of SRP from the SRP-ribosome-nascent polypeptide chain complex?
GTP-binding proteins (G proteins)
What occurs once the nascent (newly formed or in the process of being synthesized) polypeptide passes into the lumen of the ER?
the signal peptide is CLEAVED by a SIGNAL PEPTIDASE.
What is the translocon?
the translocon is a protein-lined channel embedded in the ER membrane through which the nascent polypetide can move from the ribosome to the ER lumen.
Carbohydrates are added to the nascent protein in the lumen of the ER by the enzyme __________
oligosaccharyltransferase
T/F: both oligosaccharyltransferase and signal peptidase are peripheral membrane proteins.
FASLE. Both oligosaccharyltransferase and signal peptidase are INTEGRAL membrane proteins associated with the translocon.
Function of protein disulfide isomerase (PDI)?
adds disulfide bonds to cystein.
hydrophobic proteins contain _____ transmembrane segments that interfere transfer into RER lumen.
HYDROPHOBIC
T/F: Membranes arise from preexsting membranes
True. Lipids are inserted into existing membranes.
T/F: cellular membranes are symmetric.
False. Cellular membranes are ASYMMETRIC. The two leaflets of a membrane have different compositions.
Newly synthesized phospholipids are inserted into half of the bilayer facing the _______, and then flipped into the opposite leaflet by __________
Newly synthesized phospholipids are inserted into half of the bilayer facing the CYTOSOL, and then flipped into the opposite leaflet by FLIPPASES.
Addition of sugars to an olygosaccharide chain is catalyzed by _________
glycosyltransferase
Name of the lipid carrier on which the basal segment of each carbohydrate is assembled and then transferred to specific asparagine residues of the polypeptide.
Dolichol Phosphate
Where is the dolichol phosphate located?
embedded in the ER membrane
Core segment of each carbohydrate chain is put together on __________ and then transferred to a ________
Core segment of each carbohydrate chain is put together on THE LIPID CARRIER, DOLICHOL PHOSPHATE and then transferred to a POLYPEPTIDE
The core carbohydrate is modified by ________ as the polypeptide is transferred into the ER lumen.
oligosaccharyltransferase
What does NAG stand for in glycosylation?
N-acetylglucosamine
Steps of glycosylation in the RER
- The first 7 sugars (2 NAG and 5 Mannose residues) are transferred one at a time to the dolichol phosphate on the CYTOSOLIC SIDE of the ER membrane.
- Dolichol with its attached oligosaccharide is then FLIPPED across the membrane.
- The remaining sugars (4 mannose and 3 glucose residues) are attached on the cytosolic side of the membrane to a dolichol phosphate molecule which then flips across the membrane and donates its sugar to the growing end of the oligosaccharide chain.
- Once the oligosaccharide is fully assembled, it is transferred enzymatically to an ASPARAGINE RESIDUE of the NASCENT POLYPEPTIDE.
- The dolichol phoshate is flipped back across the membrane and is ready to begin accepting sugars again.
The glycoprotein goes through a system of __________ to determine its fitness for a specific compartment.
QUALITY CONTROL
What happens to misfolded proteins?
they are tagged by a terminal glucose and recognized by CHAPERONES for refolding.
What does glucosyltransferase do to a misfolded protein?
adds a glucose to the end of the olygosaccharide chains. Glycoproteins containing MONOGLUCOSYLATED OLIGOSACCHARIDES are recognized by membrane-bound CHAPERONE CALNEXIN and given an opportunity to achieve their correctly folded (native) state. If they can’t, they are destroyed.
Where are misfolded proteins destroyed?
in PROTEASOMES in the cytosol
ER-associated degradation (ERAD)
process by which misfolded proteins and destroyed by proteasomes.
Ensures that misfolded proteins do not reach the cell surface.
What does the accumulation of misfolded proteins trigger?
UPR: unfolded protein response
Proteins that fail quality control are tagged with ____
UBIQUITIN
What keeps the misfolded protein sensors inactive?
The molecular chaperone, BiP
What causes BiP molecules to enter the ER lumen to act as chaperones for misfolded proteins? What does this influx result in?
accumulation of misfolded proteins. This results in the activation of sensors (PERK an ATF6) which monitor the concentration of misfolded proteins.
What does the activation of [misfolded proteins] sensors do?
activated sensors (PERK and ATF6) send signals to trigger proteins involved in DESTRUCTION of misfolded proteins.
What are PERK and ATF6?
- Sensor proteins.
- Sense accumulation of misfolded proteins.
- Kept inactive by BiP.
Explain what happens when sensor proteins are activated in UPR
1st strategy: Blocking translation of misfolded proteins.
-Once sensors are activated, DIMERIZATION takes place between PERK and ATF6. Once protein is DIMERIZED, it interacts with TRANSLATION FACTOR, eF2a, which gets phosphorylated.
This HALTS TRANSLATION of the misfolded protein.
2nd strategy: Increasing the transcription of chaperone mRNA and thus alleviating stress of the cell.
