1
Q
What is the endomembrane system and what does it include
A
- A network of membrane-bound organelles that function together to transport, process and sort things in the cell
- ER, Golgi, endosomes, lysosomes and vacuoles
2
Q
Why are chloroplasts and mitochondria not part of the endomembrane system?
A
- They fxn independently, possess their own DNA and are not interconnected with the membrane trafficking system
3
Q
How are materials packaged for transport in the endomembrane system
A
- In small vesicles that bud off donor compartments, transported by motor proteins and fuse with the acceptor component
4
Q
What is the role of motor proteins in vesicle transport
A
- Kinesins and Dyneins move vesicles along microtubules for precise delivery to target locations
5
Q
What is the difference between biosynthetic and secretory pathways
A
- Biosynthetic pathway = involves protein and lipids synthesis in the ER, modification in the golgi and transport to various destinations
- Secretory pathway = discharge of synthesized proteins to the cell surface
6
Q
Describe the process of constitutive secretion
A
- Involves the continuous transport of materials in secretory vesicles to the cell surface
7
Q
What triggers regulated secretion and what it is the difference between constitutive (CS)?
A
- Requires an external signal (hormones, digestive enzymes neurotransmitter) to trigger the release of stored materials and CS does not
- Occurs in endocrine cells, pancreatic acinar cells and nerve cells
8
Q
The function of sorting signals in the biosynthetic pathway
A
- they are amino acid sequences that ensure proteins and lipids are delivered to the correct place with destination-specific information
9
Q
How does the release of neurotransmitters (regulated secretion) relate to [Ca2+]?
A
- This is triggered by an increase in [Ca2+] stimulating vesicle fusion with the plasma membrane
10
Q
Why are secretory granules importing in endocrine and nerve cells?
A
- They store hormones, enzymes or neurotransmitters so they release them with specific stimuli
11
Q
What is autoradiography? how is it used to study the endomembrane system
A
- It involves using radioactively labeled molecules to visualize and track biochemical processes
- Labeled aa’s were sent into pancreatic enzymes to trace their synthesis and transport (P and J)
12
Q
What is the purpose of the “pulse-chase” experiment
A
- It identifies the pathway of secretory proteins by tracking their movement after being exposed to labeled aa’s (pulse) to see their site of synthesis and discharge
13
Q
How does GFP-tagging help visualize protein movement in living cells?
A
- GFP (green fluorescent protein) tagging involves attaching a fluorescent GTP to a protein to see its movement in living cells.
14
Q
What is the role of vesicular stomatitis virus (VSV) in protein movement
A
- The virus has a GTP-tagged viral gene to express GTP and hijack the host cell machinery for protein synthesis
- GTP is useful bcos its non-toxic, it integrates well with viral proteins, and it can fluoresce without assistance
15
Q
What is the purpose of cell homogenization in studying the endomembrane system
A
- It breaks cells open to release their contents while preserving subcellular organelles.
- You can isolate ER or golgi for analysis
- Differential centrifugation separates cellular components based on their size/density.
16
Q
How are microsomes formed
A
- Small fragments from the ER when cytoplasmic membranes are disrupted during homogenization
- Smooth microsomes from smooth ER and rough ones are from the rough ER
17
Q
What is density gradient centrifugation
A
- Separating organelles/vesicles based on their buoyant density by spinning in something of increasing density. This is for precise isolation
18
Q
What is the structure and primary function of the endoplasmic reticulum (ER)
A
- It is a network of interconnected membranes that are involves in protein synthesis, lipid metabolism, Ca2+ storage etc
- Rough and smooth ER
- Cisternae make up the rough ER by providing surface area for ribosomes to attach
- Lumen specializes in protein folding, modification and storage
19
Q
What is the difference between smooth and rough ER
A
- RER: It has ribomses on its cytosolic end and flattened sacs (cisternae) and is responsible for synthesizing membrane-bound and secretory proteins. The starting point of the biosynthetic pathway
- SER: no ribosomes and tubular membranes. Used for lipid and steroid hormone synthesis, detoxification (cyt P450) and Ca2+ storage.
20
Q
Explain the synthesis of proteins in the RER
A
- Secretory proteins have aa’s on the n-terminus of the polypeptide with a signal sequence directing it to the cisternae
- This is co-translational translocation by the signal sequence and the translocon channel
21
Q
Protein synthesis on free ribosomes
A
- The ribosome has mRNA and tRNA
- SRP binds to the ribosome to stop translation till it reached the ER membrane
- SRP binds to its receptor which allows the ribosome to be associated with the translocon with the peptide binding to its interior
- Translocon gets unplugged and the peptide gets translocated through the membrane cotranslationally to undergo folding with a chaperone (BiP)
- GTP hydrolysis triggers peptide cleavage by the SRP
- Lysosomes/vacuoles are synthesized by membrane-bound ribosomes of the RER.
