Exam 2 Flashcards
to get an A on the Exam. Hopefully. :) (101 cards)
1
Q
Are cytoplasmic organelles in eukaryotes or bacteria?
A
Only in Eukaryotes.
2
Q
What is an organelle?
A
a membrane bound compartment.
3
Q
What organelles are a part of the endomembrane system?
A
ER, golgi, endosome, lysosome, plasma membrane
4
Q
What is the general pathway of molecules in the endomembrane system?
A
ER -> golgi -> extracellular space/plasma membrane
5
Q
What is GFP?
A
Green Fluorescent Protein
6
Q
When does the trafficking of GFP-VSVG begin?
A
Temperature sensitive vesicular stomatitis virus G protein (VSVG) mutant begins to traffic at permissive temperature.
40 C to 32 C in ER
7
Q
What are the steps of biochemical assays in cell-free systems?
A
- Homogenize (homogenate + whole cells)
- Centrifuge homogenate at 20,000g for 20 min.
- Transfer postnuclear supernatant (whole cells, nuclei, mitochondria at bottom) to new tube and centrifuge at 50,000g for 2 hrs.
- Now have separated postmicrosomal supernatant(liquid) and microsomes (supernatant)
- Done to measure the amount of a target substance.
8
Q
What is RNAi?
A
RNA interference
- partial gene silencing
- off target effect
9
Q
What is CRISPR?
A
Bacteria and Archaea used for genome editing.
10
Q
How does CRISPR work?
A
- Take Cas9 (nuclease) and 20bp guide sequence and combine with human cells.
- Knocks out particular gene on human chromosome (EG. Na+ guide sequence = knockout of Na+ channel gene)
11
Q
What are the advantages of CRISPR?
A
- Complete knockout
- Highly specific
- Fast
12
Q
What are the applications of CRISPR?
A
- Study biological functions
- Disease modeling
- Therapy
13
Q
Describe the ER.
A
- Largest membrane-bound organelle
- Supply proteins to other organelles
- Approximately 1/3 of total proteins.
- One continuous organelle
14
Q
What is the rough ER for?
A
Site for protein synthesis
15
Q
What is the smooth ER for?
A
- Lipid synthesis
- Adapted to specific biological functions
- Detoxification (EG. cytochrome p450 in liver)
16
Q
What are the differences of the two ERs in terms of density?
A
- Smooth microsomes ave a low density and stop sedimenting and float at low sucrose concentration. (LOW density and LOW sucrose conc.)
- Rough microsomes have a high density and stop sedimenting and float at high sucrose concentration. (HIGH density and HIGH sucrose conc.)
17
Q
What are the two types of organelle proteins focused on?
A
- Soluble lumenal proteins
- Integral membrane proteins
- Found in all membrane-bound organelles
18
Q
How are proteins targeted to organelles?
A
- Transcription of protein includes signal sequence peptide (at N-terminus)
- Sequence corresponds to certain receptor
- Taken into specific organelle
19
Q
Are signal sequences all the same?
A
No, each organelle has its own signal sequences for targeting.
20
Q
Why is co-translocation the first goto mechanism?
A
It is faster and more efficient. Other mechanisms consume ATP and involve protein folding.
21
Q
What is co-translational translocation?
A
When the nascent protein is synthesized directly into the ER.
21
Q
What does most ER-targeting signal sequences contain?
A
Positively charged residues such as arginine and lysine PLUS hydrophobic residues.
21
Q
What is the % of total phospholipids in SM? (High, med, low)
A
Sphingomyelin
- ER, GC, and PM are medium level in percentage. ~ 20%
21
Q
What is post-translational translocation?
A
When sec62/sec63 complex and chaperone protein BiP is need to help ensure peptide moves unidirectionally into the ER lumen.
21
How do you confirm an ER signal sequence?
1. Delete the sequence (protein will end up in cytosol and die).
2. Attach sequence to a different protein that normally stays in the cytosol.
21
What are the steps for recognition of signal sequence on the ER?
