Module 1 - Cells and their components

Question 1

Cell theory

Answer 1

• Cells are the fundamental units of life
• All organisms are composed of cells
• All cells come from pre-existing cells

Question 2

Unicellular organisms and carrying out the functions of life

Answer 2

A single cell carries out all the functions of life

Question 3

Multicellular organisms and carrying out the functions of life

Answer 3

Made of many cells that are specialized for different functions – tissues and organs

Question 4

Internal structure of prokaryotes

Answer 4

• No nucleus: their DNA floats freely in the cell
• No (or rudimentary) internal membranes
• Very basic cytoskeleton

Question 5

Internal structure of eukaryotes

Answer 5

• Have a nucleus containing DNA
• A complex internal membrane system
• Extensive cytoskeleton

Question 6

Internal membranes within cells

Answer 6

• One or more membranes made up of lipid bilayers that form a physical barrier from the cytosol/other organelles
• Allows different protein contents and chemical environments to be maintained
• Allows each organelle to have a specialised function

Question 7

Characteristics of ER

Answer 7

• Network of interconnected spaces enclosed by a single membrane that is continuous with the nuclear envelope
• The entry point to the secretory pathway
• Makes: secretory and membrane proteins, and also lipids
• Very dynamic

Question 8

Two types of endoplasmic reticulum

Answer 8

Smooth ER: abundant in human cells active in lipid metabolism and in the liver for detoxification of lipid-soluble compounds

Sarcoplasmic reticulum: ER-derived calcium store in muscle cells (important role during muscle contraction)

Question 9

Characteristics of the Golgi apparatus

Answer 9

• Receives proteins and lipids as cargo from ER
• Cargo transits Golgi to the plasma membrane
• Modification of cargo e.g. glycosylation
• Sorting of cargo to the correct location

Question 10

Cytosol: what is it, how large is it, what occurs here, what is located here, and what is it not to be confused with?

Answer 10

• The soluble and aqueous portion of the cell
• Typically largest single compartment in the cell
• Site of many fundamental cellular processes: protein synthesis and degradation, intermediary metabolism
• Location of the cytoskeleton
• Not to be confused with the cytoplasm (everything except the nucleus)

Question 11

How ATP/GTP is formed

Answer 11

ADP/GDP forms a phosphoanhydride bond with an inorganic phosphate along with a proton using energy gathered from either the sunlight or consumed food, forming ATP/GTP and water

Question 12

How ATP/GTP is used - energy usage

Answer 12

ATP/GTP reacts with water, breaking the phosphoanhydride bond, and releasing the energy kept in the bond which is then used for intracellular work

Question 13

The uses of ATP/GTP - affecting proteins

Answer 13

Nucleotide binding is used to change protein shape, activity, and function

Phosphorylation by adding phosphate to serine, tyrosine and threonine

Question 14

What can phosphorylation do?

Answer 14

Affect:
Cell growth
Cell cycle
Cell division
Cell survival
Gene expression
Metabolism

Question 15

Light microscopes: how high can the magnification and resolution go, what are the prerequisites for the specimen, what do they show, and what cells can be used?

Answer 15

• Magnifies cells up to 1000 times and resolves details to a resolution of 0.2 µm.
• The specimen must be prepared in a way that allows light to pass through it.
• Shows the shape of structures, but doesn’t give molecular information.
• Suitable for live cells.

Question 16

Fluorescent dyes in fluorescent light microscopy: what do they do and how can they work?

Answer 16

Absorb light at one wavelength and release it at a longer wavelength

Some may bind organelles, may be coupled with antibodies that recognize a protein in a chemically fixed cell

Question 17

Advanced fluorescent light microscopy: what does it allow?

Answer 17

Allows for 3D imaging because of “super resolution”

Question 18

GFP

Answer 18

Green fluorescent protein can be attached to a protein as a tag to allow for fluorescent microscopy

? Molecular biology to add GFP coding sequence – produces a fusion protein
? GFP is intrinsically fluorescent, so is visible in living cells
? Potential problems: GFP is 238 amino acids = 25 kD protein. Does is alter function? Misfolding?

Question 19

Electron microscopes: what are the prerequisites for the specimen, what do they show, and what cells can be used?

Answer 19

• Thin slices of material (sections) stained with heavy metals for contrast
• Detailed subcellular structure
• Dead cells

Question 20

Cell fractionation

Answer 20

• Centrifuge to ‘pellet’ particles from suspension
• Heavier particles sediment at lower centrifugal force
• Collect supernatant (cytosol) and centrifuge again at a higher speed
• Tissue -> pellet 1 (nuclear fragments) -> pellet 2 (mitochondrial fraction) -> pellet 3 (rER and Golgi apparatus)

Question 21

SDS: what is it and what does it do?

Answer 21

Sodium dodecyl sulphate

An ionic (negative charge) detergent that binds to proteins

Denatures proteins (useful for electrophoresis)

Question 22

Polyacrylamide

Answer 22

Mesh-like gel that charged proteins move through, separating based on size

Question 23

Protein targetting

Answer 23

When there is no targetting mechanism, the protein remains in the cytosol

When the protein is instructed to go to a certain place (outside the cell), the protein’s receptor interacts with it, the protein then unfolds/loosely folds to cross the membrane, and translocation machinery is used to move the protein through membranes

The whole process uses ATP/GTP

Question 24

Signal sequences: what are they and when are they removed?

Answer 24

Stretches of polypeptide sequences made up of specific types of amino acids

May be removed after the protein reaches its target location

Question 25

The nuclear envelope: what is it made of and what does each part do?