22
Q
What role does the signal recognition particle (SRP) play in protein targeting
A
- Binds to the nascant peptide to halt translation temporarily.
- Guides the ribosome-peptide complex to the translocon to resume translation and the protein enters the ER
23
Q
Why is GTP hydrolysis important for protein trafficking in the RER
A
- This is caused by SRP and its receptor and it provides energy to release the complex to the translocon
24
Q
What is glycosylation
A
- Post-translational enzymatic addition of sugars to proteins, happens in the ER.
- Glycosyltransferases are enzymes that catalyze the addition (specialized to transfer a specific monosaccharide)
- N (Nitrogen + sugar glucosamine) and O-linked (Oxygen + sugar galacatosamine) glycosylations
25
What is the quality control mechanism for glycoproteins in the RER
- This ensures that only correctly folded glycoproteins go through the secretory pathway
- Misfolded ones remain in the ER and are tagged for degradation (ERAD)
26
Explain the glycosylation process in the RER
- Sugars are attached to dPP in the cytosol and they’re flipped into the RER lumen
- more sugars are attached one at a time to form an oligosaccharide with **glc, mannose and N-acetylglucosamine (NAG)**
- The oligosaccharide is transferred to the Asn on the nascent polypeptide
27
What is the **unfolded protein response (UPR)**
- It is a cellular stress response that is activated by the **accumulation of misfolded proteins** in the ER. (two pathways)
1
- The sensors are associated with BiP and are **inactive**
- When the # of misfolded proteins **increase**, BiP goes to help with folding and sensors are **activated** = PERK (kinase)
- PERK phosphorylates a translation factor that is **needed for protein synthesis** which stops initiation
- This gives more time for processing proteins
2
- The release of BiP allows an unbound sensor to go to the **golgi** where it cleaves the **cytosolic domain** of the sensor
- This sensor diffuses thru cytosol to the nucleus where it allows the expression of genes = proteins that reduce ER stress
28
What is the difference between the **cis** GN and the **trans** GN
- CGN: faces the **ER** and sorts proteins for either **recycling** to the ER or **forwarding** to the golgi
- TGN: faces the **plasma membrane** and is responsible for sorting proteins to their final destinations
29
Describe **vesicular transport model** and the **cisternal maturation model**
- Vesicular: Cargo moves between **static golgi cisternae** by vesicles
- Cisternal: the golgi cisternae mature, moving from **cis to trans** while carrying cargo
30
How do **coated vesicles** facilitate transport between compartments
- (COPI, COPII or clathrin-coats)
- Provide structural support for **vesicle formation** and recruit cargo molecules for transport
31
What are the differences between the **COPI, COPII and clathrin-coated vesicles**?
- **COPI**: Transport materials form the ER to the golgi (forward - **anterograde** transport)
- **COPII**: Transport materials form the **golgi to the ER** from **trans to cis** golgi cisternae (backward - **retrograde** transport
- **Clathrin-coated**: They move materials from the TGN to **endosomes and lysosomes**
32
Explain the roles of COPII coat proteins
- COPII proteins interact with ER export signals and cargo molecules to initiate vesicle formation
- Inactive Sar1 (bound to **GDP**) is recruited by GEF (guanine exchange factor) on membrane by replacing GDP for GTP = active Sar1
- This activated protein gets incorporated into the membrane = **curving**
- It recruits other protein coats = more curving and vesicle formation.