1. Signal sequence on nascent polypeptide is recognized by SRP (signal recognition particle).
2. SRP attaches to signal and then binds to SRP receptor (arrests protein translation by blocking tRNA entry).
3. Upon binding, SRP dissociates from receptor following GTP hydrolysis.
4. Ribosome is now free to bind to Sec61 complex (translocon).
5. Peptide translocates into the ER lumen
22
What is the purpose of a plug in translocon?
Prevents ions like K+ from leaving the cell.
23
What is the % of total phospholipids in PC? (High, med, low)
ER, GC, and PM are the highest in percentage. ~ 50%
24
What is the % of total phospholipids in PS? (High, med, low)
ER, GC, and PM are the lowest in percentage. ~ 10%
25
What is the % of total phospholipids in SM? (High, med, low)
Sphingomyelin
| - ER, GC, and PM are medium level in percentage. ~ 20%
26
Where are the C and N terminus ends for a type 1 membrane protein?
C term in cytosol and N term in lumen
27
Where are the C and N terminus ends for soluble lumenal protein?
Both in the ER lumen.
28
What are the steps for biogenesis of soluble lumenal proteins?
1. Signal sequence on growing polypeptide chain is recognized and brings ribosome with mRNA to the translocator.
2. SP (signal peptidase) cleaves signal peptide while translation occurs.
3. Mature polypeptide chain formed inside ER lumen and ribosome dissociates.
29
What are the steps for biogenesis of type 1 membrane protein?
1. Inserted into translocator with start-transfer sequence on n-terminus being recognized by hydrophobic start site.
2. Transfer occurs until stop-transfer sequence on polypeptide is recognized by hydrophobic stop-transfer-peptide-binding site.
3. Signal peptidase (SP) cleaves start-transfer sequence.
4. Mature transmembrane protein in ER membrane and C-terminus in cytosol.
30
After translocation into the ER, a protein:
1. Folds into its mature conformation.
2. Often acquires disulfide bonds
3. Often receives N-linked glycosylation.
31
Describe Hsp70s.
- Main protein is BiP: takes part in many aspects of ER quality control (QC).
- Binds to various nascent and newly synthesized proteins and assists their folding.
- Involved in processes of ER-associated degradation and the unfolded protein response.
- GRP170 unexplored. glucose regulated protein
32
Describe Hsp40s.
- 5 ER proteins of the hsp40 family (ERd)1-5) are known.
| - They contain luminally exposed J-domain and can stimulate BiP ATPase activity in vitro.
33
Describe Hsp90.
Only know Hsp90 member is GRP94
- Abundant in the ER, but not essential for cell viability and seems to limit its interactions to a small set of substrates.
34
Describe Calnexin and calreticulin.
Two lectin chaperones interact with and assist the folding of proteins that carry monoglucosylated N-linked glycans.
35
What is the purpose of disulfide bonds in ER proteins and are they common?
Disulfide bonds stabilize proteins and promote folding and are common among ER proteins.
- PDI = protein disulfide isomerase
36
Where is the N-glycosylation site and it's purpose?
N-X-S/T
| - Stabilizes proteins and promotes folding.
37
What is the usual cycle of polypeptides upon leaving the ER?
1. Assembles into proteins.
2. If it passes quality control (QC), it is exported to other organelles. If NOT, back to reassembly.
3. If can't be fixed (refolded), it is destroyed.
38
What type of glycosylation occurs in the golgi?
O-linked
39
How are incorrectly folded proteins targeted for degradation and where?
Ubiquitin chains will bind to the protein in the cytosol and eventually triggers its localization to the proteasome to be degraded.
40
When considering the ER stress response/unfolded protein response, what proteins are important and why does this occur?
Unfolded protein response occurs when there is a build up of unfolded or misfolded proteins in the ER lumen.
1. PERK: Translation attenuation and cell cycle arrest.
2. IREIalpha: Oligomerizes and carries out RNA splicing to remove an intron from the X-box binding protein XBP1, which allows it to become a functional transcription factor. XBP1 up regulates ER chaperones and ER associated degradation to facilitate recovery from ER stress.
3. ATF6: Goes to golgi to be cleaved by proteases to form an active ATF6 p50 transcription factor that translocates to the nucleus. Binds to stress element promoters upstream of genes that are unregulated in the UPR.
