BICD 110 midterm 1

What are the three pillars of cell theory?

1. all living organisms are composed of one or more cells

2. cells are the most basic unit of life

3. all cells arise only from pre-existing cells

How long ago was LECA?

1.5-2 billion years ago

What did Robert Hooke do?

• discovered (plant) cells

• named honeycomb pattern in “cork” cells

What did Anthonie Van Leeuwenhoek do?

• discovered microorganisms (animalcules)

• grandfather of microbiology

• discovered RBC, sperm cell → nucleus

• observed fertilization

What did Rudolphi/Link do?

• discovered cells have independent cell walls

What did Dutrochet do?

• “cell is the fundamental element of organization”

What did Schlelden do?

• discovered every part of a plant is made of cells

• incorrectly spread the idea that cells are made from “crystallization”

What did Schwann do?

• discovered every part of a plant + animal is made of cells and/or its products

What did Brown and Flemming do?

• used basic stains (hematoxylin) to discover nucleus, chromosomes, and cell division cycle

What is the “black reaction”?

A technique developed by C. Golgi that uses a silver chromate solution to stain nerve cells, identified Golgi apparatus.

What is TEM and SEM?

TEM (transmission electron microscopy): thin → stained/shadowed with heavy metals. thick → fixed dehydrated, embedded in resin, sectioned, and stained with heavy metals. uses transmitted electrons (electrons that are passing through the sample) to create an image

SEM (scanning electron microscopy): surface of sample is metal shadowed. uses electrons that that are reflected or knocked-off to create an image

What did Virchow do?

discovered cellular pathology “all cells come from other cells”

What is cryogenic electron microscopy (cryo-EM)?

hydrated, unfixed, unstained samples are plunge-frozen leading to formation of vitreous (non crystalline ice) that preserves sample.

What is a tomogram?

A 3D image obtained from combining multiple 2D slices of a sample, providing detailed structural information.

What did Porter, Claude, and Fullham do?

obtained the first electron micrograph of a cell (chicken fibroblasts)

What did George Palade do?

as Claude’s postdoc, got thinnest and most detailed image of cell via electron microscopy
grandfather of molecular cell biology

What is GFP and how is it useful?

GFP (green fluorescent protein), is a bioluminescent protein allows scientists to visualize and track proteins in live cells. FP genes can be fused to a gene of interest to produce recombinant fluorescent protein and track protein of interest

What are some ways to deliver recombinant DNA to cells?

polymers, viral transduction/transfection, microinjection, electroporation, liposomes

What is immunolabeling and how is it used?

Immunolabeling is a technique used to visualize proteins or antigens in cells by using specific antibodies tagged with a marker. first, antibodies must be generated to POI. (antigen). Then, the antibodies are applied to the sample, allowing for detection under a microscope, often using fluorescent markers.

To visualize subcellular structures or location of specific biomolecules, cells/tissue need to be:

fixed (w/ formaldehyde), embedded in paraffin, sectioned, permeabilized to make membrane permeable to detergents, and stained with a marker (fluorescence or gold) that are attached to antibodies so they gain contrast and can be seen

What are the 4 concepts of life?

molecular complementarity, polymerization, chemical equilibrium, energy

What is the order of energy of covalent/noncovalent bonds?

thermal energy < (noncov) van der Waals < (noncov) H-bond < (cov) hydrolysis of ATP < (cov) C-C < (cov) C=C

What is Kd and how does it relate to affinity

dissociation constant, lower Kd = higher affinity

What is the hydrophobic effect?

The hydrophobic effect is the tendency of nonpolar substances to aggregate in aqueous solutions to minimize their exposure to water, leading to an increase in entropy because water is liberated from the cages

What does it mean for a molecule to be amphipathic?

has both hydrophilic (water-attracting) and hydrophobic (water-repelling) regions, allowing it to interact with both polar and nonpolar substances.

How does temperature control membrane bilayer fluidity?

Temperature affects membrane fluidity by influencing the kinetic energy of the lipid molecules; higher temperatures increase fluidity while lower temperatures decrease it.

What is Tm?