Answer 25

* The inner nuclear membrane contains proteins that bind to the lamina and chromosomes * The perinuclear space between both membranes * The outer nuclear membrane is continuous with ER composition

Question 26

Crossing the nuclear envelope

Answer 26

30+ different proteins act as a gateway to the nucleus, allowing small, soluble molecules to diffuse through Larger components must be actively transported in and out of the nucleus across the nuclear pore complex

Question 27

Nuclear targetting signals

Answer 27

The nuclear localisation signal (NLS) targets proteins to the nucleus Usually made of <12 amino acids, usually positively charged (Lysine/Arginine) May be located anywhere in the nucleus and are not removed even after arriving

Question 28

Protein import

Answer 28

A receptor binds to proteins containing NLS in the cytosol Fibrils direct the receptor to the nuclear pore Cargo protein is pulled into the nucleus through the large pore (allowing folded proteins to move through)

Question 29

Ran: what is it and what does it do?

Answer 29

GTP/GDP-bound proteins which control nuclear import by binding to the receptor, releasing the cargo protein from it. This complex then passes through the nuclear pore and dissociates in the cytosol by converting the GTP to a GDP In order to ensure the cycle occurs in the right direction, there are two conformations of the Ran protein (Ran-GTP in the nucleus and Ran-GDP in the cytosol)

Question 30

The endoplasmic reticulum: what is it, what are the two types and what do they do?

Answer 30

The most extensive membrane system in eukaryotic cells made of connected sacs and tubules Smooth ER: lipid synthesis Rough ER: connected ribosomes - protein synthesis

Question 31

Types of proteins produced at the rER

Answer 31

Secretory proteins - Water soluble enzymes/hormones Membrane proteins - Embedded in the lipid bilayer

Question 32

ER signalling sequence

Answer 32

Common feature: 8+ hydrophobic amino acids (leucine/isoleucine/valine) usually near the N-terminus Targetting to the ER begins while the protein is still being synthesised (translocation occurs during translation)

Question 33

Signal recognition particle

Answer 33

SRP binds to the ER sequence as it emerges from the ribosome. SRP binds to an SRP receptor in the ER membrane Once the protein has been fully directed to the ER, SRP dissociates for reuse

Question 34

ER translocation: what is the process behind it?

Answer 34

The ER signal sequence moves through the Sec61 translocator and then loops back around and attaches to the Sec61 translocator as the polypeptide still moves through the ER membrane while translation is still occurring. Once the protein is fully through the ER membrane, a signal peptidase will snip off the ER signal sequence and the protein is then moved around the ER lumen.

Question 35

Stop-transfer sequence

Answer 35

If the protein is to be membrane-bound, then a hydrophobic stop-transfer sequence will stop translocation and the Sec61 translocator will dissociate from the polypeptide chain and the protein will be membrane-bound with the stop-transfer sequence as a transmembrane domain

Question 36

How can multiple transmembrane domains be established?

Answer 36

By having the stop-transfer sequence later in the chain

Question 37

How do mitochondria make energy?

Answer 37

Oxidative phosphorylation to produce ATP which is synthesised by the energy made from the movement of protons through ATP synthase The proton gradient is established by movement across the electron transport chain after high-energy electrons are obtained from the citric acid (Krebs) cycle

Question 38

DNP: what is it, what can it do, and is it dangerous?

Answer 38

2,4-dinitrophenol is an organic compound that is (illegally) sold in slimming aid pills It uncouples oxidative phosphorylation, reducing ATP production CAN BE LETHAL

Question 39

Thermogenin: what is it, what can it do, and is it dangerous?

Answer 39

A protein that drains the proton gradient, heating up the organism This can be dangerous as less ATP is produced as the gradient is reduced

Question 40

Where are mitochondrial proteins produced and what are the effects of the mutations?

Answer 40

99% in the nucleus but 1% in the mitochondria DNA If a mitochondrial mutation occurs, it'll only be passed on the mother's side

Question 41

IVF to remove defective mitochondria in eggs

Answer 41

Method 1: The nucleus of the fertilised egg is removed and the nucleus of the donor egg (father also fertilises) is removed and replaced with the fertilised DNA, essentially replacing the mitochondria Method 2: fertilisation occurs after the nucleus has been replaced

Question 42

Mitochondrial targeting sequences: what is the primary sequence and what is the secondary sequence?

Answer 42

Located at the N-terminus of the polypeptide chain, usually between 20-80 amino acids long and rich with Arginine, threonine, and serine Usually has an α-helix with positively charged (red) residues on one and non-polar (green) residues on the other face giving it unique amphipathic properties The MTS is cleaved off after entering the mitochondria

Question 43

The process of protein importation into the mitochondria(?)

Answer 43

A receptor in the outer membrane of the mitochondria binds to the MTS and then the MTS binds to another receptor in the inner membrane, entering the matrix.

Question 44

How is protein structure regained when in the matrix?

Answer 44

Chaperone proteins (Hsp70s) pull proteins through both membranes by ATP hydrolysis and then refolds them in the matrix

Question 45

How are proteins imported into chloroplasts

Answer 45

N-terminal chloroplast targeting sequence (CTS) made of Threonine, Serine, and other small hydrophobic amino acids which is cleaved off after entering the chloroplast The same general process as ER

Question 46

How are targetting sequences discovered?

Answer 46

By removing certain parts of the DNA, we can discover which parts are needed for proteins to go to the right place