M5: Leaving the Cell Flashcards
(56 cards)
1
Q
Is phase contrast microscopy or immunofluorescence microscopy a better technique for viewing the RER?
A
Although phase contrast microscopy shows details, immunofluorescence is a better technique because we can specifically see those membranes only on the RER.
2
Q
Two types of ER
A
rough ER and smooth ER
3
Q
Rough ER
A
- continuous
- rough appearance due to the ribosomes that dot the surface
- site of co-translational transport, protein modification and formation of vesicles
4
Q
Smooth ER
A
- continuous
- smooth appearance due to no presence of ribosomes
- site of fatty acids, phospholipid synthesis, carbohydrate metabolism occurring, and where calcium is sequestered
5
Q
Post-translational modifications in ER
A
glycosylation, protein folding, disulphide bond formation, and proteolytic cleavage
6
Q
Where do modifications to proteins targeted to the ER lumen occur?
A
along the entire length of the protein
7
Q
Where do modifications to proteins embedded in the ER membrane occur?
A
only occur on luminal portion of the protein
8
Q
Protein glucosylation
A
addition of polysaccharide or sugar group to a protein
9
Q
Where does protein glycosylation occur usually?
A
it is common on proteins embedded in the cell membrane
10
Q
N-linked glycosylation
A
- most common form of glycosylation
- addition of a polysacchardie to the NH2 of the R group of asparagine
11
Q
Lectins
A
family of proteins that recognize modified proteins and assist in protein folding in a similar manner to molecular chaperones
12
Q
BiP
A
- an ER-resident protein that is a member of HSP70 family of proteins
- BiP and its chaperones (Hsp40 and nucleotide exchange factor) is crucial for efficient ER protein folding
13
Q
Disulphide bonds
A
covalent linkages between the sulphydral groups (-SH groups) of two cysteine residues
14
Q
Where does the oxidative reaction that forms disulphide bond for protein modification occur?
A
ER lumen
15
Q
Where does the reductive reaction that reverses disulphide bonds occur?
A
cytosol in ER
16
Q
Pancreatic ribonuclease A (RNAse A)
A
- contains 4 disulphide bridges
- secreted to intestine to aid in digestion of RNA by cleaning it into small pieces
- acidic conditions of small intestine would cause most proteins to unfold but these bonds maintain the functional state of the enzyme
17
Q
Can disulphide bridges form spontaneously or non-spontaneously within the ER?
A
spontaneously
18
Q
Protein disulphide isomerase (PDI)
A
- resident ER protein that can promote oxidation
- PDI forms an intermediate with 2 cysteine residues to accelerate the rate of reaction
- can also correct inappropriate disulphide bridge formation
19
Q
Proteolytic Cleavage
A
- cleavage of the peptide backbone of a protein
20
Q
What factors cause the ER to become overwhelmed with unfolded proteins?
A
- overproduction of proteins
- delays in processing steps
- exposure to toxins, heat or other denaturing stresses
- lack of nutrients
21
Q
What is the first response to unfolded proteins?
A
trying to restore normal cell function by slowing new protein translation or removing unfolded proteins from ER for degradation through ubquitinylation. The next step is to increase production of chaperone proteins which will assist in protein folding.
22
Q
Define Unfolded Protein Response (UPR)
A
- way in which unfolded proteins are detected and then given time and tools to fold properly
- essential proteins for this process within the ER: BiP and Ire1
23
Q
How does UPR work?
A
- When BiP and Ire1 are associated, BiP is sequestered and Ire1 is inactive
- increase in unfolded proteins in ER lumen will lead to BiP dissociation from Ire1.
- Ire1 will then form homodimers in ER membrane, which are activated by endonucleases
- endonucleases make internal cuts in nucleic acids (ex. mRNA)
- specific target for Ire1 endonuclease: mRNA for gene called Hac1
- Hac1 contains an untranslated sequence (like an intron) that inhibits translation by ribosome.
- Hac1 is spliced by Ire1 endohuclease allowing for synthesis of Hac1 protein which serves as a transcription factor to activate proteins such as BiP
- essentially misfolded proteins induce the synthesis of proteins that will assist in folding.
24
Q
Anterograde transport
A
- movement from ER towards cell membrane
25
techniques for studying vesicular transport
1. Pulse-chase labelling in mammalian cells and visualization using immuno-TEM
2. Fluorescent microscopy of GFP-labelled proteins in mammalian cells
3. Genetic mutations that disrupt transport in yeast cells.
26
Pulse-chase labelling
incubating proteins or enzymes in radioactive medium and then placing them in an unlabelled medium to see where proteins go after they leave the ER
27
Acinar cells
- exocrine cells of the pancreas that produce and transport enzymes that are secreted into the digestive system
28
General protein pathway from ER to cell membrane
ER resident proteins remain in the ER however other proteins can move to other destinations via vesicles.
Some areas being golgi complex, lysosome, cell membrane or secretion out of the cell.
A proteins move from ER to cell membrane they are transported in vesicles through the golgi complex.
29
Cisternae
- series of elongated, flat sacs compromising the Golgi complex
30
Constitutive secretory pathway
- default pathway used to release proteins immediately after protein synthesis and transport.