Tm is the temperature at which a lipid bilayer transitions from a gel-like state to a more fluid state; 50% of lipids are fluid

How does fatty acid composition control membrane bilayer fluidity?

Fatty acid composition affects membrane fluidity through the degree of saturation; saturated fatty acids pack tightly, making the bilayer less fluid, while unsaturated fatty acids introduce kinks, increasing fluidity.

Which out of PC, PS, PE, PI, PG, SM, CL is a glycerophospholipid?

PC, PS, PE, PI, PG

How does cholesterol control membrane bilayer fluidity?

Cholesterol regulates membrane fluidity by inserting itself between phospholipids, increasing fluidity by disturbing lipid packing, and decreasing fluidity w/ its rigid structure. low temp: inc fluidity, high temp: dec fluidity

What is the permeability like for hydrophobic molecules, small uncharged polar molecules, large uncharged polar molecules, and ions in the phospholipid bilayer?

Hydrophobic molecules easily pass through the bilayer, while small uncharged polar molecules can also cross but more slowly. Large uncharged polar molecules and ions generally cannot pass through without assistance.

What are the special amino acids (C, G, P)?

cysteine (can form disulfide bonds, be phosphorylated)
glycine (only AA with proton as side chain, allows for tight packing within polypeptide)
profile (cyclic side chain leads to kinks and can undergo isomerization)

Which amino acids are the most major phosphorylation sites?

Ser, Thr, Tyr

What are kinases and phosphatases?

kinases add phosphate groups, phosphatases remove phosphate groups

What are the 5 protein structures?

1. primary structure: linear sequence of amino acids (peptide bond)

2. secondary structure: a helices, b sheets (h-bond)

3. tertiary strucure: peptide 3D shape

4. quaternary structure: association between multipeptide complex

5. supramolecular complex: consisting of tens to hundreds of subunits

What is coiled coil motif?

A coiled coil motif is a structural feature in proteins formed by the winding of two or more alpha helices around each other, with a hydrophobic residue at position 1 and 4. building block for spike proteins

What is the EF hand/helix-loop-helix motif

A common protein structural motif characterized by a helix-loop-helix configuration, often involved in calcium binding and regulation in various proteins.

What is a zinc-finger motif?

A zinc-finger motif is a structural domain in proteins that binds zinc ions to stabilize its fold, typically consisting of short beta strands and alpha helices, commonly involved in DNA binding and transcription regulation.

Difference between cytosol and cytoplasm?

The cytosol is the liquid component of the cytoplasm, excluding organelles and other insoluble materials, while the cytoplasm includes both the cytosol and the organelles suspended within it

Which organelles have a single bilayer of lipids?

ER, ERGIC, Golgi, TGN, PM, Endo-lysosomal system, peroxisomes

Which organelles have 2 bilayers?

Nucleus and mitochondria

Which organelle has a monolayer?

Lipid Droplets

What is an evolutionary idea as to how organelles formed?

infolding of plasma membrane → ER, nucleus
endocytosis of heterotrophic prokaryote → mitochondria
from a membrane to membrane: organelles exchange content → branches into other organelles

How does the inheritance of chromosomes differ from organelles?

chromosome inheritance is highly organized, and organelle inheritance is more variable and not guided

Contribution of organelles to cell membrane

rough + smooth ER > mitochondria > Golgi > plasma > everything else

Contribution of organelles to total volume

cytosol (50-60%) > mitochondria (20%) > rough ER (10%) > smooth ER (6%) > nucleus (6%) > peroxisome, lysosome, endosome (3%)

What is the function of the rough ER?

main manufacturer for protein synthesis, starting point of secretory pathway, place where protein undergoes folding + post translational mods, protein quality control

What is the function of the smooth ER?

lipid synthesis, cellular detoxification (p450), calcium ion storage

What is the function of the ER-Golgi Intermediate Compartment (ERGIC)?

involved in the transport and processing of proteins and lipids between the endoplasmic reticulum and the Golgi apparatus

What is the function of the Golgi apparatus?

sorting hub, lipid synthesis and transport, remodeling of n-glycans, addition of o glycans, lipidation of proteins,

