Midterm 1 Flashcards
(106 cards)
1
Q
Robert Hooke
A
Discovered using compound microscope (50X):
-plant cells
-saw “honeycomb” “pores” “cells”
2
Q
Anthonie van Leeuwenhoek
A
Discovered using single lens microscope (200-300X):
-microorganisms
-red blood cells
-cell theory
-saw “animacules” (tiny animals) from gum swabs
3
Q
Schleiden
A
-every part of a plant is made up of cells
-cells made from “crystallization process”
(Idea stolen from Dumortier)
4
Q
Schwann
A
Both plants and animals are composed of cells and their products
5
Q
Virchow
A
All cells arise only from preexisting cells
6
Q
Cell theory
A
- All living organisms are composed of one or more cells
- The cell is the most basic unit of life
- All cells arise only from preexisting cells
OBP
One Big Prison
7
Q
Brown and Flemming
A
Discovered the nucleus, chromosomes, and different stages of cell division with the help of basic stains
8
Q
C. Golgi
A
Identified Golgi apparatus using a “black reaction” (black stain)
9
Q
Scanning electron microscopy (SEM)
A
-creates an image by detecting reflected electrons
-Surface of sample is metal-shadowed
10
Q
Cryogenic electron microscopy
A
Hydrated, unfixed, untainted samples are frozen (cells not dead)
11
Q
Transmission electron microscopy (TEM)
A
-uses transmitted electrons to create an image
-thin samples: stained/ shadowed with heavy metals
-thick samples: fixed, dehydrated, embedded in resin, sectioned, and stained with heavy metals
12
Q
Last eukaryote common ancestor
A
-last common ancestor of eukaryotes and archaea
-1.5-2 billion years ago
13
Q
Biological scale
A
Individual proteins (20nm)
Macro molecular complexes (0.2 um)
Organelles (2 um)
Cells (20um)
14
Q
Palade
A
-changed the field of cell biology
-perfected electron microscopy
-picture of cell with clear organelles
15
Q
Visible spectrum of light
A
VBGYOR
Wavelength increase ->
Frequency decreases —>
Violet: 380-450 nm (70)
Blue: 450-495 (45)
Green: 495-570 (75)
Yellow: 570-590 (20)
Orange: 590-620 (30)
Red: 620-750 (130)
16
Q
Spectrum of light
A
Lower wavelength = higher energy and higher frequency
17
Q
Fluorophores
A
-Fluorescent proteins
-FP genes can be fused to a gene of interest to produce a recombinant fluorescent protein, and expressed in an organism
18
Q
Strategies for live-cell microscopy
A
Recombinant proteins: plasmid DNA + vector
GFP: allows for identification and localization of proteins in the cell
19
Q
Immunolabeling (antibodies)
A
-helps identify and localize proteins
-uses an antibody to which a probe has been covalently attached
- antibodies can be generated by injecting an animal with the protein of interest (antigen)/ generate antibodies from a cell line (monoclonal antibodies)
20
Q
Immunolabeling terms
A
- Fixation: with formaldehyde or glutaraldehyde; tissue embedded in paraffin for sectioning
- Permeabilization: with non-ionic detergent that makes the plasma membrane permeable to reagents (ex antibodies)
- Stained: with a marker (fluorescent dyes or gold particles that are covalently attached to specific antibodies
-heavy metals: stain diff. Bio molecules to gain contrast
-small fluorescent dyes: bind to membranes, DNA, etc.
