BIOL 2020 Flashcards
Briefly describe the movement of cargo through the endomembrane system
Protein transcribed in cytosol, binds to ER membrane and moves into lumen or stays on membrane, goes to golgi for processing via vesicles, then post-golgi vesicle to either PM or extracellular or lysosome.
What does pulse-chase mean in terms of the experiment?
Pulse is the incubating of the cells with radioactive proteins, and the chase is the wait time that varies to see where proteins are in the cell at different times/steps of the secretory pathway.
How is GFP used to track proteins?
GFP fusion with cell protein by incorporating GFP into corresponding DNA so that protein is green fluorescent once translated.
What are microsomes? What is an experiment that allows them to be obtained?
Microsomes are functional golgi and ER membrane vesicles that are created during the homogenization of the cell. One the homogenization and centrifugation takes place, the post-nuclear supernatant can be further centrifuged to obtain a microsome pellet.
This technique is known as sub-cellular fractionation, as these microsomes can be sorted further via a density centrifugation.
What is the principle behind sec mutants?
If you don’t know how something works, break it and see what happens.
Whose work with sec mutants allowed us to see the different classes of secretory mutations?
Randy sheckman (2013 Nobel Laureate) worked with yeast and selected heat sensitive mutant yeast to investigate where different different mutation would affect the sec pathway.
What are the 5 classes of sec mutants based on the accumulation of protein?
Class A: accumulate in cytoplasm before ER binding
Class B: accumulate in ER (no budding)
Class C: accumulate in post-ER vesicles (no golgi fusion)
Class D: accumulate in golgi (no budding)
Class E: accumulate in sec vesicles (no PM fusion or exit to extracellular).
What are 3 main function of the smooth ER?
- Calcium storage (important in cell signalling).
- Steroid hormone synthesis
- Detoxification
Where do free ribosomes and memb-bound ribosomes direct their proteins to go?
Free ribosomes: remain in cytosol, nucleus, peripheral cytosolic leaflet proteins, proteins for mitochondria.
Memb-Bound ribosomes: proteins for secretion, integral membrane proteins, proteins destined to remain in an endomembrane compartment.
How do membrane bound ribosome proteins and free ribosome proteins differ in terms of their import mechanism?
Membrane-bound ribosome proteins are involved in co-translational import.
Free ribosome proteins are imported post translationally.
What is the signal hypothesis and who created it?
Gunter Bloebel (1999 Nobel laureate) created the signal hypothesis which has 3 points:
- All ribosomes are the same.
- Amino acid signals on new proteins (the signal sequences) direct growing polypeptides where to go (tells free ribosome to go the ER or tells ER ribosome to deposit).
- That protein will then be fed into ER lumen at the same time as being translated (co-translational import).
Describe the translocons role in co-translational import.
The translocon is located on the ER membrane and has 3 regions: the SRP receptor, the pore, signal peptidase.
The SRP (signal recgongnition particle bound to peptide also binds to SRP receptor on translocon.
After this, the polypeptide is red through the pore and into the ER lumen.
Once the polypeptide is in, the singal peptidase cleaves the singal sequence on the polypeptide (allows release of polypeptide).
What is SRP?
Signal recognition particle recognizes the signal sequence on an mRNA, binds to it, and based on that signal/what SRP binds, the polypeptide can migrate accordingly via the facilitation of the SRP.
It is composed of 6 polypeptides and some RNA.
How did sub-cellular fractionation experiments provide evidence for Bloebel’s theory?
In this experiment, 2 vitro and 1 vivo condition samples were examined using gel electrophoresis monitoring the length of proteins. In one test tube (vitro 1) it contained RNA, amino acids, and ribosomes. The second test tube(vitro 2) had all the same plus microsomes. When comparing size of proteins, test tube 2 with microsomes showed polypeptides of similar length to that of the vivo sample, whereas test tube 1 lacking microsomes (and therefore the translocon) showed larger proteins.
This can be explained as the lack of microsomes/ER and golgi membranes lacked the translocon and therefore the signal sequence could not be cleaved, resulting in larger proteins.
What kind of transmembrane protein would be created from an internal stop-transfer sequence and terminal ER signal sequence?
COO- terminal on cytosolic side and amino terminal inside lumen.
What kind of transmembrane protein would be created from a single internal start-transfer sequence?