What is the function of the trans-Golgi network?

shipping + sorting station

What is the function of the plasma membrane?

physical barrier with selective permeability, fluid mosaic model, transport of solutes and macromolecules, endo/phago/exocytosis

What is the function of endo-lysosomal system?

endocytosis/phagocytosis/autopagy (engulfs damaged organelles). low pH activates digestive enzymes

What is the function of the nucleus?

defines eukaryotic cell, storage of genetic material, separates transcription from translation

What is the function of mitochondria?

produce ATP, regulate metabolic activity, involved in apoptosis

What is the function of lipid droplets?

store lipids, connected to secretory pathway

What is the function of peroxisomes?

generation/scavenging of reactive oxygen species from oxygen, breakdown of long chain lipids, D-amino acids, biosynthesis of special membrane lipids, pentose phosphate pathway

What are differences between the structures of plant and animal cells?

Plant cells have cell walls, chloroplasts, large central vacuoles, and plasmodesmata. Animal cells have microvilli

What is George Palade’s experiment about?

tracked the cellular pathway of proteins using radioactive labeling, and SDS-PAGE, revealing the role of the rough endoplasmic reticulum in protein synthesis and secretion.

Who discovered the signal sequence hypothesis and what is it?

Günter Blobel: explained how proteins are directed to their correct cellular destinations by specific sequences within their polypeptide chains

What is the pulse-chase experiment

incubate purified ER membrane briefly with radiolabeled amino acids. isolate microsome with bound ribosome. treat purified microsome with protease: -detergent → protein in ER are protected from protease. +detergent → dissolved ER membrane, protein was digested. conclusion: newly made proteins are inside lumen of rough ER after synthesis

Mechanism of cotranslational translocation (proteins made in the ER lumen)

1. N-terminal ERSS emerges from ribosome

2. SRP binds to SS - pause synthesis

3. ribosome-SRP-SS complex binds to SRPr on membrane

4. GTP binds to SRP-SRPr

5. conformational change of translocon → opens, protein enters translocon

6. GTP hydrolyzed by SRP-SRPr and it dissociates

7. protein elongation. SS cleaved by signal peptidase

8. conclude translation, translocon close

3 ways translocons preserve ER membrane integrity

1. protein plug

2. hydrophobic pore ring

3. lateral gate

Type I ER membrane protein

C in cytosol, N in lumen. has terminal SS. can be insulin, LDL, GH receptor

Type II ER membrane protein

C in lumen, N in cytosol. Lacks an N-terminal signal sequence. can be transferrin receptor, golgi ftransferase/ferase

For ER membrane proteins, which is the + sequence in the cytosol or the lumen?

cytosol (II, III)

Tail anchored proteins

are anchored to the membrane by a hydrophobic C terminus, no terminal N SS.

1. three proteins bind to hydrophobic C tail and transfer to Get3-ATP

2. protein + Get3-ATP binds to Get1/Get2 receptor on ER membrane

3. Get3-ATP hydrolysis releases tail anchored protein into membrane

4. ADP leaves Get3 and ATP is replenished

GPI anchored proteins

protein with C already in membrane is broken off by GPI transamidase in the luman side and transferred to preformed GPI anchor

Type III ER membrane protein

C in cytosol, N in lumen. no terminal SS. cytochrome P450

What amino acid is N-glycan added to

Asparagine

What is the sequence of N-glycans

Asn - Xaa - Ser/Thr

Mechanism for the synthesis of N-glycan precurser

1. dilichol phosphate + 2 GlcNAc + 5 mannose on cytosol side

2. flipped to lumen side

3. + 4 mannose residues + 3 glucose

What is the processing of the N-glycans when adding to target proteins?

minus 3 glucose one each until 1 being added/removed is cycled, last structure is -1 mannose

What is the calnexin (CNX)/calreticulin (CRT) cycle?

a quality control mechanism in the ER that assists in the proper folding of N-glycan and buys time for the polypeptide to fold. CNX/CRT repeatedly binds and releases the terminal glucose on N-glycan. after glucosidase II removes glucose. then UGGT adds CNX/CRT again. for degradation, mannosidases remove mannose until there’s 5-6 left OS-9 degrades