ERGIC
“endoplasmic reticulum Golgi intermediate compartment.”
multiple transport vesicles fused together toward the Golgi complex.
The ___ face of the Golgi complex faces the ER; the ____ face is on the opposite side of the stack.
The CIS face of the Golgi complex faces the ER; the TRANS face is on the opposite side of the stack.
CGN function
“cis Golgi network” functions to sort proteins for the ER or the next Golgi station
TGN function
‘trans Golgi network” functions in sorting proteins either to the membrane or various intracellular destinations.
Assembly of carbohydrates found in glycolipids and glycoproteins takes place in the ____
Golgi
Cisternal maturation model
- model of movement of materials through Golgi complex.
- each cistern “matures” as it moves from the cis to the trans face.
Vesicular transport model
- model of movement of materials through Golgi complex.
- cargo is shipped from CGN to TGN in vesicles
2 functions of protein coats on vesicles:
- cause membrane to curve to form a vesicle
2. select the components to be carried by vesicle
Types of coated vesicles:
- COPII-coated vesicles
- COPI-coated vesicles
- Clathrin-coated vesicles
COPII-coated vesicles:
move material from the ER “forward” to the ERGIC and Golgi complex
COPI-coated vesicles
move materials from ERGIC and GOlgi “backwards” to ER, or from trans Golgi to cis Golgi cisternae.
Clathrin-coated vesicles
move materials from the TGN to endosomes, lysosomes, and plant vacuoles
Sar1
a small G protein which plays a regulatory role in vesicle assembly.
Sar1-GTP binds to the ER
Vesicle disassembly is triggered by the hydrolysis of ______ which produces ________
Vesicle disassembly is triggered by the hydrolysis of GTP which produces Sar1-GDP
Proteins are maintained in an organelle by two mechanisms:
- RETENTION of resident molecules that are excluded from transport vesicles.
- RETRIEVAL of “escaped” molecules back to compartment where they reside
What type of coated vesicles are in charge of transporting escaped proteins BACK TO THE ER?
COPI-coated vesicles
Resident proteins of the ER contain an amino acid sequence at the C-terminus serving as a _______ signal
retrieval signal
LYSOSOMAL proteins are tagged with __________ and recognized/captured by __________
LYSOSOMAL proteins are tagged with PHOSPHORYLATED MANNOSE RESIDUES and recognized/captured by MPRs (mannose-6-phosphate receptors).
Location: RER membrane
Tag: ?
SRP
Location: ER resident proteins
Tag: ?
KDEL
Location: ?
Tag: KKXX
membrane-bound proteins in RER
Location: ?
Tag: Phosphorylated mannose residue
Lysosomes
Location: peroxisome digestive enxyme
Tag: ?
PTS (peroxisomal targeting signal)
Location: ?
Tag” presequence
Mitochondria
Location: ?
Tag: internal targetting sequence
inner mitochondrial membrane proteins
Location: Chloroplast stroma proteins
Tag: ?
stroma-targeting domain
Location: Thylakoid membrane proteins
Tags: ?
stroma-targeting domain and thylakoid transfer domain
Lysosomal enzymes are transported from the TGN in _____-coated vesicles by adaptor proteins, _____.
Lysosomal enzymes are transported from the TGN in CLATHRIN-coated vesicles by adaptor proteins, GGAs.
The coats of clathrin-coated vesicles contain:
- outer lattice composed of clathrin
2. inner shell composed of PROTEIN ADAPTORS
Funtion of ARF1
“adenosylation ribose factor 1” is a G PROTEIN required for vesicle transfer BETWEEN CISTERNAE.
Role of tethering proteins
Different tethering proteins INITIATE FUSION between different types of membranes
Role of Rab proteins
Rabs (G proteins) on the vesicle and target membrane are involved in RECRUITING TETHERING PROTEINS that mediate initial contact between two membranes.
Interactions between the ______ and ________ pull the two lipid bilayers together during fusion of a vesicle to a target membrane.
t-SNAREs (located on the target) and v-SNAREs (incorporated into vesicle)
Proteins going into the nucleus have an ___ sequence
NLS
what is a “fusion pore”
- point at which membranes are fused together.
- formed by interaction of v- and t-SNAREs
How many a-helical proteins do v- and t-SNAREs each have? What do these a-helical strands do?
- 2 each.
- a-helical strands interact and provide the mechanical force necessary to allow vesicle and target membrane to fuse.
The process of exocytosis is triggered by an increase in concentration of what ion?
Ca2+ from cytoplasmic stores
Contact between a vesicle and plasma membrane lead to the formation of
FUSION PORE
When a cytoplasmic vesicle fuses with the plasma membrane, the _____ surface of the vesicle becomes part of the OUTER surface of the plasma membrane, whereas the ______ surface of the vesicle membrane becomes part of the INNER (cytosolic) surface of the plasma membrane.
When a cytoplasmic vescle fuses with the plasma membrane, the LUMINAL surface of the vesicle becomes part of the OUTER surface of the plasma membrane, whereas the CYTOSOLIC surface of the vesicle membrane becomes part of the INNER (cytosolic) surface of the plasma membrane.