Amino end of cytosolic side and COO- end in the lumen
What is a multi-pass integral membrane protein?
Once with multiple stop/start transfer sequences so that there are multiple portions of protein on cytosolic side and inside the lumen.
What enzyme is responsible for adding sugars to dolichol phosphate on cytoplasmic side on ER?
Glycosol transferases add carbohydrates to lipid (dolichol phosphate) on cytoplasmic side.
What enzyme is responsible for flipping the sugar chain in dolichol phosphate over to the lumen side? What is this flipping process called?
Translocation is the process in which flippase coordinates the flip of the sugar chain attached to dolichol phosphate to lumen.
Sugars are continued to be added the sugar chain once
What sugars on the sugar chain thats attached to dolichol phosphate complete the chain? What can happen after this sugar chain is complete?
3 glucoses complete the sugar chain, allowing oligosaccharide protein transferase to remove the sugar chain (core oligosaccharide) off of dolichol phosphate and add it to an asparagine of a protein.
What does calnexin do? What happens if it doesn’t do its job properly the first time?
Calnexin is a molecular chaperone that ensures proper folding of the newly created protein. Once calnexin ensures proper folding, the final glucose can be removed via glucosidase 2 (recall that the final oligosaccharide on the protein has 3 glucoses, 2 are removed before calnexin, and this last one is removed after).
If calnexin “didn’t do its job properly” (i.e., protein is mis-folded, indicative of hydrophobic regions that are not tucked away) UGGT (a glycosol transferase) can recognize this improper folding and add another glucose to that calnexin can try again.
What happens if protein will not fold properly after several cycles of UGGT calnexin interactions?
A transporter will recognize this protein that won’t fold properly and will feed it back into the cytoplasm via reverse translocation to be destroyed by a proteasome.
Describe the structure of the proteasome.
A barrel shaped protein degrading machine.
Consists of a cap at each end that binds to ubiquitin-tagged proteins (meaning tagged for degradation).
Proteases that do the actual degrading of the protein.
ATPases that drive the degradation process by powering the proteases.
What enzymes are involved in tagging a protein with ubiquitin?
E3 is a ubiquitin ligase that recognizes the misfolded protein intended for degradation and will transfer ubiqutins, being carried by the other 2 enzymes: E1 and E2, onto this protein.
Once protein is polyubiquitinated it can bind to the cap(s) of the proteasome.
What molecular chaperone is involved in the unfolded protein response occuring when too many unfolded proteins are present?
Bip/Hsp70.
Briefly describe the process of protein processing in the ER, beginning at entry.
The protein is first transported into the ER lumen via the translocon. Post translational modifications taken place in the ER, such as glycosolation. N-linked glycosolation, in specific, is involved in quality control. Quality control ensures the proper folding of proteins. If a protein will not fold properly, it can exit the ER by reverse translocation to be destroyed by the proteasome.
What does Bip do when there are too many unfolded proteins in the ER?
Bip abandons the sensors on the ER membrane to help with protein folding.
What are the two ways that the Bip sensors can be activated once Bip leaves?
They can either a) dimerize, to phosphorylate elF2a which will then bind to the small subunit of ribosomes to slow translation, and/or b) Undergo proteolytic cleavage so that the cytosolic portion of the censor can go to the nucleus and act as a transcription factor for genes associated with alleviating stress of misfolded protein response.
Who was the golgi complex named after?
Camillo Golgi, 1906 nobel laureate.
What is the function of the golgi?
Further post-translation modifications of proteins, specifically glycosylation (mainly further N-linked processing occurs).
There are many different glycosolation processes taking place which is why the golgi is comprised of separate compartments.
What is the older, no longer accepted model of the golgi?
The vesicular transport model: stating that cargo proteins are moved through the golgi cisternae via vesicles, indicating that the cisternae are stationary and the cargo passes through them by continuous vesicle budding.
Explain the cisternal maturation model.
The cisternal maturation model is the currently accepted-model of the golgi, and states that the cis cisternae is created by fusion of post-ER vesicles in the ERGIC. These vesicles, at the CGN, then mature becoming medial cisternae, and eventually reaching the TGN where vesicles can bud off.
In this model the cisternae are then not stationary, rather they mature, and the only vesicles involved are at first the fusion at CGN, budding at TGN, as well as the vesicles involved in transporting the resident enzymes.