21
Q
Chemistry of life: 4 key concepts
A
- Molecular complementarity
- Polymerization
- Chemical equilibrium
- Energy
My Pretty Connie Expects (too much of my life)
22
Q
Types and energy of different bonds
A
Van der waals < hydrogen bonds < hydrolysis of ATP phosphoanhydride bond< C-C < C=C
23
Q
Dissociation constant (Kd)
A
Concentration of ligand
Higher concentration= less interaction
24
Q
Hydrophobic effect
A
Hydrophobic aggregation due to higher entropy of water (molecules released into bulk solution are less ordered)
25
How macromolecules are built
Proteins: peptide bond; amino acid combined from N to C; H2O taken out; NCrC
Nucleic acids: phosphodiester bond 3’ and 5’ sugar; H2O taken out
Carbohydrates: glycosidic bond; 1’ and 4’ bond; H2O taken out
26
L vs D
L-serine natural
D- glucose natural
27
Amphipathic molecules
Hydrophilic and hydrophobic group
28
Melting temperature of lipids
Tm: temperature at which ~50% of lipids are fluid
29
Glycerophospholipids
PC, PS, PE, PI, PG, CL
30
Sphingophospholipid
SM
31
Glycolipids
Cerebroside, LPS
32
Sterols
Cholesterol
33
Homeoviscous adaptation
-cells dynamically change their membrane lipid composition to control membrane fluidity
- to decrease transition temperature:
-unsaturated bonds and shorter acyl chains
-to increase transition temperature:
-saturated bonds and longer acyl chains
34
Special fluidity regulator
-cholesterol
-lowers membrane permeability (tight acyl chain packing)
- at low temp, it increases fluidity by preventing tight acyl chain packing
-at high temp, it decreases fluidity due to its rigid structure
35
Memorize 20 amino acids
36
Phosphorylation
-most abundant post translational protein modification in eukaryotes
-kinases transfer phosphoryl groups to side chain from ATP
-serine, threonine, tyrosine: major eukaryotic phosphorylation
- kinases phosphorylates and phosphatase dephosphorylates
37
Protein hierarchical structure:
Primary: linear sequence of amino acids
Secondary: alpha helixes and beta sheets
Tertiary: 3D shape
Quaternary: multiple peptides
Supramolecular: large scale assembly
38
Secondary structures
Alpha helix: 3.6 amino acids per turn
Beta sheet: anti parallel strands with connecting loops
39
Coiled-coil proteins
-two alpha helixes wound around each other
-heptad repeat with a hydrophobic residue at positions 1 and 4
40
Difference in plant and animal cells
-microvilli in animals
-cell wall in plants
-plasmodesmata
-vacuole
-chloroplasts
41
Monolayer vs. single bilayer vs. two bilayers
Monolayer:
-lipid droplets
Single bilayer:
-ER
-ER- golgi intermediate compartment (ERGIC)
-golgi apparatus
-trans golgi network
-plasma membrane
-lysosomes
-peroxisomes
Two bilayers:
-mitochondria
-chloroplasts
-nucleus
42
How did the first membranous organelles form?
Engulfing
43
Inheritance of membranous organelles
Every organelle stems from another organelle
44
Total cell membrane
ER (50%) and mitochondria (40%)
45
Cell volume
Cytosol > mitochondria (20%) > ER
46
1. Endoplasmic reticulum (ER)
Rough ER:
-ribosomes
-protein folding and post-translational modifications (N-glycans, S-S bonds)
-protein quality control
Smooth ER:
-lipid synthesis
-steroid hormone synthesis
-detox center
-calcium ion storage
47
2. ERGIC
-vesicular tubular clusters
-adjacent to ER exit sites
-COP 1 dependent sorting of retrograde cargo
-concentration/sorting of biosynthesis cargo toward the golgi
48
3. Golgi apparatus
-sorting hub
-lipid synthesis and transport
-remodeling of N-glycans
-addition of O-glycans
-lipidation of proteins
-3-8 disc-like cisternae
- cis, medial, trans
-stepwise modification of cargo
49
4. Trans golgi network (TGN)
-sorting hub
-clathrin coated vesicles
-extracellular matrix proteins
-protein complexes
- calcium dependent sorting
50
5. Plasma membrane
-physical barrier
-selective permeability
-transport of solutes and macromolecules
-endocytosis/phagocytosis/exocytosis
-cell signaling
-interactions
-fluid mosaic model
51
6. Endo-lysosomal system
1. Endocytosis
2. Phagocytosis
