1
Q
All cells have what features?
A
- All have a polymer chain(s) in the form of DNA
- Posses nucleotides (protein bound to sugar, bound to a nitrogenous base (A,C,T,G))
- DNA replication via complementary base pairs (templated polymerisation)
- Central dogma is DNA→RNA→Protein
- Enclosed by an amphiphilic plasma membrane
2
Q
Describe prokaryotes
A
- Exist as single cell or loose community
- Oval shape with a cilia
- Have cell wall, DNA is randomly distributed within the cytoplasm
- Much smaller than eukaryotes, plants are also eukaryotes
3
Q
- When a protein with one binding site binds with another protein only with one binding site, you end up with a ____
- When a protein with two binding sites, one up top, one on bottom, continuously bind with other proteins to form a chain, a ____ results
- When a protein with two binding sites, one being slightly askew, bonds with similarly shaped proteins a ___ will form
A
dimer
helix
ring
4
Q
What are the three types of protein binding interfaces?
A
- Surface-string
- Helix-helix
- Surface-surface
5
Q
What factors affect the shape of proteins?
A
- Things such as polar v.s nonpolar bonds as well as hydrogen bonds
- The configuration of its amino acids For example hydrophobic clustering is when the hydrophobic AAs (polar bonded) gravitate to the centre of the protein whereas the hydrophilic (non-polar bonded) ones surround the exterior
6
Q
What is hydrolase?
A
- Proteins that destroy / breakdown things. There are two types:
1. Endonuclease (destroy nucleic acid from the centre, DNA RNA)
2. Exonuclease (destroy nucleic acid from the ends)
7
Q
What is a protease?
A
- Protein that breaks down other proteins
- Binds to ligase called substrates. Acts as a catalyst that permits cells to make or break bonds controllably.
Note:
- Increase in the concentration of substrate ➔ increases the rate at which product is formed, eventually reaching a maximum value (maximum rate/Turnover Number)
8
Q
____ = the concentration at which the reaction is happening at half its maximum speed
- ____ means that even with a low conc of substrate the reaction can happen at ½ its max speed. So the binding between enzyme and substrate is strong and tight.
- ____ takes a lot of substrate to reach Vmax, so the enzyme and substrate probably bind fairly weakly
A
Km
Low Km
High Km
9
Q
How do cells regulate enzyme activity?
A
- Regulating the expression of the gene that codes the enzyme
- Confining enzymes to specific subcellular compartments
- Protein destruction
- Molecule binding
10
Q
- _____ Enzymes have two or more binding sites
- _____ site: recognizes the substrate
- _____ site: recognizes a regulatory molecule
- In both positive and negative ____ _____, the regulatory molecule has totally different shape than the enzyme’s substrate
A
Allosteric
Active
Regulatory
feedback regulation
11
Q
Describe positive enzymatic regulation
A
When inactive enzymes and substrates float around loosely. Then after, the introduction of regulatory molecule occurs which bind to the enzymes and encourage them to take up the substrate and become active
12
Q
Describe negative enzymatic regulation
A
Active enzymes float around with their substrates. Then regulatory molecules are introduced which bind to the enzymes and encourage them to release their substrates and become inactive
13
Q
Describe enzymatic transition states
A
- Substrates pass through intermediate state of altered geometry before forming the products of the reaction
- Transition state: the most unstable intermediate state
- The energy required to get to that unstable intermediate state: Activation Energy
- The activation energy is the major determinant of the reaction rate
- Goes from enzyme binds with substrate → ES → EP → Enzyme + product
14
Q
What are multienzyme complexes?
A
- The cell can increase reaction rates without raising substrate concentrations by bringing the various enzymes involved in a reaction sequence together to form a large protein assembly
15
Q
What is the difference between gated and vesicular transport?
A
Gated:
- transport between the cytosol and nucleus through nuclear pore complex in the nuclear envelope (which act as selective gates)
Viscle:
- membrane-enclosed transport -ferries proteins from one compartment to another
16
Q
What do transmembrane protein translocators do?
A
Directly transport proteins across a membrane from cytosol
17
Q
Describe the process of protein targeting.