41
The ER is the site for what?
1. Synthesis of all membrane proteins in the endomembrane system.
2. Membrane protein folding and modifications.
3. Lipid synthesis.
42
Describe the layers of the golgi.
Closest to ER -> CGN (cis golgi network) -> cis cisternae -> medial cisternae -> trans cisternae -> TGN (trans golgi network).
43
What does clathrin do?
Mediates endocytosis through the three legs (skelion: three heavy chains and three light chains)
- Forms a cage around vesicles. (lattice)
44
How does the cisternal maturation model work?
Large cargo are transported as a unit through the different layers of the golgi.
45
How does vesicular transport work?
1. Donor compartment budding
2. Vesicle travels to recipient
3. Recipient compartment fusion
46
What are the three types of coat proteins for vesicle budding?
1. Clathrin
2. COPI
3. COPII
47
What does clathrin do?
Mediates endocytosis
48
What does it mean for a system to be bidirectional?
Things can travel both ways. EG. ER to Golgi or Golgi to ER
49
What does Sar1 do?
Recruits Sec23 and Sec24 to the cargo on the ER lumen. It is also an enzyme that can hydrolyze GTP.
- When Sar1 is inactivated = uncoating
- Induces coating of COPII
50
What does COPII do?
Forms a coated vesicle (cage) and is for curvature induction.
- Dissociation of the coat (uncoating) must occur before vesicle fusion can occur.
51
Proteins destined for export are captured by what?
COPII coats
52
What are the steps for cargo packaging?
1. On the cytosolic side, ER signal is read by Sec24 and COPII coats. Soluble ER lumenal proteins need cargo receptor (adaptor).
2. Protein transferred to COPII vesicles
3. Brought to VTCs (vesicular tubular cluster)
4. Next travels through ER, golgi intermediate component (ERGIC)
5. Finishes at the cis-golgi
53
pH is slightly ______ in the glogi complex.
lower
54
What is the purpose of ER retrieval?
1. Avoid waste
| 2. Avoid aggregation
55
Where is KDEL located and functional?
ONLY in the the lumen and at C-terminus.
56
What are the steps for ER retrieval?
1. COPII vesicle escapes from rough ER
2. KDEL receptors (have both export and import signals on cytosolic side) bind to soluble ER protein to for KDEL receptor-protein complex.
3. The complex then binds to COPI vesicles and is sent back to rough ER.
4. To restore balance, KDEL receptors are sent back to cis face of golgi by COPII vesicles.
57
pH is slightly ______ in the rough ER.
higher
58
pH is slightly ______ in the glogi complex.
lower
59
What are the three general steps for vesicle fusion?
1. Tethering
2. Docking
3. Fusion
60
What is the purpose of v-SNARE?
Engine for fusion
61
What are the active and inactive forms of Rab?
Inactive: Rab-GDP
Active: Rab-GTP
62
What is Rab and its purpose?
Rab is a G-protein that when active helps the vesicle tether to the Rab effector (tethering protein) located on the target membrane.
63
What does t-SNARE do?
t-SNARE interacts with the v-SNARE and allows docking of the vesicle.
64
What are the three general steps for vesicle fusion?
1. Tethering
2. Docking
3. Fusion
65
What does a trans-SNARE complex do?
Applies pressure to force fusion of vesicle to target membrane.
- Exerts inward force between vesicle membrane and target membrane.
66
What are the steps for the SNARE cycle?
SNAREs only involved during docking and fusion.
1. Zippering of v-SNARE and t-SNARE
2. trans-SNARE complex applies pressure for fusion.
3. cis-SNARE complex formed on membrane and cargo is released.
4. Disassembly of SNARE complex. Due to it's stable form, process requires NSF, alphaSNAP, and ATP hydrolysis.
5. Budding process repeats cycle.
Total of 35 SNAREs in the cell.
67
Does a Cis-SNARE complex exert force?
No, because there is already a single, fused membrane.
68
What are the two types of exocytosis?
1. Constitutive exocytosis (all cell types)
| 2. Regulated exocytosis (neurotransmitter release in neurons: EG insulin secretion from pancreas)
69
What is the difference between early and late endosome?