33
What is ERGIC
- The **Endoplasmic Reticulum-Golgi Intermediate Compartment**
- Facilitates the sorting and transport of vesicles between the **ER and the golgi**
34
How are **escaped proteins** transported back to the ER
- the escaped molecules are **retrieved** back to their normal compartments, **retention** of resident molecules are excluded from transport vesicles
- Resident proteins have **retrieval signals** at their **C-terminus**, they’re captured and brought to the ER in COPI-coated vesicles
35
What is the role of mannose-6-phosphate (M6P) in protein sorting
- M6P is a **molecule address tag** that makes sure lysosomal enzymes are packaged and sent to lysosomes
- Their receptors (MPRs) recognize them and direct the enzymes to **clathrin-coated vesicles** for their delivery to lysosomes
36
Explain the process of vesicle fusion with target compartments
- This involves tethering proteins bring the vesicles close to their target membranes (microtubules and motor proteins), then SNARE proteins of both membranes interact to facilitate the **lipid bilayers merging** = cargo delivery
37
What is the function of SNARE proteins in vesicle formation
- They mediate vesicle docking and fusion by forming tight complexes between the vesicle and targent membranes
- v-SNARES are found in **transport vesicles** and t-SNARES are found in the **target compartment**
38
What triggers the process of **exocytosis** in cells
- This is triggered by and **increase in intracellular [Ca2+]**. The Ca2+ influx activates the proteins that mediate **vesicle fusion** with the membrane
- Contacts btw the vesicle and plasma membranes = **fusion pore** (temp. opening at the plasma-vesicle interface allowing contents to be released to the ECM)
39
t- and v-SNARES
- The plasma membrane has **two** t-SNARES (syntaxin and SNAP-25)
- The synaptic vesicle membrane hand **one** v-SNARE (synaptobrevin)
- They interact to form **Four-stranded bundles** that are parallel, they zip to form a complex that pulls the two bilayers closer
40
What types of enzymes are found in lysosomes
- They have **hydrolytic enzymes** (proteases, lipases) that peak down proteins. Function well in **acidic environment** to degrade **macromolecules**
41
Explain the process of **autophagy**
- It involves encapsulating damaged organelles/proteins in a **double membrane vesicle (autophagosome)** which **fuses** with a lysosome for degradation (autolysosome)
- It helps to protect an organism agains **intracellular threats** and prevents aging and prevention of some cancers
42
What are the **storage** functions of plant cell vacuoles
- They **store water, solutes, macromolecules and toxic compounds** creating **turgor** that keeps the cell rigid
- They’re resorvoirs for enzymes involved in metabolism
43
How does the **tonoplast** regulate ionic concentration within the vacuole
- Has active transporters that pump ions into the vacuole, creating an electrochemical gradient that draws water into the vacuole by **osmosis**
44
What is **phagocytosis**, what cells specialize in it
- The process of engulfing large particles into vesicles called **phagosomes** which then fuses with a lysosomes = **phagolysosome**
- Macrophages, neutrophils
45
What is the difference between **receptor-mediated endocytosis (RME)** and **bulk-phase endocytosis**
- RME: selectively internalizes **specific** macromolecules on the membrane = clathrin-coated proteins
- Bulk-phase: (**Pinocytosis**), **nonspecifically** takes up ECF and solutes
46
How do clathrin-coated pits facilitate endocytosis
- They form specialized regions on the membrane where receptors bind to ligands, the pits **invaginate** = vesicles to deliver cargo to **endosomes**
- Dynamin (G-protein) needed for the **fission** of the vesicle from the membrane, inhibit GTP hydrolysis and **fission wont occur**
47
What is the role of adaptors (AP2) in selecting cargo for endocytosis
- These adaptors link clathrin to the cytoplasmic tails of receptors, selecting specific astro for inclusion in clathrin-coated vesicles
- 4 subunits
48
What is a **triskelion**
- It is a **three-legged** clathrin structure that assembles into a **lattice** to drive membrane curvature and vesicle formation
49
LDLS
- **Low-density lipoproteins (LDLs)** have cholesterol ad proteins
- Their receptors are transporter to the membrane an **bound to a coated pit**
- These are taken up by RME to the **lysosomes** to realize their cholesterol
50
How does protein targeting differ between the ER and other organelles like mitochondria
- Proteins for the ER are translocated **co-translationally**, with ribosomes attached to the membrane
- Proteins targeted for mitochondria are synthesized in the cytosol and imported **post-translationally**, guided by specific signal sequences
51
Explain how protein are imported into **peroxisomes**
- Proteins for the peroxisomes have specific PTS that are recognized by cytosolic receptor proteins to guide the protein to the peroxisome& facilitate their translocation across the membrane
52
Explain protein import into a **mitochondrion**
- A protein that is targeted to the mitochondria bcos of **N-terminus sequence** gets unfolded by a chaperone which allows it to go through the **TOM complex** into the **outer** mitochondrial membrane
- The protein is directed to the **TIM complex** into the matrix where it **either** gets bound to a chaperone to unfold it and cleave its **pre sequence enzymatically** or it assumes its native unfolded conformation by itself
53
What is the difference between between a TOM and TOC complex
- **TOM**: a complex responsible for recognizing and importing mitochondrial proteins across the outer membrane
- **TOC**: a complex responsible for recognizing and importing proteins across the outer chloroplast membrane
54
How is RNA interference used to study gene expression
- RNAi uses small interfering RNAs to bind to mRNA, leading to their **degradation**, this allows to study specific genes by silencing others
55
What is the role of siRNAs in identifying genes involved in secretion
- siRNAs target and silent genes potentially involves in secretion pathways, the roles of the genes are then analyzed
56
What is the structure of Clathrin
- Outer lattice of clathrin
- Inner shell of GGA adaptor proteins interacting with M6P, G-proteins and clathrin
- G-protein Arf-1
BIOL-3221
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