Topic 3

Question 1

how are different cells organized

Answer 1

Prokaryote cells generally lack membrane bound organelles, but they do have organization

Eukaryotic cells have organized organelles

Question 2

how does differential centrifugation work

Answer 2

Increases gravity on the solutes, causing the sediments to precipitate out of solution based on sedimentation velocity. Heavier sediments are easier to remove, particles with large sedimentation coefficients precipitate out earliest, then those with intermediate, then small.

Differential centrifugation splits pellet and supernatant

Question 3

how does density gradient centrifugation work

Answer 3

In density gradient centrifugation a mixture of particles is placed on a liquid with a gradient of density from top to bottom. The lower down the more dense. Particles are separated into bands of different particles based on density. Ex peroxisomes are in a layer, mitochondria in a layer, lysosomes in a layer, etc. Also called equilibrium centrifugation

Question 4

how does separation work

Answer 4

Separation is based on size and density

organelles are enriched in each fraction, but not purified.

Question 5

what is the origin of the nucleus and the ER

Answer 5

An ancient prokaryotic cell had DNA and membrane bound ribosomes

Plasma membrane folds inward and branches

the infolding eventually breaks off and creates a membrane around the DNA to form the nucleus and various passages to form the ER

Question 6

what is the origin of the mitochondria

Answer 6

An anaerobic pre-eukaryotic cell with or without a nucleus envelops an aerobic prokaryotic cell with a membrane. It surrounds it in another membrane. This becomes the mitochondria.

Question 7

what’s the mitochondria

Answer 7

• Up to 1um wide and 10 um long
• most cells have hundreds
• double membrane leads to multiple compartments
• in both plants and animals

Question 7

What is cytoplasm and cytosol

Answer 7

Cytoplasm = everything inside the cell membrane

Cytosol = Cytoplasm - organelles

Inside the nucleus is chemically similar to cytosol, but not considered part of it.

In liver cells:

Cytosol takes up 54%, followed by mitochondria at 22%, then Rough ER at 9%, Smooth ER at 6%, the nucleus at 6% and Peroxisomes, Lysosomes and Endosomes at 1% each

Question 8

what are mitochondria fun facts

Answer 8

• maintain their own DNA but it is incomplete
• Require imported proteins from cytosol
• DNA replication and protein translation more closely resemble bacteria than eukaryotes
• replication of mitochondria DNA is not limited to S phase
• in humans, mitochondrial DNA is almost exclusively maternally inherited

Question 9

what catabolism does the mitochondria do

Answer 9

Sugars and polysaccharides → sugars → glucose → pyruvate → Acetyl CoA

Fats → fatty acids → Acetyl CoA

Happens in the cytosol then mitochondria

Question 10

how is the mitochondria organized

Answer 10

• The inner membrane is folded into cristae
• inside the inner membrane is the matrix

Question 11

what is glycolysis

Answer 11

Process where glucose is oxidized to pyruvate

glucose + 2 NAD(+) + 2ADP + 2Pi → 2 Pyruvate + 2 NADH + 2H + 2 ATP

Glycolysis occurs in the cytosol

Ancient Eukaryotes could perform glycolysis for energy, but not the rest of the process

Under Anaerobic exercise we can use glycolysis to produce ATP

Pyruvate moves into the mitochondria after glycolysis

Question 12

what is redox

Answer 12

Oxidation involves losing electrons

Reduction involves gaining electrons

The most oxidized form of carbon is CO2

Question 13

what is NAD+

Answer 13

NAD+ is an electron carrier that takes 2 e- to become NADH

Question 14

What is the TCA cycle

Answer 14

Aka the Tricarboxylic acid cycle, Krebs cycle, TCA cycle

Pyruvate must be converted to Acetyl CoA

Conversion is carried out by pyruvate dehydrogenase which produces Acetyl CoA, NADH, CO2

The citric acid cycle completely oxidizes the 2 carbons in Acetyl CoA and produces 1 ATP

Glucose → 2 Pyruvate + 2ATP → 2 Acetyl CoA → 2 ATP

Also produces NADH, FADH2 as electron carriers

Question 15

what are the products of the TCA

Answer 15

Products of TCA cycle

Acetyl CoA + 3 NAD(+) + FAD + ADP + Pi → 2 CO2 + 3 NADH + FADH2 + CoA-SH + ATP

From one glucose molecule:

Glucose + 10 NAD(+) + 2 FAD + 4 ADP + 4 Pi → 6 CO2 + 10 NADH + 2 FADH2 + 4 ATP

Question 16

describe the e- transport chain

Answer 16

• Empties electron carriers
• electrons move through electron transport chain
• eventually end up on oxygen to be reduced to water
• also creates an H+ gradient
• Products from whole process at this point are Co2 and H2O (same as combustion, another type of oxidation)
• The e- chain uses the energy to actively transport protons from the matrix into the intra membrane space (energy from redox reaction)

Question 17

how do cells convert redox energy to mechanical energy

Answer 17

proton pumps create a proton gradient across the inner mitochondrial membrane.

ATP synthase lets H+ ions pass through to drive the reaction of

ADP + Pi → ATP

Question 18

What are the steps of the e- transport chain

Answer 18

The etc is made from four complexes

NADH transfers 2 electrons onto Complex 1

Succinate transfers 2 electrons onto Complex 2

Both complex 1 and 2 transfer electrons onto CoQ

CoQ transfers electrons to complex 3

Complex 3 tranfers electrons to Cyt C

Cyt C transfers electrons onto complex 4

complex 4 transfers electrons onto O2

All steps are energetically favourable movements, allow drive of H+

Question 19

what is the terminal electron acceptor

Answer 19

O2 is the terminal electron acceptor, it gets reduce to water at the end of respiration

(in our type of respiration at least, not all the time)

Question 20

how many protons does each complex move

Answer 20

The electrons from NADH goes through complexes 1, 3, and 4

The electrons from FADH2 goes through complexes 2, 3, and 4

Complexes 1 and 3 move 4 protons

Complexe 4 moves 2 protons

Question 21

What is the chemiosmotic hypothesis

Answer 21

In 1961 Peter Mitchell proposed the following hypothesis:

• transfer of electrons in the ETC creates a proton gradient
• Proton gradient leads to chemical gradient ([H+] or pH) and charge gradient → proton motive force
• Redistribution of the gradient is coupled to ATP synthesis

Proton motive force (∆p) = chemical gradient (∆pH) + charge gradient (∆ψ)

Question 22

what is the definitive experiment

Answer 22

• made synthetic membrane vesicle
• added bovine heart ATP synthase but no electron transport chain
• added Bacteriorhodopsin which pumps protons into the vesicle powered by photons
• Light caused ATP production

Therefor, ATP synthase is independent of the e- transport chain

Question 23

what is the stator and rotor

Answer 23

Stator = no move

Rotor (C ring) = moves

Question 24

what does the stator do

Answer 24

Stator has partial passage way to middle. From their it binds to one position of the rotor. The rotor rotates picking up protons until it reaches another partial passage that leads to the other side, where the proton is released into the cell Each proton must travel in a full circle before being released into the matrix

Question 25

How is ATP synthesized

Answer 25

the c ring is attached to the gamma stalk which rotates. The gamma stalk is lodged in a ring of static alpha and beta subunits that form a hexagon Has 3 phases: O = Open phase T = Tense phase L = Loose phase 3 ATP are produced per turn of C ring Open phase takes in ADP + Pi Loose phase aligns ADP and Pi to prepare for reaction Tense phase turns it into ATP Open phase then releases ATP

Question 26

how much ATP is produced for what

Answer 26

a 360º rotation of the gamma subunit yields 3 ATP if the c ring has 10 subunits, then each ATP needs 3.33 protons to be transported Actual value is closer to 4 protons per ATP 10 go through ATP synthase, 12 through the e- transport chain for one full rotation So Complex 1 = 4H+ = 1 ATP Complex 3 = 4 H+ = 1 ATP Complex 4 = 2 H+ = 0.5 ATP 1 NADH = 2.5 ATP 1 FADH2 = 1.5 ATP 1 Glucose = 30-36 ATP

Question 27

what is oxidative phosphorylation

Answer 27

This process is called Chemiosmotic coupling 2 stage process: 1. High energy e-s are used to pump H+ across a membrane 2. H+ flows down the gradient through ATP synthase

Question 28

How do you uncouple oxidative phosphorylation

Answer 28

- Poisons such as cyanide, uncouple oxidative phosphorylation, resulting in a loss of ATP production - Cyanide acts by binding cytochrome oxidase - Dinitrophenol can shuttle protons across membranes - In brown fat cells, the process is uncoupled on purpose to generate heat - Protons move through Thermogenin (UCP1 = uncoupling protein 1)