3. Autophagy: ER engulfs damaged organelles
Lysosomes: ph 4.5-5, containers enzymes that degrade polymers
52
7. Nucleus
-storage of blue prints
-separates transcription from translation
-continuous with the ER
-nuclear pore complexes
-nucleoplasm
-inner mmebrane is reinforced by lamin
-nucleolus: site of rRNA production and assembly of ribosome components
Ribosomes: help with mRNA translation; contained rRNA and protein
53
8. Mitochondria
-production of ATP
-have their own genome
- two bilayers
54
9. Lipid droplets
-energy storage
-neutral lipids and cholesterol esters
-monolayer
-biogenesis within the bilayer of the ER
-size can be dynamically controlled
-membrane homeostasis
55
10. Peroxisomes
-generation and scavenging of reactive oxygen species from oxygen
-breakdown of long chain lipids, etc
-biosynthesis of special membrane lipids
-pentose phosphate pathway
-bile acid synthesis
56
SDS polyacrylamide gel electrophoresis (SDS-PAGE)
1. Denature proteins with ionic detergent (SDS)
2. Acrylamide acts as a sieve; proteins move toward the anode (+)
57
Palade’s famous pulse-chase experiment
3 minute pulse: amino acid in rough ER
7 minute chase: in golgi
120 minute chase: secretory granules
58
Gunter Blobel signal sequence hypothesis
Proteins contain signal sequences that can direct them to the rER
59
Translation and translocation occur simultaneously
60
3 things required to direct proteins to the ER
1. Signal sequence
2. Signal recognition particle (SRP)
3. Translocon (sec61) + SRP receptor
61
Cotranslational translocation
1. N terminal ER signal sequence emerges from the ribosome
2. Signal recognition particle binds to signal sequence, arrests protein synthesis
3. SRP nascent polypeptide chain ribosome complex binds to the SRP receptor in the ER membrane
4. Ribosome transfers to the translocon, opens it to admit the growing polypeptide
5. Elongating polypeptide chain passes through the translocon channel. SS cleaved by signal peptidase
6. Growing peptide chain translated toward the 3’ end
7. Translation completes at mRNA stop codon and ribosome is released
8. Nascent proteins folds into native conformation. Translocon closes
62
Sec61
-translocon; preserves integrity of the ER membrane by three gating mechanisms
1. - peptide: channel closed by plug
2. +peptide: ring of isoleucine residues form a gasket
3. Lateral exit: for the signal sequence and for hydrophobic transmembrane domains
63
Type 1 membrane protein
1. Translocation initiation and SS cleavage
2. Peptide elongates
3. Elongation continues until a hydrophobic stop transfer anchor sequence enters the translocon
4. Stop transfer anchor sequence moves laterally through a hydrophobic cleft between translocon subunits and becomes anchored in bilayer; translocon closes
5. Synthesis continues in cytosol
6. Synthesis completes at stop codon, releasing ribosome
64
Classes of ER membrane proteins
Type 1: cleaved signal sequence; COO- in cytosol, NH3+ in ER lumen
Type 2: NH3+ outside, COO- inside
Type 3: COO- outside, NH3+ Inside
Tail anchored proteins: NH3+ outside and COO- is embedded in the membrane
Type 4: many transmembrane domains
GPI anchored protein: anchor with NH3+ tail inside
65
Type 2 and 3 single spanning membrane proteins
Positive inside rule: positively charged residues anchored in the cytosol side
66
Tail anchored proteins
-hydrophobic C terminus
1. Sgt2, get4, get 5 sequester nascent protein hydrophobic C terminal tail anchor sequence and transfers it to get3-ATP
2. Get3 ATP nascent protein complex docks onto the ER membrane get1/get2 receptor
3. Get3 ATP hydrolysis, release of tail anchored protein into the get1/get2 receptor, releases it into the ER membrane
4. Get 3 release of ADP and binding of ATP releases it from get1/get2
67
GPI anchored proteins
GPI molecule anchor covalently attached protein in membrane
68
N-glycans are added to proteins in the ER lumen
-N-glycans are attached to asparagine residues
-Asn-X-Ser/Thr
- N glycan precursor is synthesized at the ER membrane
-promotes protein folding, stability, adhesion, and recognition