The golgi resident enzymes move via retrograde vesicle back to their home cisternae as the cisternae matures.
What are the 3 main evidence points for the cisternal maturation model?
- Labelling cargo proteins only ever reveal them in cisternae, never in vesicle while in the golgi network. This indicates maturation rather than vesicle transport.
- Golgi-resident enzyme labelling shows them moving in vesicle and in cisternae in a retrograde fashion, suggesting they have a home compartment they are constantly being moved from.
- Sec mutants involved in the inhibition of ER vesicle budding show that the golgi disappears, suggesting that the budding vesicles (which can’t occur in these mutants) creates the golgi cisternae, explaining why the golgi disappears.
What are the 2 main functions of coat proteins?
- Membrane curvature: assembly of coat proteins creates a mechanical force driving membrane to curve into vesicle shape.
- They dictate what can enter the vesicle (via receptors) and what can interact with the membrane.
Different coat proteins are involved with different types of vesicles.
Explain what vesicles are involved with COP2, COP1, and clathrin coats.
COP2: ER to golgi movement, anterograde.
COP1: Golgi to ER, or TGN to CGN, retrograde.
Clathrin: TGN to out of cell, or PM to endosomes.
What monomeric G protein is involved with COP2 vesicles?
Monomeric G protein (a.k.a GTPase) involved wit COP2 vesicle formation is a Sar1.
What are the 2 adaptor proteins involved with COP2 vesicles?
Sec 23 and sec24, they form a heterodimer that binds the cytoplasmic tail of the transmembrane cargo receptors, Sar1 (it recruited them), and binds the outer-coat polypeptides.
What are the 2 outer coat polypeptides on COP2 coated vesicles?
Sec 13 and Sec31, which enable the vesicle to bud off.
How do the coat proteins fall off the vesicle?
The monomeric G protein hydrolyzes GTP.
Where would protein accumulate in a sec 23, 24, 13, or 13 mutant?
Sec 23, 24, 13, and 31 proteins are all involved in COP2 vesicle formation, which are the vesicle that move from the ER to the golgi. So if these proteins were mutated, they would not be able to bud from the ER and therefore they would accumulate there.
What is the GTPase (monomeric G protein) and outer coat proteins involved in COP1 vesicle formation?
Arf 1 is the GTPase, and there are 7 different outer coat proteins that form a coatamer with triskellion shape (b1 and alpha components).
Where do COP1 vesicles move in the cell?
They move retrogradely either from TGN to CGN, or from golgi to ER (i.e., when returning ER proteins back after COP2 delivered them).
How do proteins know to move retrogradely in COP1 vesicles?
The ER enzymes that need to move retrograde has a specific KDEL sequence (lys-asp-glu-lue) that can bind to specific receptors that allow them to move back to home compartment via COP1 vesicles.
Describe the structure of clathrin coated vesicles (outer coat).
Triskellion shape with 3 heavy chains and 3 light chains that can form a hexagon or pentagon shape. Hexagon shape is flat and can convert to pentagon shape to get rounded formation. Triskellion forms lattice around vesicle.
What are the 2 types of adaptor proteins that can recruit clathrin?
- GGAs: vesicles going from TGN to endo/lysosomes via anterograde motion. The GTPase for this is Arf1.
- AP2: vesicles coming from PM into endo/lysosomes via retrograde movement. The GTPase for this is no specified.
What is dynamin and what is its function?
Dynamin is a monomeric G protein that forms a ring around the clathrin coated vesicles. Once hydrolyzed, it pinches off the vesicle to be released.
What do GTP gamma S do?
Dynamin bound to GTP gamma S is activated, similarly to when its activated bound to normal GTP, but with gamma S it cannot hydrolyze and therefore the vesicle cannot be pinched off. This forms a neck like structure that the vesicle and membrane are attached to.
Describe the function of the lysosome and its part in the secretory pathway.
The lysosome is involved in intracellular digestion. It contains acid hydrolyases (meaning they are optimal in acidic conditions) and they degrade all of the main macromolecules.
They contain proton pumps to make themselves acidic.
Acid hydrolyases are made in ER, tagged with mannose-6-phopsphate in the golgi, and transported to lysosome due to their tag.
The low pH of the late endosome (right before it is converted to the lysosome) dissociates the receptor from the acid hydrolase (tagged with mannose 6 phosphate), so that the receptor can be recycled.