Question 29

How much energy does 1 glucose produced

Answer 29

From Glycolysis: Glucose + 2 NAD(+) + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H(+) + 2 ATP From TCA: Glucose + 10 NAD(+) + 2 FAD + 4 ADP + 4 Pi → 6 CO2 + 10 NADH + 2 FADH2 + 4 ATP Via Oxidative Phosphorylation: 10 NADH + 10 H(+) + 2FADH2 + 6O2 + ~30ADP + ~30Pi → 10NAD(+) + 2FAD + 12H2O + ~30ATP ATP is the cellular currency of energy 30 ATP molecules generated per glucose

Question 30

what is mitochondrial migration

Answer 30

- observed in many organisms including yeast and rodents, but not yet observed in humans - 3 ways to go: - tunneling nanotube - extracellular vesicle - Freely floating

Question 31

what is the nucleus

Answer 31

The Nucleus contains almost all the DNA in a eukaryotic cell site of DNA replication, RNA transcription, RNA processing and ribosome assembly Surrounded by nuclear envelope that is continuous with ER

Question 32

Describe the nucleus

Answer 32

- Surrounded by double membrane - topologically equivalent to the cytosol - further organization within nucleus (not compartmentalized though), ex nucleolus - Surface may have up to 400 nuclear pores - each can move up to 500 macromolecules a second - can move things in and out simultaneously

Question 33

what is chromatin

Answer 33

Nucleus about 5-10 um in diameter single chromosome may measure 10 cm or more DNA must be compacted

Question 34

How is chromatin compacted

Answer 34

Core unit is called a nucleosome consists of histones (2 units each of H2A, H2B, H3, and H4) and a linker region Looks like beads on a string attached by H1 proton to linker Wrapped into a loop called packed nucleosomes (30 nm wide) This chromatin fibre gets looped into a domain 300 nm wide Then into heterochromatin (dense areas) and euchromatin (less dense areas) These form chromosomes Histones have tails that can be modified. Ex by attaching Me, P, Ac, etc. Affects ability to compact

Question 35

describe epigenetics

Answer 35

How DNA is packaged can regulate gene expression Heterochromatin vs euchromatin epigenetics Genetics talk about the physical order of nucleotides in DNA, epigenetics ignores that and talks about inheritability. Some traits are seemingly passed along without a basis in the nucleotide order the compaction of DNA can influence the expression of genes. The compaction of DNA is inheritable

Question 36

describe death metal chickens

Answer 36

Divided chickens into two populations control had normal, healthy conditions death metal chickens were exposed to flashing lights and heavy music. The death metal chickens had trouble getting food, even if it was given to them Two generations of the offspring later had trouble foraging, even when they lived their whole lives without music and lights no change in genes -. different inherited traits Epigenetics Environmental changes can affect gene expression in following generations

Question 37

describe the nuclear pore complex

Answer 37

Systems of gated transport where small, water soluble molecules (<5kDa) diffuse passively while larger molecules must be actively transported Proteins have a sequence within them that tells them if they should be taken into or out of the nucleus NLS → Nuclear localization sequence NES → Nuclear export sequence Sequences within the primary sequence of a protein mRNA, tRNA, ribosome subunits → leave nucleus Proteins needed for transcription, chromosome replication, or building ribosomes → enter nucleus

Question 38

Describe how Ran works

Answer 38

Monomeric GTPase: essentially an “on/off” switch a cell has around 70 of these switches The one we care about is called “Ran” Takes GTP and speeds up transformation to GDP + Pi Ran binds to GTP (Ran-GTP) and uses water to hydrolyse it into Ran-GDP + Pi Ran-GTP + H2O → Ran-GDP + Pi Ran-GTP = On Ran-GDP = Off Sped up by an enzyme called “Gap” Gap = GTPase activating protein Ran-Gap is gap for Ran To get back to Ran-GTP (on) a brand new GTP takes the phase of the GDP, releasing the GDP and forms Ran-GTP exchange happens because of GEF GEF = Guanine nucleotide exchange factor In summary: RAN-GTP + H2O → RAN-GDP + Pi (RAN-GAP) RAN-GDP + GTP → RAN-GTP + GDP (RAN-GEF)