69
Synthesis of N-glycan precursor
1. 2 GlcNac and 5 mannose residues added on the ER membrane cytosolic face
2. Flipped to ER membrane luminal face
3. Additional sugars added by ER enzymes to complete the n-glycan precursor
=Glc(3)Man(9)GlcNAc(2)
70
Addition of N-glycans to target proteins and initial processing
1. Precursor is transferred to asparagine residues in a Asn-X-ser/thr sequence on a nascent protein while coming out of the translocon
2. Glucose residue removed
3. Additional and removal cycles of glucose residue
4. Removal of one mannose residue
(Glc)3(Man)9(GlcNAc)2 —>(Man)8(GlcNAc)2
-glucose
- 1 mannose
71
Calnexin/ calreticulin cycle
-bind N-linked oligosaccharides and 1 glucose added
-retain protein in the ER until properly folded
-if proteins misproperly folded, mannose trimming
-OS-9 binds to Man5-6(GlcNAc)2= dislocation of the misfolded protein out of the ER, degrades
=Glc(3)Man(9)GlcNAc(2) —-> Man(9)GlcNAc(2)
72
Formation of covalent disulfide bonds
Reduced substrate protein + oxidized PDI protein disulfide isomerase —> reduced PDI + oxidized substrate protein
Reduced PDI gives electrons to oxidized Ero1 —> oxidized PDI
73
Rearrangement of disulfide bonds
Reduced PDI (SH-SH) forms transient disulfide bonds to break improper disulfide and protein is correctly folded
74
BiP chaperone
-membrane protein folding
-binds to exposed hydrophobic regions of the nascent chain and stabilizes them until the protein is folded correctly
75
Unfolded protein response
Ire 1: transmembrane ER protein, its luminal domain binds excess BiP, its cytosolic domain is a specific RNA endonuclease
1. Unfolded proteins bind available BiPs, BiPS released from Ire1= activation of endonuclease activity
2. Endonuclease cuts unsliced mRNA
3. Two cut mRNA joins together
4. Translation of functional mRNA moves into the nucleus and activated transcription of gene encoding more BiPs
76
ER golgi interface
-30% of all proteins encoded in the genome
-50% of ER volume exported every 40 mins
- 150 COP2 vesicles per second
-90% of membrane leaving the ER is recycled
77
Temperature sensitive (Ts) mutant genes
Permissive temperature (24C): protein folds
Restrictive temperature (37C): protein does not fold
Several classes of mutants were identified
78
Rothman’s simplest hypothesis
Specificity dictates anatomy
79
Vesicle trafficking
Requirements:
1. Machinery to attract coat
2. Coat proteins
3. Uncoating of carrier
4. Machinery for fusion
80
GTPase
-molecular switches
GTPase (GDP) off — GTPase (GTP) on— effector
GEF= GTP binding
GAP= GTP hydrolysis
81
Bidirectional transport at the ER-Golgi interface
COP2 carriers: anterograde transport (away from ER)
COP1: retrograde transport (return to ER)
82
Assembly and disassembly of COP2 coats
1. sar1-GDP interacts with the ER membrane protein Sec12, sec12 GEF activity, sar1 integrates its hydrophobic N terminus into the ER membrane
2. Sar1-GTP recruits the Sec23/Sec24 coat protein complex; sec13/sec31 coat complex assembles into coat
3. Sec23 GAP activity stimulates Sar1 GTP hydrolysis
4. Release of Sar1-GDP from the vesicle membrane causes disassembly of the coat
COP2 carriers form at ER exit sites
83
The inner layer of the COP2 coat recognizes cytoplasmic signals on membrane cargo proteins
Di-acidic signals: DXE, EXE, DXD
DI-hydrophobic signals: FF, YY, FY
84
Luminal cargo is clustered into COP2 carriers via the pectin ERGIC-53
Lectin: a carbohydrate binding protein
ERGIC 53: type 1 transmembrane protein, dihydrophobic motif in cytoplasmic tail; binds high mannose N-glycans on cargo proteins made in the ER
PH neutral= glycans bound= ON
PH acidic= cargo released at ERGIC= OFF
85
Uncoating prepares the COP2 carrier for the target membrane (ERGIC)
1. Vesicle budding from donor membrane; vesicle-snares captured in budding vesicle membrane
2. Vesicle fusion with target membrane: interaction of specific V-snare with specific target membrane T-snares
86
Docking and fusion of transport vesicles with their target membrane