Mechanism for PDI (protein disulfide isomerase)

1. reduced substrate protein comes out of translocon

2. oxidized PDI with thiols locates thiols on protein

3. reaction makes reduced PDI and oxidized substrate protein with intramolecular S-S bond

4. Oxidized Ero1 oxidizes PDI

Mechanism for UPR (unfolded protein response)

1. unfolded protein in lumen bind to BiPs

2. BiP leaves Ire1 monomer its attached to to help protein fold

3. Ire1 becomes a dimer

4. Ire1 dimer cuts unspliced Hac1 mRNA

5. translation of Hac1 mRNA

What the requirements for vesicle trafficking

1. machinery to attract vesicle coat

2. coat proteins

3. uncoating of carrier

4. machinery for fusion

What turns GTPase off?

GAP (GTPase Activating Protein), hydrolyzes GTP into GDP

What activates GTPase?

GEF (Guanine Nucleotide Exchange Factor), exchanges of GDP for GTP

COPII assembly/disassembly mechanism

1. Sar1-GDP binds to Sec12-GEF on membrane to convert to Sar1-GTP. Sar1 has a conformational change and anchors into membrane

2. Sec23/Sec24 + Sec13/Sec31 are recruited to make coat protein complex and becomes vesicle

3. Sec23 GAP hydrolyzes Sar1 GTP

4. Sar1-GDP is released from vesicle and causes disassembly of coat

COPII fusion mechanism

1. Rab1 on COPII vesicles bind to Rab effects on target membrane to melt through “cocoon”

2. v-SNARE protein on COPII interacts with t-SNARE on membrane to form stable coiled-coil

3. fusion of vesicle to membrane

4. NSF ATPase separates 4 helix bundle

COPI assembly/disassembly mechanism

1. Arf1-GDP binds to p23/p24 ER membrane protein

2. GBF1 GEF converts to Arf1-GTP to activate and Arf1 has conformational change

3. Arf1-GTP recruits coatomer (heptameric protein) and begins curving ERGIC/Golgi membrane

4. Coatomer disassembles when Arf1 is hydrolyzed to Arf1-GDP, releasing the vesicle

What is KDEL?

KDEL is a C-terminal signal sequence found on proteins that ensures they remain in the ER, preventing their transport to the Golgi apparatus. It consists of the amino acids K, D, E, L

Mechanism for remodeling of N-glycan in Golgi apparatus

cis: - 3 mannose
medial: + 1 GlcNAc, - 2 mannose, + 2 GlcNAc, + 1 fucose
trans: + galactose, + 3 NANA

What kinds of lipids are at the cis vs trans Golgi?

cis: PC
trans: sphingolipid + cholesterol

Are retrograde signals acidic or basic?

basic

Are anterograde signals acidic or basic?

acidic and hydrophobic

Which direction does COPI and COPII go

COPII: ER → Golgi (anterograde)
COPI: Golgi → ER (retrograde)

Which mechanism do the cargo-binding proteins ERGIC-53 and the mannose-6 phosphate (M6Pr) have in common?

pH dependent cargo release. reduced pH in target membrane triggers vesicle release

TGN: What are the 5 main types of cargo and their destinations?

1. retrograde transport → trans Golgi

2. lysosomal enzyme → lysosome

3. lysosomal enzyme → late endosome

4. constitutive secretory protein (PM/ECM protein)

5. regulated secretory protein (hormone, enzyme, NT)

Which protein is present in regulated exocytosis but not in constitutive exocytosis?

synaptotagmin-1

What does M6P do and what is its mechanism?

M6P (mannose 6 phosphate) is a tag that signals to the TGN that it is going to the lysosome.
1. in TGN, M6P binds to M6Pr
2. CCV will bud off with M6Pr and carry to the late endosome
3. M6Pr will dissociate with the acidic pH of endosome
4. endosome delivers enzymes to lysosome

What does BiP do?

it binds to exposed hydrophobic regions of the nascent chain and stabilizes them until the protein is folded correctly