Question 39

describe Nuclear import

Answer 39

NIR is an importin protein that moves proteins with an NLS sequence into the nucleus When RAN is GDP it can’t bind to NIR, when it is RAN-GTP it can NIR can only bind to one of RAN-GTP or Cargo at a time RAN-GEF is found inside the nucleus. This means RAN will be bound to GTP in the nucleus RAN-GAP is found in the cytosol. This means RAN will be bound to GDP in the cytosol NIR outside the nucleus will bind to cargo protein and import it into the nucleus In the nucleus, NIR will bind to RAN-GTP releasing the cargo RAN-GTP-Importing complex exits the nucleus RAN-GAP causes the complex to fall apart

Question 40

describe nuclear export

Answer 40

GAN-GTP is able to bind exportin (NER) and this allows exportin to bind the cargo protein. This complex is exported out of the nucleus Ran-GAP in the cytosol hydrolyzes GTP to GDP. Ran-GAP no longer binds to the exportin. The exportin is released and it now releases the cargo. The exportin and Ran-GDP are returned to the nucleus

Question 41

describe the ER

Answer 41

The ER is continuous with the nucleus Important for intracellular calcium storage production of transmembrane proteins and lipids for most organelles makes most proteins destined for secretion of the lumen of ER, golgi, or lysosomes 2 types: Rough and Smooth

Question 42

describe the Rough ER

Answer 42

- Ribosomes embedded in the ER give the rough appearance - Site of protein production and modification and lipid synthesis

Question 43

describe the smooth ER

Answer 43

- Involved in lipid metabolism, production of lipoprotein particles (carry lipids via bloodstram to other parts of the body) and detoxification of lipid soluble drugs and other harmful products - transition ER are areas of the smooth ER which form transport vesicles for delivery to the golgi - important for intracellular Ca2+ storage

Question 44

how can we study the smooth vs rough ER individually

Answer 44

Homogenization of the ER causes it to reseal to form microsomes of either smooth or rough types. Smooth microsomes have low density and stop sedimenting and float at low sucrose concentration Rough microsomes have a high density and stop sedimenting and float at high sucrose concentration

Question 45

how are proteins imported

Answer 45

Co-translational translocation = ribosome pushed protein into ER Post-translational translocation = after seperation with ribosome - ER uses a signal sequence to mark proteins for import - while the signals are different, all have 8 or more non-polar amino acids - signal sequence discovered in 1970s using in vitro translation studies - SRP - signal recognition particle

Question 46

What is glycosylation

Answer 46

- 1/2 of eukaryotic proteins are glycosylated, although very few cytosolic proteins are glycosylated - precursor oligosaccharide composed of N-acetylglucosamine, mannose and glucose makes a 14 sugar entity which is attached en bloc to proteins by oligosaccharide transferase - N-linked (asparagine) - All diversity in glycosylation is obtained in later processing steps, some in the ER, and some in the Golgi - N-linked accounts for 90% of the glycosylation - O-linked glycosylation occurs in the golgi

Question 47

How does glycosylation work

Answer 47

1. oligosaccharide transferred from lipid to newly synthesized protein, and trimmed 2. Glycoprotein packaged into vesicle and sent to golgi 3. Based on protein structure, specific sugars are added by transferase enzymes 4. Based on its overall structure, each glycoprotein can be recognized, packaged into a vesicle, and sent to a different compartment

Question 48

describe the Golgi

Answer 48

Stained nervous tissue with metallic impregnations in a method later know as the “black reaction” First noticed Golgi bodies in 1898. These were not confirmed until the mid 1950s Shared 1906 Nobel Prize in Physiology or Medicine with Cajal (not for work on Golgi though) The Golgi is a series of flattened sacs / compartments The cis cisternae is towards the ER. The trans cisternae is furthest from the ER. EAch cisternae does different modifications to proteins. Lumen of Golgi is similar to Lumen of ER, which is similar to extracellular space

Question 49

What are some Golgi facts

Answer 49

- major site of carbohydrate synthesis - sorts and dispatches proteins from the ER - attached oligosaccharides to many of the proteins and lipids it receives form the ER - in animals, usually located close to the nucleus, mediated by microtubules - All resident proteins are membrane-bound - all single-pass transmembrane proteins - O-linked glycosylation vis S and T