What kind of vesicles are destined to go to the lysosome?
Clathrin coated vesicles that contain acid hydrolyases tagged with mannose 6 phosphate.
Their adaptor protein GGA binds mannose 6 phosphate/receptor, Arf1 GTP, and clathrin outer coat.
What are Rabs and SNAREs?
Rabs are small GTP binding proteins that specify vesicle destination, approximately 60 types of them. They associate via a lipid anchor and recruit tethering proteins to loosely attach the vesicle.
SNAREs are membrane proteins that mediate vesicle fusion with alpha helix domains (coil to coiled): t SNARE on target membrane that will associate with v SNARE on vesicle membrane. This is the docking step of vesicle fusion.
Briefly describe the process of vesicle fusion that RABs and SNARES are involved in.
- Target transport vesicle via lipid anchor.
- Tethering occurs when rabs recruit tethering proteins to loosely attach the vesicle (tethering proteins connect to vesicle holding it in place).
- Docking, now that vesicle is close by the help of tethering proteins, SNAREs coil together to pull vesicle close to PM.
- Fusion is promoted by SNARE interaction .
- Dissociation occurs when NSF uses ATP to untwist the SNAREs, release them along with Rab.
What vesicles are formed during receptor-mediated endocytosis and what coat proteins mediate this?
Clathrin coated vesicles are created in receptor-mediated endocytosis. There is no GTPase, rather ligand and receptor interaction causes diffusion to make a coated pit, where an invagination occurs and clathrin begins coating on cytoplasmic side.
Adaptor protein AP2 (4 polypeptide complex) facilitates this by connecting to the clathrin, the receptor (that extend through PM to connect to ligand.
What are the 2 main differences in receptor mediated endocytosis vesicle formation?
After this recap different dissociation of coat proteins in the other types of vesicles.
- There is no monomeric G protein (rather ligand receptor interaction that diffuses to form coated pit).
- In the other types of vesicles, the GTPase was what was responsible for the uncoating, since there is no GTPase here, and ATPase needs to come in and bind to coat proteins, hydrolyze, and allow dissociation.
Recall:
Arf1 invovled in clathrin coats from TGN is GTPase that can hydrolyze at end to dissociate coat proteins.
Arf 1 involved in COP1 coated vesicles undergoing retrograded movement is a GTPase that can hydrolyze to dissociate coat proteins (but no adaptor protein specified, rather their KDEL sequence on these proteins allow binding to various receptors).
Sar1 involved in COP2 coated vesicles undergoing anterograde movement from ER to golgi is a GTPase that can hydrolyze to dissociate coat proteins.
How are receptor-ligand complexes released after receptor mediated endocytosis?
After the ATPase uncoats the clathrin coat (releases clathrin and AP2), you are left with vesicle containing the cargo, but the cargo is still attached to the receptors from the initial coated pit formation. When the vesicle fuses with the early endosome, the pH increases slightly which reduces receptor ligand affinity, receptors are released back to PM.
After the clathrin coated vesicle fuses with late endosome what does it fuse with?
Can then fuse to form lysosome, sometimes fuses with clathrin vesicles coming from TGN, where pH increases and allows hydrolyases to be activated.
What are methods of acidification for the lysosomes and endosomes?
- Proton pump in the lysosome.
2. Endosome fuses with lysosome that can take on new cargo.
What is phagocytosis?
The engulfment of relatively large particles, NOT facilitated by receptor-ligand interaction like in receptor mediated endocytosis. Here, pseudopods form to engulf materials which fuses with early endosome, and eventually lysosome for digestion.
What is autophagy and who studied it?
Yoshinori Ohsumi won nobel prize in 2016 for his work with autophagy, which is the destruction of organelles by isolation in a double membrane followed by fusion with a lysosome. He showed a mitochondria being sequestered in a autophagosome.
How do proteins get to the mitochondria?
They get there due to transit sequences that direct them there by binding to Hsp 70 and Hsp 90, which delivers them in an unfolded state.
What are the 2 fates once a protein reaches the mitochondria for entry?
Once it enters into the IM via the TOM (Transport outer membrane), it can either go through TIM 22 if it wants to reside in the IM, or through TIM 23 if it wants to go to the matrix.
Once in the matrix it binds to Hsp 60 chaperone, and mitochondrial transit peptidase will cleave its transit sequence.