Early: Endocytosed material reaches here first
Late: Matured early endosomes (become more acidic, larger, removal of recycling molecules).
70
What do toxins do to SNAREs?
Toxins can cleave SNAREs which leads to the inhibition of synaptic transmission.
- EG. Tetanus toxin
71
Enzymes only function at _____ pH.
Low. pH ~ 5.0
| aka Acid Hydrolases: proteases, nucleases, lipases
72
What are the steps for a lysosomal enzyme turning into a lysosome?
1. From rough ER, lysosomal enzyme transported to cis Golgi cisterna.
2. Phosphorylation of lysosomal enzyme and moved to trans Golgi network.
3. Binds to adaptor and budded off in vesicle containing Mannose 6 phosphate receptors.
4. Dissociation of lysosomal enzyme from mannose 6 phosphate receptors (MPR).
5. MPR recycled in the TGN and lysosomal enzyme moved to endosome.
6. Enzyme carried to lysosome.
73
What does MPR do?
Mannose 6 phosphate is a key targeting signal for acid hydrolase precursor proteins that are destined for transport to lysosomes.
- Added by a reaction involving N-linked oligosaccharide.
74
What unit sets up protein degradation in the lysosome?
Ubiquitin
75
Enzymes only function at _____ pH.
Low. pH ~ 5.0
| aka Acid Hydrolases: proteases, nucleases, lipases
76
What are the main characteristics of fluorescence microscopy?
1. Only fluorophore labeled structures are visible.
2. Fluorophore labeled proteins may not reach their correct target.
3. Some fluorophore are part of clonable peptides.
77
The Golgi is the site for what?
1. Modification of N-linked glycosylation (occurs in ER too)
2. Site for O-linked glycosylation (only here)
3. Sorting station for distributing ER-synthesized proteins to other organelles (ER too).
4. Cleavage of peptides (ER too).
78
What does Sec23 and Sec24 do?
Act as the inner coat for cargo and recruits Sec13/31 as the outer coat.
82
What is the best microscopy method to observe a single motor protein actively walking along its track?
TIRF: Total internal reflection fluorescence microscopy
83
What does the nucleus do?
Separates chromatin and all DNA related processes such as transcription, repair etc. from the cytosol. Communicates with the cytosol through nuclear pores.
84
What is a nucleolus?
Made up of proteins and ribonucleic acids. It transcribes ribosomal RNA and combines it with proteins to form incomplete ribosomes.
85
What are karyopherins?
Group of proteins (importins and exportins) that transport molecules between the cytoplasm and the nucleus.
86
What do importins do?
Interact with a NLS peptide
87
What are the advantages of Oil-Emersion?
1. Allows collecting highly scattered light to be collected by front lens.
2. High-resolution imaging
3. Eliminates refraction between glass.
88
What is the f-number in photography?
Affects focal depth and amount of light
89
What is the use of transmission election microscopy?
1. Works light a slide projector
2. Atomic resolution possible
3. Produces 2D projections
90
What is scanning electron microscopy?
1. Produces surface topography data
| 2. Scans a sharply focused electorn beam over the specimen surface.
91
Advantages of light microscope?
1. Simple specimen preparation procedures.
| 2. DIC allows to study molecular processes
92
Advantages of electron microscope?
1. High resolution
2. Study molecular depth
3. Cryo-techniques allow to freeze distinct conformational changes of macromolecules.
93
What is fluorescence microscopy?
Detects the signal of fluorophore.
94
What is deconvolution microscopy?
A fluorescnece image is made up of a large number of small fluorescent light sources.
2. High spatial resolution
3. Cheaper that confocal microscopy
95
Why is it so difficult to see life cells in a n electron microscope (EM)?
Because an EM operates at high vacuum conditions.
96
What are hemidesmosomes?
Rivet-like links between cytoskeletons and extracellular matrix components = anchoring junctions.
97
What are gap junctions?
Allow for direct chemical communication between adjacent cellular cytoplasm through diffusion without contact of the extracellular fluid.
98
What are connexin proteins?
A cylinder with a pore in the center found at gap junctions.
99
What do exportins do?
Interact with a NES peptide.