Question 50

what does the Golgi do

Answer 50

In ER, oligosaccharides added to proteins to assist with folding and ensure proper folding these can be modified in the golgi Oligosaccharide processing occurs in organized sequence in the golgi stack

Question 51

what are lysosomes

Answer 51

- Digest macromolecules - Resident proteins modified by Golgi apparatus, trafficked via vesicles - contain acid hydrolases which require an acidic environment to function - pumps out macromolecuels that can either used for metabolism or secreted - can degrade all cellular components

Question 52

how do lysosomes take in material

Answer 52

- Unlike most organelles, which have relatively uniform structures, lysosomes are heterogenous - this is a because of their wide diversity of function and the way they are formed - they are formed by the fusion of a vesicle (late endosome, phagosome or autophagosome) with an existing lysosome There are 3 pathways deliver materials to endosomes - Endocytosis - Phagocytosis - Autophagy

Question 53

describe peroxisomes

Answer 53

- Single membrane organelles - found in all eukaryotic cells - Peroxisomes use molecular oxygen and hydrogen peroxide to perform oxidation reactions - site of beta oxidation, where fatty acids are broked down to acetyl coA (also occurs in mitochondria of mammals)

Question 54

what do peroxisomes do

Answer 54

RH2 + O2 → R + H2O2 But H2O2 is highly toxic, therefore peroxisomes perform “peroxidation”: H2O2 + R’H2 → R’ + 2 H2O Catalase can also eliminate H2O2 by converting it to H2O 2H2O2 → 2H2O + O2

Question 55

what is Zellweger syndrome

Answer 55

Peroxisomal biogenesis disorder Abnormalites in brain, liver, kidneys, and other physical deformities most infants do not survive past 6 months

Question 56

what is chloroplast

Answer 56

Site of photosynthesis harvet energy from light and fix Co2 as carbohydrates may be ancient trapped cyanobacteria

Question 57

how is Co2 fixed

Answer 57

In general: Light + CO2 + 2H2A → (CH2O) + 2A + 2H2O Where H2A is the electron donor Oxygenic photosyntehsis (produces oxygen) utilizes H2O as the electron donor: light + 6CO2 + 6H2O → C6H12O6 + 6O2

Question 58

how is chloroplast structured

Answer 58

- 2 membranes - inside is called the stroma - have thylakoids that can be stacked into granum - inside thylakodis is the thylakodi lumen

Question 59

what does the chloroplast do

Answer 59

Light reactions: Photosynthetic e- tranfer Dark reactions: carbon fixation (calvin cycle)

Question 60

how does the chloroplast harvest light

Answer 60

Pigments such as chlorophyll are embedded in the thylakoid membrane organized into light harvesting complexes and photosystems special chlorophyll a in PS converts solar energy to chemical energy Different pigments absorb different wavelengths Electrons harvested from H2O, which has a very low ∆Eo’ not enough energy would be produced to keep the cell alive

Question 61

what does PSII do

Answer 61

PSII removes electrons from water and passes them sequentially to a manganese-stabilizing protein (MSP), a tyrosine residue (Tyr) on protein D1, a sepcial pair of chlorophyll a molecules (P680), pheophytin (Ph), and two plastoquinones (Qa and Qb) oxygen is released as water is oxidized.

Question 62

what does cytochrome b6/f do

Answer 62

The cytochrome b6/f complex recieves electrons from PSII and passes them to PS-I via plastocyanin (PC). Simultaneously, protons are pumped from the chloroplast stroma into the thylakoid lumen.

Question 63

what does PSI do

Answer 63

PSI accepts the electrons from PC and passes the to ferreoxin (Fd) via another special pair of chlorophyll a molecules (P700) a modified chlorophyll a molecules (Ao) phylloquinone (A1) and three iron sulfer centres Sometimes electrons can be cycle in PSI multiple times moving more protons

Question 64

where is the e- chain in the chloroplast

Answer 64

e- transport chains in the chloroplasts are located on the thylakoid membrane, not the inner membrane

Question 65

what is the Calvin cycle

Answer 65

- Melvin Calvin, Andrew Benson, James Bassham - Calvin awarded Nobel Prize in Chemistry, 1961 - Also called Calvin-Benson cycle or reductive pentose phosphate cycle - this cycle is used by most organisms that get their carbon from Co2 - each turn of the cycle fixes one carbon - start with 5-C carbohydrate add CO2 making 6-C carbohydrate. This is then split into 2 3C carbohydrates