Module 1 - Cells and their components Flashcards
Cell theory
- Cells are the fundamental units of life
- All organisms are composed of cells
- All cells come from pre-existing cells
Unicellular organisms and carrying out the functions of life
A single cell carries out all the functions of life
Multicellular organisms and carrying out the functions of life
Made of many cells that are specialized for different functions – tissues and organs
Internal structure of prokaryotes
- No nucleus: their DNA floats freely in the cell
- No (or rudimentary) internal membranes
- Very basic cytoskeleton
Internal structure of eukaryotes
- Have a nucleus containing DNA
- A complex internal membrane system
- Extensive cytoskeleton
Internal membranes within cells
- 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
Characteristics of ER
- 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
Two types of endoplasmic reticulum
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)
Characteristics of the Golgi apparatus
- 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
Cytosol: what is it, how large is it, what occurs here, what is located here, and what is it not to be confused with?
- 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)
How ATP/GTP is formed
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
How ATP/GTP is used - energy usage
ATP/GTP reacts with water, breaking the phosphoanhydride bond, and releasing the energy kept in the bond which is then used for intracellular work
The uses of ATP/GTP - affecting proteins
Nucleotide binding is used to change protein shape, activity, and function
Phosphorylation by adding phosphate to serine, tyrosine and threonine
What can phosphorylation do?
Affect:
Cell growth
Cell cycle
Cell division
Cell survival
Gene expression
Metabolism
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?
- 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.
Fluorescent dyes in fluorescent light microscopy: what do they do and how can they work?
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
Advanced fluorescent light microscopy: what does it allow?
Allows for 3D imaging because of “super resolution”
GFP
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?
Electron microscopes: what are the prerequisites for the specimen, what do they show, and what cells can be used?
- Thin slices of material (sections) stained with heavy metals for contrast
- Detailed subcellular structure
- Dead cells
Cell fractionation
- 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)
SDS: what is it and what does it do?
Sodium dodecyl sulphate
An ionic (negative charge) detergent that binds to proteins
Denatures proteins (useful for electrophoresis)
Polyacrylamide
Mesh-like gel that charged proteins move through, separating based on size
Protein targetting
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
Signal sequences: what are they and when are they removed?
Stretches of polypeptide sequences made up of specific types of amino acids
May be removed after the protein reaches its target location
The nuclear envelope: what is it made of and what does each part do?
- 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
Crossing the nuclear envelope
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
Nuclear targetting signals
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
Protein import
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)
Ran: what is it and what does it do?
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)
The endoplasmic reticulum: what is it, what are the two types and what do they do?
The most extensive membrane system in eukaryotic cells made of connected sacs and tubules
Smooth ER: lipid synthesis
Rough ER: connected ribosomes - protein synthesis
Types of proteins produced at the rER
Secretory proteins - Water soluble enzymes/hormones
Membrane proteins - Embedded in the lipid bilayer
ER signalling sequence
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)
Signal recognition particle
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
ER translocation: what is the process behind it?
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.
Stop-transfer sequence
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
How can multiple transmembrane domains be established?
By having the stop-transfer sequence later in the chain
How do mitochondria make energy?
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
DNP: what is it, what can it do, and is it dangerous?
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
Thermogenin: what is it, what can it do, and is it dangerous?
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
Where are mitochondrial proteins produced and what are the effects of the mutations?
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
IVF to remove defective mitochondria in eggs
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
Mitochondrial targeting sequences: what is the primary sequence and what is the secondary sequence?
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
The process of protein importation into the mitochondria(?)
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.
How is protein structure regained when in the matrix?
Chaperone proteins (Hsp70s) pull proteins through both membranes by ATP hydrolysis and then refolds them in the matrix
How are proteins imported into chloroplasts
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
How are targetting sequences discovered?
By removing certain parts of the DNA, we can discover which parts are needed for proteins to go to the right place