A
A signal sequence is located on the n-terminus or middle of the polypeptide chain and designates a specific destination. So pretty much tells the protein where to go. These sequences are used to get proteins to their destinations (google maps)
18
Q
Describe the process of transport through the ER
A
- A protein is made with an ER signal sequence (ERSS)
- One end of a signal recognition particle (SRP) binds to ERSS to guide it towards destination, the other end blocks the elongation factor binding site so that synthesis pauses
- The SRP receptor which is attached to ER, binds to SRP opening a translocator allowing for the SRP to unbind from the elongation factor site and for protein synthesis to resume, feeding into the ER lumen
- The SRP then unbinds from the SRP receptor and is recycled to repeat the process
19
Q
Mitochondrial proteins are first fully made as mitochondrial ____ ____ in the cytosol then translocated into the mitochondria via a ___ ____ mechanism
A
precursor proteins
post-translational
20
Q
Describe the transport of a protein into the mitochondria
A
- Precursor protein is marked by a mitochondrial signal sequence (MSS)
- The MSS binds to a receptor protein in the TOM complex on the outer membrane
- The TOM complex then acts as a gate and inserts the protein into the membrane
- The MSS after fed through into the membrane, binds to the TIM23 complex on the inner membrane
- The TIM complex translocates the protein into the matrix, where it has it’s MSS cleaved from it by peptidase
21
Q
Describe protein import into the matrix
A
- Chaperone protein hsp70 helps keep mitochondrial precursor proteins unfolded as it arrives to the translocator of the outer membrane (TOM)
- ATP hydrolysis separates hsp70 protein from the precursor protein
- The signal sequence of precursor protein has entered the outer membrane and is guided to translocator (TIM) situated between the inner membrane and the matrix
- Mitochondrial hsp70 binds tightly to an imported protein chain as soon as it emerges from the TIM translocator in the matrix space and pulls protein through the translocation channel using ATP hydrolysis
- Once fully in matrix mitochondrial hsp60 uses ATP hydrolysis to fold the protein
22
Q
What are Pore-Forming 𝛃-barrel Proteins?
A
Occurs when:
1. Protein is bound for the outer membrane
2. Protein passes through TOM
3. Chaperone proteins bring protein to a SAM complex
4. SAM complex folds and bends protein into this straw-like shape which remains on the outer membrane and acts as a pore
23
Q
Describe the import of proteins into the intermembrane space
A
- Protein has a intermembrane signal sequence (ISS) and immediately following that stop-transfer sequence (STS)
- Protein arrives at TIM where translocation occurs
- Once the ISS passes through the TIM complex into the matrix it is cleaved
- Now that the STS is getting processed the TIM complex recognises it and stops translocation
- The STS is anchored into the inner membrane and the protein remains there like an inflatable man
- Sometimes the TIM fully translocates the full protein, if this happens:
5. The STS acts as a signal sequence and the protein gets taken from the matrix to an OXA complex on the inner membrane
6. OXA complex ejects the protein back into the intermembrane space where it anchors itself in the inner membrane - Note that some mitochondrial synthesised proteins can also pass through OXA
24
Q
Describe the import of proteins into a chloroplast
A
- Thylakoid precursor protein is tagged with thylakoid signal sequence (TSS) and then a chloroplast signal sequence (CSS)
- CSS binds to receptor protein in the TOC complex on the outer chloroplast membrane
- TOC complex translocates protein into intermembrane space
- Protein gets translocated through the TIC complex on the inner chloroplast membrane (requires GTP or ATP)
- CSS gets cleaved, leaving TSS exposed
- TAT complex translocates the protein into the thylakoid membrane
- TSS is cleaved from protein
25
ER translocators need accessory proteins called ____, a binding protein, to feed the polypeptide chain. This then associates with ____ ATP-dependent cycles.
____ = A translocator working in tandem with an accessory protein(s)
BiP
Sec61 (core of translocator)
Translocon
26
____ = when protein is fully synthesised by free-ribosmes in cytosol before entering ER lumen
Post-Translational Translocation
27
What is the process for a stop transfer of a single pass from cytosol to ER lumen?
1. Post-Translationally made protein has a signal sequence at the beginning of the chain and a stop-transfer sequence near the middle
2. The signal sequence brings the protein to a protein translocator found on the er membrane
3. The signal sequence is anchored into the membrane and the protein begins to be translocated into the er lumen
4. Once the stop-transfer signal is reached the signal sequence is cleaved from the protein by peptidase, leaving half of the protein in the the cytosol (5' end) and half the protein in the ER lumen (3' end)
28
What is the process for a stop transfer of a multipass from cytosol to ER lumen?
1. Post-Translationally made protein has a start-transfer sequence at about 1/3 of the chain and a stop-transfer sequence near 2/3
2. The signal sequence brings the protein to a protein translocator found on the er membrane
3. The start-transfer sequence is bound to the protein translocator and gets anchored into the membrane and then translocation into the er lumen begins, in this case the 3' end remains in the cytosol and translocation goes towards the stop transfer sequence (5' end)
5. The stop-transfer sequence is reached and translocation finishes this results in both ends being exposed to the cytosol and within the er lumen a loop is formed, called a mature double-pass transmembrane protein
29
How do proteins undergo quality control?
- At the ER protein glycolysation occurs and oligosaccharides (many monosaccharides) are added to proteins which ensures that the proper folding happens by binding to incompletely folded proteins and keeping them in the ER.