What are lysosomal enzymes called?
acid hydrolases
The inside of lysosomes has a ____ pH (high or low?)
Low (acidic)
What maintains the low pH optimum for acid hydrolases in lysosomes?
ATPase proton pumps on the membrane
What does it “lysosomes play a key role in organelle turnover” mean?
it means that lysosomes play a role in the regulated DESTRUCTION of the cell’s OWN ORGANELLES and their replacement (ie. autophagy)
Describe the general steps of autophagy
an organelle is surrounded by a double membrane structure (PHAGOPHORE) to produce a double-membrane sequestering vesicle called a AUTOPHAGOSOME.
The autophagosome outer membrane fuses with a lysosome, generating an AUTOLYSOSOME, in which both the inner membrane of the autophagosome and the enclosed contents are degraded.
T/F: plant vacuoles contain acid hydrolases
true
2 categories of endocytosis:
- pinocytosis
- receptor-mediated endocytosis
Endocytosis vs. phagocytosis
Endocytosis: uptake of cell surface receptors and BOUND extracellular ligands.
Phagocytosis: uptake of patriculate matter
Pinocytosis:
NONSPECIFIC uptake of extracellular fluid or ions
Receptor-mediated endocytosis
uptake of SPECIFIC extracellular ligands following their binding to receptors
Substances that enter the cell through RME (receptor-mediated endocytosis) become bound to ____ on the plasma membrane
COATED PITS
What shape do clathrin-coated pits resemble?
polygons (like a honeycomb)
Clathrin contains ___ (number) chains that form a _____
Clathrin contains THREE chains that form a TRISKELION
Clathrin-coated vesicles contain ____ between clathrin and membrane
adaptors
ex: AP2
What is dynamin?
dynamin is a G protein required for the release of a clathrin-coated vesicle from the membrane where it forms.
Dynamin acts as an enzyme that uses energy from GTP to provide mechanical force.
Explain dynamin’s role in the formation of clathrin-coated vesicles
Steps:
1. clathrin-coated vesicle forms connected to the plasma membrane by a stalk.
2. dynamin subunits undergo POLYMERIZATION to form a ring around the stalk.
3(a). GTP hydrolysis causes changes in ring conformation which leads to FISSION of the coated vesicle from the plasma membrane and DISASSEMBLY of the dynamin ring.
OR
3(b). If GTPγS is present, dynamin polymerization continues, producing a narrow tubule.
Following internalization, vesicle-bound materials are transported in vesicles and tubules known as _______
endosomes
Where are early endosomes located?
near the periphery of the cell, where materials are sorted and sent bound to the late endosomes
Where are late endosomes located?
near the nucleus.
LDLs stands for
low-density lipoproteins
LDLs are a complex of ______ and _____
cholesterol and proteins
LDL receptors are transported to the _____________ and bound to a ___________
LDL receptors are transported to the PLASMA MEMBRANE and bound to a COATED PIT
How are LDLs taken up by the cell?
by Receptor-Mediated Endocytosis (RME)
What do LDLs release?
cholesterol
What is the role of HDLs?
“High-density lipoproteins” transport CHOLESTEROL from tissues to the LIVER for excretion.
Which is associated with low blood cholesterol? HDLs or LDLs?
HDLs are associated with LOWERING cholesterol.
LDLs are associated with HIGH blood cholesterol
What does the accumulation of LDLs lead to?
plaque formation on the inner walls of blood vessels.
Can lead to a HEART ATTACK
In phagocytosis, the plasma membrane takes up large particles and pinches off to form a ____________
phagosome
The phagosome fuses with a __________ and the material is digested within the ________
The phagosome fuses with a LYSOSOME and the material is digested within the PHAGOLYSOSOME
The engulfment of particles by PHAGOCYTSIS is driven by _______________
actin-containing MICROFILAMENTS
Uptake of proteins into a peroxisome requires a ________ _______ signal
peroxisomal targeting signal (PTS)
Mitochondrial proteins are tagged with a
PRESEQUENCE
Peroxisomes
membrane-bound organelles that contain enzymes which degrade hydrogen peroxide and free radicals
T/F: movement of proteins into the mitochondria is voltage-dependent.
TRUE
What is the name of the protein-import complex on the OUTER mitochondrial membrane?
TOM complex
What is the name of the protein-import complex on the INNER mitochondrial membrane and what 2 major complexes does this complex include?
TIM complex: includes TIM22 and TIM23
the protein containing a presequence interacts with a _____ protein which unfolds it and sends it into the mitochondria via a _____ complex on the outer mitochondrial membrane.
the protein containing a presequence interacts with a CHAPERONE protein which unfolds it and sends it into the mitochondria via a TOM complex on the outer mitochondrial membrane.
Once inside the mitochondria, ____ proteins work to refold the protein
CHAPERONE
Proteins headed to the chloroplast contain a sequence called:
TRANSIT PEPTIDE sequence