What are each of the 3 main cytoskeletal elements made of (what are they polymers of?)
- Microtubules are polymers of tubulin
- Microfilaments are polymers of actin
- Intermediate filaments are polymers of varying proteins
All polymers have non covalent linkages necessary for their dynamic nature.
Compare alpha and beta tubulin.
Similar amino acid sequence and 3D conformation allowing for easy linking, however, b tubulin has ability to hydrolyze GTP to GDP, so b tubulin may have GDP or GTP cap and alpha tubulin will only ever have GTP cap.
This creates a plus and minus end on MT. Plus end is b tubulin end, and minus end is alpha tubulin end.
Explain the role of Tau in the cell and what can happen if it is mutated.
Tau is a structural/classical MAP that helps maintain the structure of MTs. If mutated, MT structure can unstable, resulting in tangled MTs and Tau clumps. Clinically speaking, this can lead to frontotemporal dementia.
How were motor proteins first discovered.
Motor proteins were first discovered when studying the squid giant axon and vesicles were seen moving in both directions along the axon.
Compare dynein and kinesin.
Dynein moves toward minus end of cell and kinesin moves toward positive end of cell.
Both have head regions that hydrolyze ATP and tail regions that bind cargo.
Dynein requires dynactin to help bind MT and vesicle.
Explain the hand over hand mechanism seen with Kinesin.
- ATP binding to leading head
- This triggers the trailing head to swing in front (power stroke)
- MT binding of new leading head takes place
- ATP hydrolysis of new trailing head and ADP is released from leading head.
What are the 2 exceptions to kinesin that do not move toward plus end.
Kinesin 13 doesn’t move.
Kinesin 14 moves toward minus end.
Explain how kinesin motility assays work.
First secure the kinesin tails to coverslip preventing movement of kinesin.
Then add MT stained.
Then watch MT slide along kinesin head, this occurs because kinesin can move so MT does instead as a result of the head hand over hand mechanism.
Where are MTOCs in the cell and what is their function?
They are located perinucleur and near centre of cell.
The function to organize MT and as a result orient kinesin-mediated and dynein-mediated transport of vesicles, organelles, and vesicular tubular clusters.
What are centrosomes made of?
2 perpendicular centrioles and pericentriolar material (PCM).
Centrioles are composed of 9 triplet microtubules.
What is PCM and what is its significance in the cell.
PCM is a diffuse granular matrix that surrounds the centrioles. It seems to be recruited by centrioles to aid in MT nucleation.
It nucleates MT by providing a basis for growth: first has the base called gamma-tubulin ring complex, which is topped with a gamma tubulin ring that allows for alpha and beta tubulin to begin attaching.
Describe the structural hierarchy of MTs.
Tubulin comprises MT, and exists as a heterodimer of alpha and beta tubulin. These heterodimers form protofilaments, which then can make up a MT when 13 are linked together.
Explain dynamic instability model associated with MTs.
Growth can only occur at the beta/plus end where GTP can be hydrolyzed.
Plus end begins with GTP cap.
Caps promotes addition of dimers and MT will grow.
As it grows, GTP hydrolyzed to GDP, converting the GTP cap to a GDP cap.
Beta tubulin bound to GDP more prone to shrinkage (catastrophe).
What is the “9 + 2 array” ?
The microtubule structure seen in the axoneme of cilia and flagella. It has 9 doublet MT and 2 single central MT when looking at a cross section.
What would happen if the doublets weren’t linked?
Axonemal dynein attached to one MT on a doublet walks along a MT of an adjacent doublet, this results in bending. Bending occurs because they are attached. If they weren’t attached, dynein would just continue to walk along carry the MT with it and they would slide past each other.
What is plectin?
An IF associated protein that forms bridges between MFs, MTs, and IFs.
Briefly describe the hierarchical structure of intermediate filaments.
- Coiled regions of two proteins linked together forms dimer, amino and c terminus are aligned (both on same side, therefore polar).
- Two dimers join to form a tetrameric protofilament that align anti-parallel making this non polar
- Protofilaments join together in bundles making up the IF (overall non polar).
How is the assembly of IF different from MT and MF assembly?
- New tetramers are incorporated into middle of filament not the ends.
- No ATP involved, instead phosphorylation dictates growth: phosphotases remove phosphate causing assembly, and kinases add phosphate causing disassembly.