- they move straight from transport to Golgi to cell membrane
31
Regulated secretory pathway
- used by proteins that are kept in the cell until a signal trigger releases
32
Secretory granules
- secretory vesicles held in the cell during the regulated secretory pathway
33
Germ agglutinin
- lectin that recognizes N-linked polysaccharides found in golgi cisternae
- used to recognize golgi complex (i.e. differentiate them)
34
Anterograde transport
- movement of proteins from ER to cell membrane
| - moving in normal/forward direction
35
two models that describe transport through golgi
Model A - Vesicular transport model
| Model B - Cisternal maturation Model
36
Model A - Vesicular transport model
- vesicles carrying proteins move from cis- to medial- cisternae and from medial- to trans-cisternae
- in anterograde movement of vesicles containing proteins from one cisternae to the next
37
Model B - Cisternal maturation model
- proteins stay in cisternae, but the cisternae themselves are moving forward through Golgi complex
- requires movement of empty vesicles in backward direction (retrograde)
38
How do you define cisternae?
Base on their location in the golgi complex - cis-Golgi become medial-Golgi, and medial-Golgi become trans-Golgi
39
Implications of cisternae maturation model
- new cis-Golgi are formed by vesicles from ER
- trans-Golgi cisternae dissipate into transport vesicles
- golgi resident proteins must be resorted in the anterograde direction
40
Four steps of vesicular trafficking
1. Vesicle form by a process called budding; buds arise from the membrane of the 'donor' compartment
2. Cargo proteins are loaded into buds via. cargo signal sequences and receptors.
3. Vesicle formation and release
4. Vesicle docking and fusion to membrane of the 'recipient' compartment.
41
Three types of coated vesicles
Clathrin-coated vesicles, COP I coated vesicles, and COP II coated vesicles
42
What does GTPase accelerating protein (GAP) do?
it assists in the GTPase mediated hydrolysis
| of GTP to GDP which helps in the conversion of active G-protein to inactive G-protein
43
What does guanine exchange factor (GEF)?
it assists in the displacement of GDP and its replacement of GTP which helps in the conversion of inactive G-protein to active G-protein
44
Where in the vesicular transport process is clathrin vesicles required?
- transport away from trans-Golgi network to endosomes and to cell membrane
- used in endocytosis that uses the cell membrane to transport macromolecules into the cell
45
Where in the vesicular transport process is COP I vesicles required?
- retrograde transport from Golgi back to ER
46
Where in the vesicular transport process if COP II vesicles required?
- transport from RER to cis-Golgi network
47
Describe step 1: vesicle formation using COP II as a model example
- Sar1 (GTPase protein required for vesicle budding)
- Sec12 is a transmembrane protein found on the membrane of a donor compartment (ER) which serves as a GEF that will assist in exchange of GDP to GTP on Sar1
- activation (Sar1-GTP) involves conformational change that reveals the hydrophobic N-terminus that will anchor Sar1 in membrane of ER
- Sar1 on ER interact with COP II protein
- COP II proteins include Sec23 (binds directly to Sar1) and Sec24 (binds indirectly to Sar1) and Sec13 and Sec31
- curvature of membranes only occurs when COP II proteins are present
48
Are vesicles formed for clatharin and COP I proteins in the same way they are formed for COP II proteins?
yes except ARF-G proteins replace Sar1
49
Describe step 2: loading cargos using COP II model as an example.
- cargo accumulates within the curved bud on the ER membrane to mediate loading
- cargo receptors accumulate in the bud and pick up soluble proteins
- transmembrane proteins may also be cargo and these accumulate in the budding membrane
- this accumulation is accomplished by the interaction of cystolic domains of the receptors of transmembrane cargo with coat proteins
50
Describe step 3: vesicle release using COP II model as an example.
- once cargo is loaded, vesicle must be released from donor membrane which is done by converting Sar1-GTP into Sar1-GDP
- this allows for release of Sar1 and coat protein in a process called uncoating
- uncoated vesicles is loaded with cargo and ready to be transported - recognized by a motor protein and carried along microtubules from donor to recipient membrane
51
Preventing of uncoating allows for a collection of accumulated coasted vesicles. This can be done in 3 ways..
1. Nonhydrolyzable form of GTP
2. Mutation in G-protein
3. Lodish MCB in fig. 17.10
52
Describe step 3: vesicle release using clatharin protein model as an example.
- tri-scallion which is comprised of 3 heavy and 3 light chains, interact with one another on surface of budding membrane to form clathrin coat
- dynamin (G-protein required for release of the clatharin-coated vesicles from the budding membrane) changes from GTP-bound to GDP-bound which changes its structure and shape leading to vesicle release
53
Clatharin coat
- forms a polyhedral lattice using a collection of different clathrin proteins: clathrin heavy chains, clatharin light chains and adaptor proteins
54
Two models for how dynamin might use its structure to induce vesicle release
1. Poppase Model
| 2. Pinchase Model
55
Poppase Model
- dynamin helices elongate and push the vesicle away from donor membrane
56
Pinchase Model
- dynamin helices constrict and squeeze the membrane to initiate release