1. Rab1 GTPase on COP2 vesicles interacts with Rab effectors in golgi to melt through the cocoon
2. 1 V snare proteins forms stable coiled coil interaction with 3 t-Snares
NSF unwinds this coiled coil
87
Assembly and disassembly of COP1 coats
1. Arf1-GDP interacts with the p23/p24 membrane proteins in ERGIC/cis golgi; GBF1 GEF activity, Arf1 N terminal integrated into membrane
2. Arf1 GTP recruits the heptameric coatomer coat protein complex; coatomer binds specific short sequences in membrane cargo protein cystosolic domains
3. Arf GAP activity stimulates Arf 1 GTP hydrolysis
4. Release of Arf1-GTP from the vesicle membrane= disassembly of coat
88
COP1 coat recognizes cytoplasmic signals on membrane cargo proteins
Basic signals: KKXX, K/HDEL, RR, RXR, RRR
89
Retrieval of ER-resident luminal proteins via the KDEL receptor
-prevents depletion of ER luminal proteins needed for proper folding (ex BiP)
90
Golgi
-cis, medial, trans
-ribbon, mini stacks
-flat centers: where enzymes are; fenestrated rims: transport machinery and cargo
91
Intra-golgi transport models
Maturation model: cisternae are dynamic compartments, mature from cis to trans, COP1 vesicles move enzymes to former cisternae to convert into new cisternae, cargo stays put
Vesicular transport model: cisternae are static compartments, each cisternae has a specific set of resident enzymes which stay put, cargo is transported via tubular or vesicular carriers from one cisternae to the next
92
Processing of N-glycans in the golgi
1. Cis golgi: Man8(GlcNAc)2 —> Man5(GlcNAc)2
2. Medial: 1 GlcNAc added
3. Medial: -2 mannose
4. 5. Medial: 2 GlcNAc added, 1 fucose added
6. Trans: 3 galactose added
7. Trans: 3 NANA residues added
93
What are N-glycans used for?
-promotes specific interactions (with proteins and lectins)
-a shield/barrier (resistance to proteases)
-to monitor protein folding
-N glycan remodeling can be used to measure protein transport (N glycan in the ER is D-resistant, in the cis golgi it’s D-sensitive)
94
N glycan remodeling can be used to measure protein transport (N glycan in the ER is D-resistant, in the cis golgi it’s D-sensitive)
Temperature sensitive VSV G protein:
-permissive temp: 32C = protein folds and moves through secretory pathway
-restrictive temp: 40C = misfolded protein retained in the ER
95
O-glycosylation
- in ser/thr in the cis golgi
-no consensus sequence
-linear
96
N glycan and O glycan similarities
-can be convalently attached
-linked to nitrogen and oxygen atoms of amino acid residues
-often capped with negatively charged skaldic acid
97
Different liquid composition from cis to trans golgi
-plasma membrane is more fluid in the cis; neutral charge
-plasma membrane is more tightly packed in the trans; negative charge
-cholesterol and spingolipids are enriched towards the PM
98
OSBP
Feeds cholesterol from the ER into the trans-golgi
99
Covalent attachment of a lipid to proteins: S-palmitoylation
S-palmitoylated cargo in golgi —> concentration at cisternal rim —> anterograde transport
100
Trans golgi network: 5 destinations
1. Retrograde back to trans golgi
2. Lysosomes
3. Late endosomes
4. Constitutive secretory vesicles
4. Regulated secretory vesicles
101
Clathrin coated vesicles
-bud from trans golgi and PM
-need AP complexes
-tryskelion (3 heavy chains, 3 light chains)
102
Arf1 recruits AP complexes
-hydrophobic and acidic motif
-AP complexes recruit clathrin = curvature bends membrane
-dynamin pinches off the bud
-uncoating of CCVs by Hsc70 and auxilin
103
M6P signal sorts lysosomal enzymes from the TGN to the lysosome
-recognizes QSHEY sequences in newly synthesized lysosomal enzymes
-M6P signal + M6P receptor
- receptor lets go of enzyme at late endosome bc low pH
104
Proteolytic processing or pro proteins at the TGN
Constitutive secretory pathway: fur in endoprotease
Regulated secretory pathway: PC2 and PC3 endoprotease
105
Ca2+ dependent sorting at the TGN
106
Cycling of synaptic vesicles in axon termini
Synaptotagmin: prevents membrane fusion and release of NT