- Uses ER chaperone proteins calnexin and calreticulin
- Catalysed by oligosaccharyltransferase (glucosyl transferase) which adds a glucose to those oligosaccharides that have lost their last glucose ONLY if it is attached to an unfolded protein
- Glucose trimming is done by glucosidase
- Glucose addition is done by glucosyl transferase
30
Describe the process of retrotranslocation
1. Proteins leave calnexin and then the attempt to pass from the er lumen to the cytosol
2. Chaperone protein brings a misfolded protein to a protein translocator complex on the er membrane
3. Protein is fed through and recognised to be improperly folded by the E3 ubiquitin ligase which then attaches polyubiquitin tags to unfolded proteins as they emerge into the cytosol - marking them for destruction
4. AAA-ATPase uses ATP so that the protein is folded enough to be broken down by proteasome
5. N-glycanase removes the oligosaccharide tree from the protein
6. Protein is brought to proteasome and broken down
31
What is the Heat-Shock Response?
Bodily response that stimulates the transcription of genes encoding cytosolic chaperones that help to refold the proteins
32
What is the Unfolded Protein Response?
Increased transcription of genes encoding proteins involved in:
- Retrotranslocation
- ER chaperones
33
What are the three types of coated vesicles and their destinations?
1. Clathrin-coated = Golgi to Plasma membrane transport
2. COPI-coated: Golgi to ER
3. COPII-coated: ER to golgi
34
Describe the process of transport from ER to golgi
1. Proteins leave the ER towards golgi in COPII-coated vesicles
2. The vesicles bud from specialised ER exit sites
3. Soon after the coat is shed and the vesicles fuse with one another to form a vesicular tubular cluster
4. Clusters move on microtubule tracks to the golgi which endogenously assimilates them
35
Describe the KDEL sequence and receptor
KDEL is a target peptide sequence in mammals and plants located on the C-terminal end of the amino acid structure of a protein. The KDEL sequence prevents a protein from being secreted from the endoplasmic reticulum (ER) and facilitates its return if it is accidentally exported
36
Describe the gogli apparatus
- Cis face = entry face
- Trans face = exit face
- Some proteins live and function within golgi or they leave to their next destination
- Sorts and distributes proteins
37
Describe the pathway of proteins along the trans gogli network (TGN) to lysosomes
1. Manose has a acid hydrolase (AH) precursor added to it in the cis golgi network
2. A phosphate is added
3. M6P signal on complex is uncovered
4. Complex binds to an M6P receptor in a budding vesicle in the trans golgi network
5. Vesicle buds off and heads towards endosome
6. Vesicle assimilates in endosome and the phosphate is removed from the complex
38
Explain endosome maturation process
1. Early endosome matures to a late endosome which contains hydrolase and intraluminal vesicles
2. Endosome fuses with a lysosome which contains only hydrolase
3. The fusion results in the formation of a endolysosome
4. Within the endolysosome the hydrolase interacts with the the intraluminal vesicles and breaks them down
5. After all are broken down you're left with an endolysosome that contains only hydrolase, which makes it a lysosome
39
What is exocytosis and it's two pathways?
- Exocytosis: Fusion of the vesicles with the plasma membrane
- Constitutive Secretory Pathway: operates continuously
- Regulated secretory pathway: soluble proteins and other substances are stored secretory vesicles for later release by exocytosis
39
What are regulated secretory vesicles?
Cells that are specialised for secreting some of their products rapidly on demand concentrate and store these products in regulated secretory vesicles
(also known as: dense-core secretory granules)
39
What does the Endocytic-Exocytic Cycle entail?
The same amount of membrane being removed by endocytosis is being added to the cell surface by the exocytosis
40
As endosomes mature, patches of their membrane ____ into the endosome lumen and pinch off to form ____ _____. Endosome with ____ _____ is called a _____ body
invaginate
intraluminal vesicles
intraluminal vesicles
multivesicular
41
How are vesicles coated when they begin budding off?
1. Clathrin coated using triskelions which assemble into "cage-like" coated buds (clathrin cage)
2. Adaptor proteins which form the inner layer of the coat and bind the clathrin coat to the membrane
3. Dynamin which is a cytoplasmic protein that seals the forming vesicle right before it buds off
42
What is the job of membrane-bending proteins?
- Have crescent-shaped domains (BAR Domains)
- Bind to and impose their shape on the underlying membrane to help a clathrin-coated vesicle to form
43
LDL (low-density lipoprotein) cholesterol, sometimes called “____” cholesterol, makes up most of your body's cholesterol. High levels of LDL cholesterol ____ your risk for heart disease and stroke
bad
raise
44
What is a phagosome?
A large endocytic vesicle used to ingest large particles
45
COPII-coated vesicle formation – _______
COPI-coated vesicle formation- ______
Sar1 involved
Arf involved
46
What are the functions of GEFs and GAFS
- Protein bound to GTP = ACTIVE (GTP-bound state) to
- Protein bound to GDP = INACTIVE (GDP-bound state)
- GEFs are molecular switches that activate the protein (GDP-->GTP)
- GAFs are molecular switches that deactivate the protein (GTP-->GDP)
47
Coat disassembly process?
1. Vesicle pinches off, protein becomes inactivated by GAP and hydrolyzes GTP->GDP
2. COPI & Clathrin – sheds coat right away
3. COPII – sheds coat closer to the target membrane
4. Coat disassembles and vesicle ready for fusion
Year 2 Sem 1
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